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WO2022125673A1 - Cell-penetrating peptides and peptide complexes and methods of use - Google Patents

Cell-penetrating peptides and peptide complexes and methods of use Download PDF

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Publication number
WO2022125673A1
WO2022125673A1 PCT/US2021/062422 US2021062422W WO2022125673A1 WO 2022125673 A1 WO2022125673 A1 WO 2022125673A1 US 2021062422 W US2021062422 W US 2021062422W WO 2022125673 A1 WO2022125673 A1 WO 2022125673A1
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WO
WIPO (PCT)
Prior art keywords
seq
peptide
cell
penetrating
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2021/062422
Other languages
French (fr)
Inventor
Steven Chen
Zachary CROOK
Natalie Winblade Nairn
Scott Presnell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Blaze Bioscience Inc
Fred Hutchinson Cancer Center
Original Assignee
Blaze Bioscience Inc
Fred Hutchinson Cancer Center
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Publication date
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Publication of WO2022125673A1 publication Critical patent/WO2022125673A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43522—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from scorpions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • a major objective of drug development is ensuring that a therapeutic agent can reach its intended target. Frequently, this means designing therapeutics that can reach the cytosol, nucleus, or other subcellular compartments of a cell. Drugs with poor cell permeation may not reach an intended cytosolic, nuclear, or other intracellular drug target, resulting in decreased efficacy. Moreover, drugs with poor cell permeation may also require higher dosages than those with more targeted or more successful cell permeation, resulting in a higher incidence of toxicity or side effects. Biologic drugs, such as those made of peptides, proteins, knotted peptides, or miniproteins, RNA, and DNA frequently are unable to enter the cytosol and therefore these classes of drugs are not able to drug intracellular targets. There is a need for methods to deliver drugs with low permeation or absorption across cellular membranes to reach cytosolic and nuclear drug targets.
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising a sequence that has: at least 80% sequence identity with any one of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20 - SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 44 - SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 75, SEQ ID NO: 77, or SEQ ID NO: 80, or a fragment thereof; at least 85% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:
  • the peptide complex comprises a sequence that has at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, or a fragment thereof. In some aspects, the peptide complex comprises a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84. In some aspects, the peptide complex comprises a sequence of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 8; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 40; SEQ ID NO: 41; or SEQ ID NO: 42. In some aspects, the cell-penetrating peptide is fused or linked to a cargo molecule.
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof.
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, SEQ ID NO: 195 - SEQ ID NO: 254, or a fragment thereof, and wherein the cargo molecule is a cystine-dense peptide, a DNA- binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or combinations thereof.
  • the cargo molecule is
  • the cell-penetrating peptide comprises a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • the peptide complex comprises a sequence of: SEQ ID NO: 195; SEQ ID NO: 197; or SEQ ID NO: 198.
  • the cellpenetrating peptide comprises a sequence of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 8; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 40; SEQ ID NO: 41; or SEQ ID NO: 42.
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence of any one of SEQ ID NO: 325 - SEQ ID NO: 342 or SEQ ID NO: 343 - SEQ ID NO: 351, and wherein the cargo molecule wherein the cargo molecule is a cystine-dense peptide, a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or combinations thereof.
  • the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, at least 3.5, at least 3.7, or at least 4.0 arginine amino acid residues per ten amino acid residues.
  • the cell-penetrating peptide comprises no more than 0.5, no more than 0.8, no more than 0.9, no more than 1.0, no more than 1.1, no more than 1.2, no more than 1.3, no more than 1.4, no more than 1.5, no more than 1.6, no more than 1.7, no more than 1.8, no more than 1.9, no more than 2.0, no more than 2.1, no more than 2.2, no more than 2.3, no more than 2.4, no more than 2.5, no more than 3.0, no more than 3.3, no more than 3.5, no more than 3.7, or no more than 4.0 arginine amino acid residues per ten amino acid residues.
  • the cell-penetrating peptide comprises no arginine amino acid residues.
  • the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, at least 3.5, at least 3.7, or at least 4.0 lysine amino acid residues per ten amino acid residues.
  • the cell-penetrating peptide comprises no more than 0.5, no more than 0.8, no more than 0.9, no more than 1.0, no more than 1.1, no more than 1.2, no more than 1.3, no more than 1.4, no more than 1.5, no more than 1.6, no more than 1.7, no more than 1.8, no more than 1.9, no more than 2.0, no more than 2.1, no more than 2.2, no more than 2.3, no more than 2.4, no more than 2.5, no more than 3.0, no more than 3.3, no more than 3.5, no more than 3.7, or no more than 4.0 lysine amino acid residues per ten amino acid residues.
  • the cell-penetrating peptide comprises no lysine amino acid residues. [0012] In some aspects, the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least
  • the cell-penetrating peptide comprises no more than 4.0, no more than 3.9, no more than 3.8, no more than 3.7, no more than 3.6, no more than 3.5, no more than 3.4, no more than 3.3, no more than 3.2, no more than 3.1, no more than 3.0, no more than 2.9, no more than 2.8, no more than 2.7, no more than
  • the cellpenetrating peptide comprises at least 0.03, at least 0.05, at least 0. 1, at least 0.2, at least 0.5, at least 0.8, at least 0.9, at least 1 .0, at least 1 .
  • the cell-penetrating peptide comprises a ratio of positively charged amino acid residues to negatively charged amino acid residues of at least 1.0, at least 1.5, at least 2, at least 2.5, at least 2.75, at least 3, at least 3.5, at least 4, at least 5, at least 6, or at least 7.
  • the cell-penetrating peptide comprises a ratio of negatively charged amino acid residues to positively charged amino acid residues of no more than 1.0, no more than 1.5, no more than 2, no more than 2.5, no more than 2.75, no more than 3, no more than 3.5, or no more than 4. In some aspects, the cell-penetrating peptide comprises at least 1, at least 2, at least 3, or at least 4 negatively charged amino acids.
  • the cell-penetrating comprises at least 1, at least 2, at least 3, or at least 4 histidine amino acid residues. In some aspects, the cell-penetrating comprises at least 1, at least 2, at least 3, or at least 4 proline amino acid residues. In some aspects, the cell-penetrating peptide comprises an amphipathic a-helix. In some aspects, the cell-penetrating peptide comprises at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues. In some aspects, the cell-penetrating peptide comprises no cysteine amino acid residues.
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising: at least 1.0, at least 1.8, at least 1.9, at least 2.0, at least 2.2, at least 2.5, at least 3.0, at least 3.3, or at least 3.5 arginine amino acid residues per 10 amino acid residues; and at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues; wherein the cell -penetrating peptide is fused or linked to a cargo molecule.
  • the cell-penetrating peptide comprises at least four cysteine amino acid residues. In some aspects, the cell-penetrating peptide comprises no more than 3.3, no more than
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising: at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, or at least 2.7 arginine amino acid residues per 10 amino acid residues; and no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than
  • the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising: at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, or at least 3.5 arginine amino acid residues per 10 amino acid residues; and no more than 3.3, no more than 3.2, no more than 3.1, no more than 3.0, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, or no more than 2.0 positively charged amino acid residue per 10 amino acid residues at a pH of 7.4; wherein the cell-penetrating peptide is fused or linked to a cargo molecule.
  • the positively charged residues are arginine, lysine, or any combination thereof.
  • the cell-penetrating peptide comprises no cysteine amino acid residues.
  • the cell-penetrating peptide comprises at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues.
  • the cell-penetrating peptide comprises a disulfide through disulfide knot.
  • the cell-penetrating peptide comprises a plurality of disulfide bridges formed between cysteine residues.
  • the cell-penetrating peptide comprises three or more disulfide bridges formed between cysteine residues, wherein one of the disulfide bridges passes through a loop formed by two other disulfide bridges. In some aspects, the cell-penetrating peptide comprises at least 1.1 arginine amino acid residues per 10 amino acid residues. In some aspects, the cell-penetrating peptide comprises no more than 2.7 positively charged amino acid residue per 10 amino acid residues.
  • the cell-penetrating peptide is derived from maurocalcin, imperatoxin, hadrucalcin, hemicalcin, opicalcin-1, opicalcin-2, midkine, MCoTI-II, chlorotoxin, huwentoxin, vejocalcin, intrepicalcin, or urocalcin.
  • the cell-penetrating peptide or the fragment thereof comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least, 8, at least
  • the cell-penetrating peptide or the fragment thereof comprises no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than
  • no more than 11 no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, no more than 30, no more than 31, no more than 32, no more than 33, no more than 34, no more than 35, no more than 40, no more than 50, no more than 60, no more than 70, or no more than 80 residues.
  • the cellpenetrating peptide comprises an isoelectric point within a range from about 6.0 to about 12.0, from about 6.0 to about 10.0, from about 6.5 to about 7.5, from about 7.0 to about 10.0, or from about 8.0 to about 10.0.
  • the cell-penetrating peptide is stable at pH of from 6.5 to 7.5.
  • the cell-penetrating peptide is stable at pH values within a range from pH 5.0 to pH 7.0.
  • the cargo molecule comprises a cargo peptide comprising four or more cysteine amino acid residues and at least two disulfide bonds.
  • the cargo molecule is fused or linked to the cell-penetrating peptide at an N-terminus or a C-terminus of the cell-penetrating peptide.
  • the cargo molecule comprises at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors.
  • the cargo molecule comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors.
  • the cargo molecule comprises a molecular weight of at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, or at least 1000 Da.
  • the cargo molecule comprises a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0.
  • logP partition coefficient
  • the cargo molecule comprises an antibody, an antibody fragment, an Fc domain, a single chain Fv, an intrabody, or a nanobody.
  • the cargo molecule comprises a cystine-dense peptide, an affibody, a B-hairpin, an avimer, an adnectin, a stapled peptide, a nanofittin, a kunitz domain, a fynomer, or a bicyclic peptide.
  • the cargo molecule comprises an immunomodulatory imide drug, a Boc3Arg tag, an adamantyl group, or a carborane.
  • the cargo molecule comprises a target-binding molecule.
  • the target-binding molecule comprises a target-binding peptide.
  • the target-binding peptide comprises a cystine-dense peptide.
  • the target-binding peptide is capable of binding a transcription factor or a tyrosine kinase.
  • the target-binding peptide is capable of binding TEAD, cold-inducible RNA-binding protein, androgen receptor, ikaros, aiolos, a nuclear receptor, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, tau, AKT,
  • the target-binding peptide comprises a sequence with at least 90% or at least 95% sequence identity to any one of SEQ ID NO: 295, SEQ ID NO: 298, or SEQ ID NO: 364 - SEQ ID NO: 407. In some aspects, the targetbinding peptide comprises a sequence of any one of SEQ ID NO: 295, SEQ ID NO: 298, or SEQ ID NO: 364 - SEQ ID NO: 407. In some aspects, the target-binding peptide comprises a sequence of SEQ ID NO: 295.
  • the peptide complex comprises a sequence of SEQ ID NO: 307 or SEQ ID NO: 308.
  • the target-binding molecule binds a ubiquitin ligase.
  • the ubiquitin ligase comprises cereblon, cellular inhibitor of apoptosis protein 1, MDM2, DCAF15, DCAF16, cullin-4A, a Cul2-Rbxl-EloN/C-VHL E3 ubiquitin ligase, APC/C activator protein CDH1, or von Hippel -Lindau protein.
  • the target-binding molecule comprises an immunomodulatory imide drug.
  • the target-binding molecule comprises a thalidomide, a pomalidomide, a lenalidomide, a methyl bestatin, a bestatin, a nutlin-3, or a VHL ligand 1.
  • the cargo molecule comprises an anticancer agent, a transcription factor binding agent, an inhibitor of protein-protein interactions, a promoter of protein-protein interactions, a transcription factor, an RNA, a CRISPR component, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or an immunomodulating agent.
  • the oligonucleotide comprises a DNA, an RNA, an antisense oligonucleotide, an aptamer, an miRNA, an alternative splicing modulator, an mRNA-binding sequence, an miRNA-binding sequence, an siRNA-binding sequence, an RNaseHl -binding oligonucleotide, a RISC-binding oligonucleotide, a polyadenylation modulator, a gapmer, a RIG-I ligand, an mRNA, an antisense RNA, a small interfering RNA, a guide RNA, a U1 adaptor, a micro RNA, or a combination thereof.
  • the oligonucleotide comprises: a sequence of any one of SEQ ID NO: 488 - SEQ ID NO: 573; a sequence that binds to any one of SEQ ID NO: 574 - SEQ ID NO: 611; a sequence of any one of SEQ ID NO: 574 - SEQ ID NO: 611, or a fragment thereof; or a sequence targeting or encoding a gene target provided in TABLE 12.
  • the CRISPR component is a guide RNA, a tracrRNA, a crRNA, or a Cas nuclease.
  • the cargo molecule comprises a detectable agent or a therapeutic agent.
  • the detectable agent is a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a radioisotope, or a radionuclide chelator.
  • the peptide complex further comprises one or more chemical modifications.
  • the chemical modification extends the half-life or modifies a pharmacokinetics of the peptide complex.
  • the chemical modification blocks an N-terminus of the peptide complex.
  • the chemical modification comprises methylation, acetylation, or acylation.
  • the chemical modification comprises: methylation of one or more lysine residues or analogue thereof; methylation of the N-terminus; or methylation of one or more lysine residue or analogue thereof and methylation of the N- terminus.
  • the peptide complex further comprises a half-life modifying agent, a nuclear localization signal, or an endosomal escape motif.
  • the half-life modifying agent comprises a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin.
  • PEG polyethylene glycol
  • a hydroxyethyl starch polyvinyl alcohol
  • a water soluble polymer a zwitterionic water soluble polymer
  • a water soluble poly(amino acid) a water soluble poly(amino acid)
  • the peptide complex further comprises an additional active agent.
  • the additional active agent is fused to the cell-penetrating peptide or the cargo molecule at an N-terminus, at the epsilon amine of an internal lysine residue, at the carboxylic acid of an aspartic acid or glutamic acid residue, or a C-terminus of the cell-penetrating peptide or the cargo molecule by a linker.
  • the linker comprises an amide bond, an ester bond, a carbamate bond, a carbonate bond, a hydrazone bond, an oxime bond, a disulfide bond, a thioester bond, a thioether bond, or a carbon-nitrogen bond.
  • the linker comprises a peptide linker.
  • the peptide linker comprises a sequence of any one of SEQ ID NO: 255 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485.
  • the linker is a cleavable linker or a pH sensitive linker.
  • the cleavable linker comprises a cleavage site for matrix metalloproteinases, thrombin, cathepsins, or betaglucuronidase.
  • the linker is a hydrolytically labile linker.
  • the linker is a stable linker.
  • the additional active agent comprises at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors. In some aspects, the additional active agent comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors. In some aspects, the additional active agent comprises a molecular weight of at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, or at least 1000 Da. In some aspects, the additional active agent comprises a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0.
  • logP partition coefficient
  • the additional active agent is a detectable agent or a therapeutic agent.
  • the detectable agent is a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a radioisotope, or a radionuclide chelator.
  • the cell-penetrating peptide is a membrane-penetrating peptide. In some aspects, the cell-penetrating peptide is a nuclear envelope-penetrating peptide. In some aspects, the cell-penetrating peptide is a blood brain barrier-penetrating peptide. In some aspects, the cell-penetrating peptide is arranged in a multimeric structure with at least one other peptide. In some aspects, the cell-penetrating peptide lacks an immunogenic sequence. In some aspects, the cell-penetrating peptide is modified to increase homology to a human protein sequence. In some aspects, the cell-penetrating peptide is modified to increase resistance to degradation.
  • the cell-penetrating peptide is modified to reduce an affinity of the peptide for a human leukocyte antigen complex, a major histocompatibility complex, or both. In some aspects, the cell-penetrating peptide is an active agent.
  • the present disclosure provides a pharmaceutical composition comprising a peptide complex of the present disclosure, or a salt thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated for administration to a subject.
  • the pharmaceutical composition is formulated for oral administration, intravenous administration, subcutaneous administration, intramuscular administration, or a combination thereof.
  • the present disclosure provides a method of delivering a cargo molecule across a cellular layer of a cell, the method comprising: contacting the cell with a peptide complex comprising a cell-penetrating peptide fused or linked to cargo molecule, wherein the cell-penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof; penetrating the cellular layer with the cell -penetrating peptide; and delivering the cargo molecule across the cellular layer.
  • the present disclosure provides a method of delivering a cargo molecule across a cellular layer of a cell, the method comprising: contacting the cell with a peptide complex of the present disclosure comprising a cell-penetrating peptide fused or linked to cargo molecule; penetrating the cellular layer with the cell-penetrating peptide; and delivering the cargo molecule across the cellular layer, thereby treating the condition.
  • the present disclosure provides a method of treating a condition in a subject in need thereof, the method comprising: administering to the subject a composition comprising a peptide complex comprising a cell-penetrating peptide fused or linked to cargo molecule, wherein the cell -penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof; penetrating a cellular layer of a cell of the subject with the cell-penetrating peptide; and delivering the cargo molecule across the cellular layer, thereby treating the condition.
  • the present disclosure provides a method of treating a condition in a subject in need thereof, the method comprising: administering to the subject a composition comprising a peptide complex of the present disclosure comprising a cell-penetrating peptide fused or linked to cargo molecule; penetrating a cellular layer of a cell of the subject with the cell-penetrating peptide; and delivering the cargo molecule across the cellular layer.
  • the disease or condition is cancer, a neurological disorder, an inflammatory disorder, an immune disorder, a neurodegenerative disorder, or a genetic disorder.
  • the cancer is liver cancer, breast cancer, colon cancer, lung cancer, prostate cancer, brain cancer, skin cancer, pancreatic cancer, leukemia, or lymphoma.
  • the composition is administered by inhalation, intranasally, orally, topically, intravenously, subcutaneously, intramuscularly administration, intraperitoneally, intratumorally, intrathecally, intravitreally, or a combination thereof.
  • the composition is administered intravenously as a bolus, injection, infusion, or prolonged infusion.
  • the cellular layer is a cell membrane, a nuclear envelope, an endosomal membrane, a lysosomal membrane, or a blood brain barrier.
  • the method comprises penetrating a cell membrane of the cell. In some aspects, the method comprises penetrating a nuclear envelope of the cell. In some aspects, the method comprises delivering the cargo molecule to a cytosol of the cell. In some aspects, the method comprises producing a cargo molecule concentration of at least 10 nM, at least 50 nM, at least 100 nM, at least 300 nM, at least 500 nM, at least 700 nM, at least 1000 nM, at least 1200 nM, at least 1400 nM, or at least 1600 nM in the cytosol of the cell. In some aspects, the method comprises delivering the cargo molecule to a nucleus of the cell.
  • the method comprises producing a cargo molecule concentration of at least 10 nM, at least 50 nM, at least 100 nM, at least 300 nM, at least 500 nM, at least 700 nM, at least 1000 nM, at least 1200 nM, at least 1400 nM, or at least 1600 nM in the nucleus of the cell.
  • the cargo molecule comprises an oligonucleotide that binds a target sequence
  • the method further comprises modulating alternative splicing of the target sequence, dictating the location of a polyadenylation site of the target sequence, inhibiting translation of the target sequence, inhibiting binding of the target sequence to a secondary target sequence, recruiting RISC to the target sequence, recruiting RNaseHl to the target sequence, inducing cleavage of the target sequence, or regulating the target sequence upon binding of the oligonucleotide to the target sequence.
  • the method comprises delivering the cargo molecule into an intracellular space or a paracellular space.
  • the intracellular space is a nanolumen.
  • the cell has uncontrolled or dysregulated cell growth.
  • the cell is a cancerous cell or a tumor cell.
  • the cell is a pancreatic cell, liver cell, colon cell, smooth muscle cell, ovarian cell, breast cell, lung cell, brain cell, skin cell, ocular cell, blood cell, lymph cell, immune system cell, reproductive cell, reproductive organ cell, prostate cell, fibroblast, kidney cell, adenocarcinoma cell, glioma stem cell, tumor cell, or any combination thereof.
  • the method further comprises binding the cargo molecule to a target molecule.
  • the target molecule comprises a transcription factor or a tyrosine kinase.
  • the target molecule comprises TEAD, cold-inducible RNA-binding
  • -l i protein, androgen receptor ikaros, aiolos, a nuclear receptor, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-5, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, tau, AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, P-caten
  • the method further comprises inhibiting the target molecule.
  • the method further comprises binding the cargo molecule to a ubiquitin ligase.
  • the ubiquitin ligase comprises cereblon, cellular inhibitor of apoptosis protein 1, MDM2, DCAF15, DCAF16, cullin-4A, a Cul2-Rbxl-EloN/C-VHL E3 ubiquitin ligase, APC/C activator protein CDH1, or von Hippel -Lindau protein.
  • the cargo molecule comprises an immunomodulatory imide drug, a thalidomide, a pomalidomide, a lenalidomide, a methyl bestatin, a bestatin, a nutlin-3, or a VHL ligand 1.
  • the method further comprises ubiquitinating the target molecule upon binding of the cargo molecule to the target molecule and the ubiquitin ligase.
  • the cargo molecule comprises a detectable agent.
  • the method further comprises imaging the cell.
  • the method further comprises detecting a presence, absence, location, or a combination thereof of the detectable agent in the cell.
  • the method comprises delivering at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% of the peptide complex across the cellular layer.
  • FIG. 1A schematically illustrates a process of covalently tagging peptides with a benzylguanine SNAP substrate by using BG-GLA-NHS.
  • the NHS-ester of a benzylguanine SNAP substrate (BG-GLA-NHS) reacts with a reactive amine group, such as at the N-terminus or at a lysine residue, of a peptide to generate a SNAP substrate-tagged peptide (referred to as a BG-peptide, a BG-peptide complex, a BG-cystine-dense peptide, or a BG-cell-penetrating peptide).
  • FIG. IB schematically illustrates a SNAP penetration assay (SNAPP A) to quantify cell penetration of a BG-peptide.
  • SNAPP A SNAP penetration assay
  • BG-fluorophore a SNAP substrate- tagged fluorophore that can diffuse across the cell membrane
  • Free SNAP -tag protein in the cell will bind to the BG-fluorophore, while SNAP -tag protein that is bound to BG-peptide cannot bind to the fluorescent SNAP substrate because the BG-peptide already occupies the SNAP -tag.
  • a cell penetrant SNAP substrate-tagged peptide (BG-peptide)
  • BG-peptide cell penetrant SNAP substrate-tagged peptide
  • a cell penetrant peptide blocks the SNAP -tag from binding fluorescent SNAP substrate and therefore reduces the fluorescent signal in the assay.
  • This assay may also be used to assess penetration into other cellular compartments, such as the nucleus, by expressing a SNAP -tag protein fusion that localizes to the cellular compartment of interest.
  • FIG. 2 shows representative images of reversed-phase high performance liquid chromatography (RP-HPLC, top) and size exclusion chromatography (SEC, second, third, and bottom) associated with a typical SNAP substrate conjugation reaction with peptide and subsequent purification process.
  • the bottom two images show RP-HPLC traces of concentrated fractions following SEC, demonstrating separation and purification of BG-peptides and unreacted peptides. Peaks in the top panel correspond to, from left to right, BG-GLA-NHS hydrolyzed to BG-GLA-OH, BG-GLA-NHS ligand, KR CTX (SEQ ID NO: 71), and KR CTX BG-peptide (BG-SEQ ID NO: 71).
  • FIG. 3 shows representative fluorescence images of the SNAPPA assay after contacting various BG-peptides comprising cystine-dense peptides to HeLa cells stably transfected with pSNAPf and expressing SNAP -tag, followed by addition of BG-fluorophore and washing away unbound BG-fluorophore.
  • BG-peptides comprising cystine-dense peptides, KR_KTxl5.8 (BG-SEQ ID NO: 74), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG-SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59), were assayed for cell penetration.
  • Phosphate buffered saline (PBS) was used as a negative control.
  • FIG. 4A shows the results of a SNAP penetration assay measuring cellular penetration into the cytosol by various BG-peptides comprising cystine-dense peptides exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag protein in the cytosol.
  • the BG-peptides comprising cystine-dense peptides were assayed for cell penetration to reach the cytosol.
  • FIG. 4B shows the results of a SNAP penetration assay measuring cellular penetration into the nucleus by various BG-peptides comprising cystine-dense peptides exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag protein in the nucleus.
  • the BG-peptides comprising cystine-dense peptides were assayed for cell penetration to reach the nucleus.
  • FIG. 5 schematically illustrates BG-peptides complexes comprising cell-penetrating peptides and cargo peptides assayed by SNAPPA in FIG. 6A - FIG. 8D.
  • FIG. 6A shows results of a SNAP penetration assay to measure cellular penetration into the cytosol of the cell-penetrating peptide complexes illustrated in FIG. 5 exposed to NIH3T3, HeLa and HEK293 cells.
  • BG-MCa-KTx3.10 Cellular penetration into the cytosol of penetrating peptide complexes BG-MCa-KTx3.10 (BG-SEQ ID NO: 312), BG-MCa-elafin (BG-SEQ ID NO: 316), BG- KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) and BG-KR_IpTxa-elafin (BG-SEQ ID NO: 317) was measured, along with BG-cargo peptides without cell-penetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297).
  • BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration of these cargo peptides with the addition of further cell-penetrating peptides.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
  • FIG. 6B shows results of a SNAP penetration assay to measure cellular penetration into the nucleus of the cell-penetrating peptide complexes illustrated in FIG. 5 exposed to NIH3T3, HeLa and HEK293 cells.
  • BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG- substrate) is hydrolyzed, was used as a positive control and for normalization.
  • BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
  • FIG. 7A shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-KTx3.10, with increasing concentrations of a BG- MCa-KTx3.10 (BG-SEQ ID NO: 312) cell-penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 7B shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR_IpTxa-KTx3.10, with increasing concentrations of a BG-KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) cell-penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 7C shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-elafin, with increasing concentrations of a BG- MCa-elafin (BG-SEQ ID NO: 316) cell-penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm.
  • the data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 7D shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR IpTxa-elafin, with increasing concentrations of a BG-KR_IpTxa-elafin (BG-SEQ ID NO: 317) cell -penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 8A shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-KTx3.10, with increasing concentrations of a BG- MCa-KTx3.10 (BG-SEQ ID NO: 312) cell-penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • the data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 8B shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR_IpTxa-KTx3.10, with increasing concentrations of a BG-KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) cell-penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 8C shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-elafin, with increasing concentrations of a BG- MCa-elafin (BG-SEQ ID NO: 316) cell-penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • the data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 8D shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR IpTxa-elafin, with increasing concentrations of a BG-KR IpTxa-elafm (BG-SEQ ID NO: 317) cell -penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • the data was normalized to cells exposed to 10 pM BG-GLA-OH.
  • FIG. 9 schematically illustrates BG-cargo peptides and BG-cell penetrating peptide- cargo peptide complexes assayed by SNAPPA in FIG. 10A and FIG. 10B.
  • FIG. 10A shows results of a SNAP penetration assay to measure cellular penetration to reach the cytosol of the cell-penetrating peptide complexes illustrated in FIG. 9 in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm.
  • BG-peptide comprising CDP peptides complexes BG-MCa(loop)-KTx3.10 (BG-SEQ ID NO: 314), BG-peptide of MCa(loop)-elafin (BG-SEQ ID NO: 318), BG-C3A_MCa(l-9)-KTx3.10 (BG-SEQ ID NO: 315), and BG-C3A_MCa(l-9)-elafin (BG-SEQ ID NO: 319) was measured, along with BG-cargo peptides without cell-penetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297).
  • BG-KTx3 10 (BG-SEQ ID NO: 296) and BG-elafm (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration of these cargo peptides with the addition of further cell-penetrating peptides.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • FIG. 10B shows results of a SNAP penetration assay to measure cellular penetration to reach the nucleus of the cell-penetrating peptide complexes illustrated in FIG. 9 exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • BG-peptide comprising CDP peptides complexes BG-MCa(loop)-KTx3.10 (BG-SEQ ID NO: 314), BG-MCa(loop)-elafm (BG-SEQ ID NO: 318), BG-C3A_MCa(l-9)-KTx3.10 (BG- SEQ ID NO: 315), and BG-C3A_MCa(l-9)-elafin (BG-SEQ ID NO: 319) was measured, along with BG-cargo peptides without cell-penetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafm (BG-SEQ ID NO: 297).
  • BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafm (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration of these cargo peptides with the addition of further cell-penetrating peptides.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • FIG. 11 schematically illustrates BG-peptides complexes comprising a TEAD-binding peptide (“TEAD-binder”), with or without a cell-penetrating peptide, assayed by SNAPPA in FIG. 12A, FIG. 12B, and FIG. 13
  • FIG. 12A shows results of a SNAP penetration assay to measure cellular penetration to reach the cytosol of the cell-penetrating peptides conjugated to a TEAD-binding peptide illustrated in FIG. 11 contacted to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm.
  • SNAP substrate-tagged peptide comprising CDP TEAD-binding peptides complexes BG-C3A_MCa(l-9)-TEAD-binder (BG-SEQ ID NO: 320) and BG- C5A_Had(l-l l)-TEAD-binder (BG-SEQ ID NO: 321) was measured, along with BG-TEAD- binding peptide without cell-penetrating peptides BG- TEAD-binder (BG-SEQ ID NO: 298).
  • BG- TEAD-binder (BG-SEQ ID NO: 298) was used as a comparator to assess the basal level of penetration of this cargo peptides with the addition of further cell-penetrating peptides.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • the SNAP substrate-tagged peptide (BG- peptide) comprising BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
  • FIG. 12B shows results of a SNAP penetration assay to measure cellular penetration to meet the nucleus of the cell-penetrating peptides conjugated to a TEAD-binding peptide illustrated in FIG. 11 exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • a SNAP substrate-tagged peptide comprising CDP TEAD-binding peptides complexes BG-C3A_MCa(l-9)-TEAD-binder (BG-SEQ ID NO: 320) and BG- C5A_Had(l-l l)-TEAD-binder (BG-SEQ ID NO: 321) was measured, along with BG-TEAD- binding peptide without cell-penetrating peptides (BG-SEQ ID NO: 298).
  • BG-TEAD-binder (BG-SEQ ID NO: 298) was used as a comparator to assess the basal level of penetration of this cargo peptides with the addition of further cell-penetrating peptides.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
  • FIG. 13 shows results of a SNAP penetration assay to measure cellular penetration into the nucleus of the cell-penetrating peptides conjugated to a TEAD-binding peptide (“TEAD- binder”) illustrated in FIG. 11 exposed to primary GSC cells transiently transfected with pSNAPf-H2B and expressing SNAP -tag in the nucleus.
  • TEAD- binder TEAD-binding peptide
  • SNAP substrate-tagged peptide comprising CDP TEAD-binding peptides complexes BG-C3A_MCa(l-9)-TEAD- binder (BG-SEQ ID NO: 320) and BG-C5A_Had(l-l l)-TEAD-binder (BG-SEQ ID NO: 321) was measured, along with BG-TEAD-binding peptide without cell-penetrating peptides BG- TEAD-binder (BG-SEQ ID NO: 298).
  • BG-TEAD-binder (BG-SEQ ID NO: 298) was used as a comparator to assess the basal level of penetration of this cargo peptides with the addition of further cell-penetrating peptides.
  • FIG. 14A shows the results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR_IpTxa in the presence of a variety of endocytosis inhibitors to assess mechanisms responsible for uptake of a BG-KR_IpTxa cellpenetrating peptide (BG-SEQ ID NO: 59).
  • Cytoplasmic penetration of BG-KR IpTxa was measured when exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm while various inhibitors were added.
  • FIG. 14B shows the results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide MCa_varl in the presence of a variety of endocytosis inhibitors to assess mechanisms responsible for uptake of a BG-MCa_varl cellpenetrating peptide (BG-SEQ ID NO: 67).
  • BG-MCa_varl Cytoplasmic access of BG-MCa_varl was measured in NIH3T3, HeLa and HEK293 cells expressing SNAP-tag in the cytoplasm.
  • BG-MCa_varl with no inhibitor was used as a comparator to assess the level of penetration in the absence of inhibitor.
  • BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control for normalization.
  • FIG. 15A shows the results of a SNAP penetration assay of various SNAP substrate- tagged peptide (BG-peptide) comprising cell-penetrating peptides to measure cellular penetration into the cytosol of the cell-penetrating peptides fused to additional peptides that may further promote endosomal escape.
  • BG-peptide SNAP substrate- tagged peptide
  • BG-KR_IpTxa-PAS BG-SEQ ID NO: 322
  • BG-KR_IpTxa-S19 BG-SEQ ID NO: 323
  • BG-KR_IpTxa BG-SEQ ID NO: 59
  • BG-GLA-OH formed when BG-GLA-NHS (BG- substrate) is hydrolyzed, was used as a positive control and for normalization.
  • FIG. 15B shows the results of a SNAP penetration assay of various SNAP substrate- tagged peptides (BG-peptides) comprising cell-penetrating peptides to measure cellular penetration to the nucleus of the cell-penetrating peptides fused to additional peptides that may further promote endosomal escape.
  • BG-peptides SNAP substrate- tagged peptides
  • BG-KR_IpTxa-PAS nuclear penetration of BG-KR_IpTxa-PAS (BG-SEQ ID NO: 322) and BG-KR_IpTxa-S19 (BG-SEQ ID NO: 323) was measured in NIH3T3, HeLa and HEK293 cells expressing the SNAP-tag in the nucleus, along with BG-KR_IpTxa (BG-SEQ ID NO: 59).
  • BG-KR_IpTxa without an endosomal escape peptide was used as a comparator to assess the baseline level of penetration in the absence of an endosomal escape peptide.
  • the cell penetrant moiety BG-GLA-OH formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
  • FIG. 16A schematically illustrates various types of cargo peptides that may be conjugated to cell -penetrating cystine-dense peptides or peptide fragments.
  • FIG. 16B schematically illustrates various types of antibody fragments that may be conjugated to cell -penetrating cystine-dense peptides or peptide fragments.
  • FIG. 17 illustrates a multiple sequence alignment of calcin variants MCa_varl (SEQ ID NO: 67), KR Urocalcin (SEQ ID NO: 39), KR IpTxa (SEQ ID NO: 59), KR Hemicalcin (SEQ ID NO: 34), KR_Opicalcin-l (SEQ ID NO: 37), KR_Opicalcin-2 (SEQ ID NO: 38), KR Vejocalcin (SEQ ID NO: 35), and KR Intrepicalcin (SEQ ID NO: 36).
  • FIG. 18 shows the results of mass spectrometry analysis identifying tryptic fragments of the SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR_IpTxa (SEQ ID NO: 59; BG-KR_IpTxa) conjugated to SNAP -tag following a cell penetration assay in HeLa cells.
  • HeLa cells expressing GFP-SNAP-tag protein were incubated with 10 pM BG- KR IpTxa before subsequent trypsinization, lysis in mammalian protein extract reagent, and immunoprecipitation with magnetic beads. The recovered GFP-SNAP-tag conjugates were then submitted for mass spectrometry analysis.
  • FIG. 18 discloses SEQ ID NO: 616, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
  • FIG. 19 shows the results of mass spectrometry analysis identifying tryptic fragments of BG-KR_IpTxa conjugated to SNAP -tag following a cell penetration assay in HeLa cells to determine limits of sensitivity.
  • HeLa cells expressing GFP-SNAP-tag were exposed to either BG-KR_IpTxa or BG-GLA-OH.
  • One seventh of the cells were incubated with BG-KR_IpTxa and six sevenths were incubated with BG-GLA-OH.
  • the cells were pooled and prepared as above for mass spectrometry analysis, demonstrating the ability to detect uptake of BG-KR- IpTxa at this ratio.
  • FIG. 19 discloses SEQ ID NO: 617, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
  • FIG. 20 shows the results of mass spectrometry analysis identifying tryptic fragments of the SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR_IpTxa (SEQ ID NO: 59) (BG-KR_IpTxa) conjugated to GFP-SNAP-tag protein.
  • BG-peptide cell-penetrating peptide KR_IpTxa
  • BG-KR_IpTxa cell-penetrating peptide KR_IpTxa conjugated to GFP-SNAP-tag protein.
  • HeLa GFP-SNAP cells were lysed with M-PER supplemented with protease and phosphatase inhibitors before incubating with 10 pM BG-KR_IpTxa for 2 hours at 4C.
  • FIG. 20 discloses SEQ ID NO: 617, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
  • FIG. 21 shows the results of mass spectrometry analysis identifying additional tryptic fragments of the SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR IpTxa (SEQ ID NO: 59; BG-KR_IpTxa) conjugated to GFP-SNAP-tag protein in a cell free environment.
  • BG-peptide SNAP substrate-tagged peptide
  • KR IpTxa SEQ ID NO: 59
  • BG-KR_IpTxa cell-penetrating peptide KR IpTxa conjugated to GFP-SNAP-tag protein in a cell free environment.
  • Prototypic peptides from BG-KR_IpTxa were also detected under the previously described conditions with the tryptic fragment highlighted detected and identified by MS2, indicating the presence of intact BG-KR_IpTxa.
  • FIG. 21 discloses SEQ ID NO: 618 (ADNDCCGR
  • FIG. 22 shows the results of mass spectrometry analysis identifying tryptic fragments of BG-KR_IpTxa (SEQ ID NO: 59) in a cell free environment.
  • HeLa GFP-SNAP cells were lysed with M-PER supplemented with protease and phosphatase inhibitors before incubating with IpM BG-KR_IpTxa for 2 hours at 4 °C.
  • the modified GFP-SNAP-tag was detected and identified by MS2.
  • FIG. 22 discloses SEQ ID NO: 619, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
  • FIG. 23 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with PBS in the SNAPPA. Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP -Cell TMR-Star dye).
  • FIG. 24 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with 2 pM hydrolyzed BG-GLA-NHS (BG-GLA-OH) in the SNAPPA. Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP-Cell TMR-Star dye).
  • FIG. 25 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with 10 pM BG-KR_IpTxa in the SNAPPA.
  • Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP- Cell TMR-Star dye).
  • FIG. 26A shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the cytosol of 3T3 cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP -substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the cytosol, which was quantified as 936 nM based on these results.
  • the maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 26B shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the cytosol of HeLa cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP -substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the cytosol, which was quantified as 700 nM based on these results. The maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 26C shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the cytosol of HEK-293 cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the cytosol, which was quantified as 1378 nM based on these results.
  • the maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 27A shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the nucleus of 3T3 cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the nucleus, which was quantified as 1632 nM based on these results.
  • the maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 27B shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the nucleus of HeLa cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the nucleus, which was quantified as 471 nM based on these results.
  • the maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 27C shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the nucleus of HEK-293 cells.
  • SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the nucleus, which was quantified as 1523 nM based on these results.
  • the maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
  • FIG. 28A shows the results of SNAP penetration assays measuring the cytosolic uptake of BG-SEQ ID NO: 320 in the presence of various chemical inhibitors of different mechanisms of endocytosis.
  • Uptake was measured in 3T3 (“3T3 GFP-SNAP”), HeLa (“HeLa GFP-SNAP”), and HEK-293 (“293 GFP-SNAP”) cell lines expressing GFP-SNAP-tag protein in the cytosol. Uptake is shown as the fraction of GFP-SNAP-tag protein that was occupied, where a fraction of 1.0 indicates 100% of the protein was occupied.
  • the cell penetrant moiety BG-GLA-OH was used for normalization to 100% and PBS was used for normalization to 0%.
  • FIG. 28B shows the results of SNAP penetration assays measuring the cytosolic uptake of BG-SEQ ID NO: 321 in the presence of various chemical inhibitors of different mechanisms of endocytosis.
  • Uptake was measured in 3T3 (“3T3 GFP-SNAP”), HeLa (“HeLa GFP-SNAP”), and HEK-293 (“293 GFP-SNAP”) cell lines expressing GFP-SNAP-tag protein in the cytosol. Uptake is shown as the fraction of GFP-SNAP-tag protein that was occupied, where a fraction of 1.0 indicates 100% of the protein was occupied.
  • the cell penetrant moiety BG-GLA-OH was used for normalization to 100% and PBS was used for normalization to 0%.
  • FIG. 29A illustrates examples of components for use in building a targeted degradation complex for targeted degradation of TEAD.
  • Components include a cell-penetrating peptide, a TEAD-binding cystine-dense peptide (CDP), a linker, and an immunomodulatory imide drug (IMiD).
  • the TEAD-binding CDP is capable of binding TEAD and the IMiD is capable of binding cereblon (CRBN), an E3 ubiquitin ligase.
  • FIG. 29B illustrates construction of a targeted degradation complex for targeted degradation of TEAD.
  • the IMiD is linked to the cell-penetrating peptide/TEAD-binding CDP complex via a linker, thereby forming a targeted degradation complex.
  • FIG. 29C illustrates binding of the targeted degradation complex described in FIG. 29B to TEAD.
  • FIG. 29D illustrates ubiquitination and subsequent degradation of TEAD facilitated by the targeted degradation complex described in FIG. 29B.
  • the targeted degradation complex forms a ternary complex with TEAD and CRBN. Recruitment of CRBN to TEAD facilitates ubiquitination of TEAD, which targets TEAD for degradation.
  • FIG. 30 illustrates examples of molecules capable of binding an E3 ubiquitin ligase that may be incorporated into a targeted degradation complex.
  • Molecules capable of binding an E3 ubiquitin ligase include thalidomide or pomalidomide which bind cereblon (CRBN), methyl bestatin or bestatin, which bind cellular inhibitor of apoptosis protein 1 (cIAPl), nutlin-3 which binds MDM2, and VHL ligand 1 which binds von Hippel-Lindau protein (VHL).
  • FIG. 31 shows a western blot of cell lysates generated from HeLa GFP-SNAP cells that were treated with BG-peptides and then washed and lysed, where the blot was stained with an anti-GFP antibody.
  • Intact cells were incubated with 10 pM BG-SEQ ID NO: 320 or 10 pM BG- SEQ ID NO: 321 for 15, 30 or 60 minutes prior to washing, lysis, and sample preparation.
  • the band slightly below the 55 kDa marker may represent GFP-SNAP protein.
  • the band between the 55 kDa and the 72 kDa markers may represent the BG-peptide of SEQ ID NO: 320 or SEQ ID NO: 321 occupying the GFP-SNAP protein, thus causing a shift upward in molecular size versus the GFP-SNAP protein alone.
  • the fainter band between 95 and 130 kDa may represent a dimer of two GFP-SNAP proteins linked and occupied by a BG-peptide that contains at least two BG moieties (Lys residues and the N-terminus may react with BG-GLA-NHS). Occupancy of GFP-SNAP protein indicates the peptides of SEQ ID NO: 320 and SEQ ID NO: 321 penetrated the cells and entered the cytosol.
  • the loss of the shifted GFP-SNAP band suggests some intracellular degradation of exposed BG-peptide (where the cleavage could be on the N-terminal GS, the cell penetrating MCa or Had sequences, the GGS linker, or the TEAD binding peptide) or residues of the SNAP protein proximal to the reactive site may be occurring, resulting in the continued occupation of GFP-SNAP -tag protein and preventing subsequent BG-fluorophore binding.
  • FIG. 32 illustrates examples of structures of various peptide oligonucleotide complexes (e.g., a CPP-oligonucleotide complexes in which the peptide portion comprises a CDP or a fragment thereof) containing alternative and nonconventional bases, as represented in singlestranded, double-stranded, and hairpin structures.
  • oligonucleotides include an aptamer, a gapmer, an anti-miR, an siRNA, a splice blocker ASO, and a U1 adapter.
  • the CPP portion of the CPP-oligonucleotide complex can be used to guide the oligonucleotide sequence to a specific tissue, target, or cell, or to cause endocytosis, cytosolic or nuclear penetration, or endosomal escape of the oligonucleotide sequence by a cell.
  • FIG. 33A - FIG. 33E illustrate incorporation of the shown groups on RNA or DNA.
  • FIG. 33A illustrates structures of oligonucleotides containing a 5 ’-thiol (thiohexyl; C6) modification (left), and a 3 ’-thiol (C3) modification (right).
  • FIG. 33B illustrates an MMT-hexylaminolinker phosphoramidite.
  • FIG. 33C illustrates a TFA-pentylaminolinker phosphoramidite.
  • FIG. 33D illustrates RNA residues incorporating amine or thiol residues.
  • FIG. 33E illustrates oligonucleotides with aminohexyl modifications at the 5’ (left) and 3’ ends (right).
  • FIG. 34 illustrates generation of a cleavable disulfide linkage between a peptide (e.g., a cell-penetrating peptide of SEQ ID NO: 1) and a cyclic dinucleotide.
  • a peptide e.g., a cell-penetrating peptide of SEQ ID NO: 1
  • a cyclic dinucleotide e.g., SEQ ID NO: 1
  • Drug design is often limited by the ability of a drug to access intracellular targets.
  • Small molecule therapeutics can often diffuse through the cell membrane to reach intracellular targets, but small molecules may be unable to specifically and potently inhibit protein-protein interactions, or those interactions lacking well-defined binding pockets.
  • Protein- or peptide- based therapeutics such as antibodies, antibody fragments, and peptides, are more capable of specifically and potently disrupting protein-protein interactions, targeting poorly-defined binding pockets, or drive the formation of protein-protein interactions or otherwise have a therapeutic effect, but protein- or peptide-based drugs may have a difficult time reaching the intracellular milieu. Proteins and peptides are often too large or too hydrophilic to diffuse across the cell membrane.
  • RNA and DNA are large, hydrophilic, and charged and are often unable to diffuse across the cell membrane or unable to exit the endosome in order to access the cytosol, nucleus, or other subcellular compartments at levels sufficient for therapeutic effect
  • cell -penetrating peptides capable of penetrating cellular layers and accessing intracellular spaces, such as the cytoplasm, nucleus, or other subcellular compartments and in some aspects, intercellular compartments, such as nanolumen.
  • These cell-penetrating peptides can carry cargo molecules, such as peptide-based therapeutics (e.g., cystine-dense peptide-based therapeutics), protein-based therapeutics, nucleotide-based therapeutics, or small molecule therapeutics with low permeation or absorption, across cell membranes, thereby delivering the cargo into intracellular or intercellular spaces.
  • cell-penetrating peptides of the present disclosure and complexes or conjugates thereof, penetrate cells and reach an intracellular concentration, or reach an intracellular space, at a sufficient amount to exert a prophylactic or therapeutic effect.
  • peptides e.g., proteins, peptides, or cystine-dense peptides
  • cargos e.g., target-binding molecules, small molecules, RNAs, mRNAs, DNAs, active agents, macromolecular agents, detectable agents, therapeutic agents, or drugs
  • delivery of therapeutic agents across the cell membrane may increase access of the therapeutic agent to intracellular drug targets.
  • Many therapeutically relevant targets have been deemed “undruggable targets” in part due to their location in the nucleus as well as the nature of their functional interactions.
  • transcription factors responsible for driving many types of cancer such as KRAS, MYC, MYB, FOS, JUN, ABL, NF-KB, RAS, RHO, RAN, RAB, and TEAD reside in the nucleus, and their functional protein-protein and protein-nucleic acid interactions may be difficult to target using small molecule therapeutics, which may penetrate cells but may not specifically or potently interact with the transcription factor or block proteinprotein interactions (PPIs).
  • PPIs transcription factor or block proteinprotein interactions
  • Transcription factors residing in the nucleus may be inaccessible to protein and peptide drugs that are capable of specifically and potentially interacting with the transcription factor or blocking protein-protein interactions.
  • Exemplary transcription factors used for targeting the cell -penetrating peptides of the disclosure include transcription factors disclosed in “Targeting Transcription Factors for Cancer Treatment” Molecules. 2018 Jun; 23(6): 1479, which is hereby incorporated by reference. Dysregulation of these transcription factors is responsible for driving many cancers and have been difficult to target with small molecules as they interact and coordinate large protein complexes. Peptide therapeutics that modulate transcription factor interactions may be delivered to the nucleus using the cellpenetrating peptides of disclosed herein, enabling modulation of targets previously considered “undruggable”. Scaffold proteins in the cytoplasm coordinate multiple proteins into complexes through similar mechanisms and are also difficult to target using small molecule therapeutics.
  • the cell-penetrating peptides of the present disclosure can be used to deliver agents that modulate these protein complex interactions to drug targets in the cytoplasm.
  • a cell-penetrating peptide of the present disclosure may deliver a therapeutic agent into the cytoplasm to target the inflammasome, which may be responsible for activating inflammatory responses and coordinated by the Nod-like receptor (NLR) family of proteins.
  • NLR Nod-like receptor
  • Low-grade inflammation driven by NLR family pyrin domain-containing protein 3 (NLRP3) contributes to diabetes and aging.
  • the ability to modulate inflammasome activation is an appealing cytoplasmic target.
  • the cell-penetrating peptides of the present disclosure may facilitate delivery of small peptides or other therapeutic molecules into intracellular cellular compartments (e.g., the cytoplasm, the nucleus, lysosomes, or other subcellular compartments) or intercellular compartments (e.g., nanolumen, intercellular space, or paracellular space).
  • intracellular cellular compartments e.g., the cytoplasm, the nucleus, lysosomes, or other subcellular compartments
  • intercellular compartments e.g., nanolumen, intercellular space, or paracellular space.
  • Drug makers often adhere to the “rule of five” to design small molecule drugs with acceptable pharmacological properties, including permeation or absorption, restricting drug design to molecules with five or fewer hydrogen bond donors, ten or fewer hydrogen bond acceptors, a molecular weight of less than 500 Da (i.e., 500 g/mol), and a log of the partition coefficient of less than 5.
  • the cellpenetrating peptides of the present disclosure may be used to deliver drugs that do not adhere to the “rule of five” and may not otherwise be able to reach intracellular drug targets.
  • the peptides of the present disclosure may be used to deliver heterobifunctional targeted degradation molecules, such as PROTAC molecules, which often do not follow the rule of five.
  • a targeted degradation molecule may be around 1 kDa in size and comprise an E3 ubitiquitin ligase recruiting ligand linked to a target-binding molecule that binds to a protein of interest. These targeted degradation molecules can recognize and promote the ubiquitination and subsequence degradation of specific proteins.
  • cystine-dense peptides including knottins and hitchins and cyclotides
  • adnectins including knottins and hitchins and cyclotides
  • adnectins including knottins and hitchins and cyclotides
  • affibodies including knottins and hitchins and cyclotides
  • affilins includingcalins and atrimers, avimers, fynomers, kunitz domains, humabodies, Obodies, nanofittins, centyrins, DARPins, stapled peptides, cyclic peptides, bicyclic peptides, pronectins, macroclycics, solomers, VNARs, IgNARs, and lasso peptides
  • PPIs protein-protein interactions
  • CRISPR Cas9 proteins ⁇ 160kDa
  • CRISPR components e.g., guide RNAs, tracrRNAs, or crRNAs
  • Delivery of antisense RNA (>30kDa) using the cellpenetrating peptides of the present disclosure could be used to inhibit translation of problematic proteins.
  • the active agent is an anti-cancer agent, a molecule that binds a transcription factor, a molecule that blocks intracellular protein-protein interactions, a molecule that causes the formation or stabilization of intracellular protein-protein interactions, a transcription factor, an RNA, a Cas enzyme or other CRISPR component, an immunomodulating agent, a molecular glue, a targeted degradation molecule, an inhibitor of protein-protein interactions, an inhibitor of enzymatic activity, or a neurotransmitter.
  • a cell-penetrating peptide may be complexed with a RIG-I ligand or a receptor for a RIG-I-like receptor (e.g., MDA5 or TLR3).
  • the ligand may be a double stranded RNA (dsRNA) and may be delivered to a desired cellular compartment (e.g., the cytoplasm or an endosome) to activate the receptor. Delivery of RIG-I ligands across a cellular layer may promote anti -tumor or anti-viral activity in a subject and may be used to treat a cancer or viral infection in a subject.
  • dsRNA double stranded RNA
  • the cell-penetrating peptides of the present disclosure may deliver agents (e.g., cargos, cargo peptides, cargo proteins, or cargo molecules) that drive the formation of protein-protein interactions or that block the formation of protein-protein interactions.
  • the cell-penetrating peptides may deliver agents to the cytoplasm.
  • the cell-penetrating peptides may deliver agents to the nucleus.
  • the cellpenetrating peptides may deliver agents to a nanolumen.
  • a cellpenetrating peptide may deliver an agent to the nucleus that promotes formation of a transcription factor complex or disrupts formation of a transcription factor complex.
  • the agent may activate or inhibit the transcription factor.
  • a cellpenetrating peptide of the present disclosure may deliver an agent to the nucleus that promotes or disrupts protein-protein interactions within transcription factor complexes or protein-nucleic acid interactions AP-2, ARID/BRIGHT, ARID/B RIGHT (RFX), AT hook, BED ZF, bHLH, Brinker, bZIP, C2H2 ZF, C2H2 ZF (KRAB), C2H2 ZF (non-KRAB), C2H2 ZF (AT hook), C2H2 ZF (BED ZF), C2H2 ZF (Homeodomain), C2H2 ZF (Myb/SANT), CBF/NF-Y, CCCH ZF, CENPB, CG-1, CSD, CSL, CUT (Homeodomain), CxxC, CxxC (AT hook), DM, E2F, EBF1, Ets, Ets (AT hook), FLYWCH,
  • a cell-penetrating peptide of the present disclosure may deliver an agent to the nucleus that promotes or disrupts protein-protein interactions with MLL, GABP, RUNX1, KLF8, SIX1, RUNX2, PML, RARu, FOXO, TALI, or Myc [oni]
  • the cell-penetrating peptides of the present disclosure may deliver agents (e g., cargos, cargo peptides, cargo proteins, or cargo molecules) that can cause targeted protein degradation, for example by functioning as molecular glues, proteolysis-targeting chimeras (PROTACs), inhibitors of protein-protein interactions, or targeted degradation complexes.
  • agents e g., cargos, cargo peptides, cargo proteins, or cargo molecules
  • PROTACs proteolysis-targeting chimeras
  • a cell-penetrating peptide may deliver a thalidomide-like molecule to the cytosol.
  • Thalidomidelike molecules that may function as immunomodulatory imide drugs (IMiDs), such as thalidomide, pthalidomide (a-(N-phthalimide)glutarimide), phthalimide derivatives, thalidomide analogues, thalidomide hybrids (e.g., N-phenyl-phthalimide sulfonamides (3a-e), isosters biphenyl -phthalimide amides (4a-e)), pomalidomide, and lenalidomide, can bind to cereblon, cause a conformational change in cereblon, and drive the formation of a complex between cereblon and proteins such as Ikaros and Aiolos, whereby they are ubiquitinated by the CRL4 complex and undergo degradation by the ubiquitin-proteosome system (UPS).
  • IiDs immunomodul
  • IMiDs may be called molecular glues, which cause the formation of PPIs and cause the targeted degradation of proteins.
  • the cell-penetrating peptides of the present disclosure may deliver agents to the cytosol that bind to cereblon, VHL, DCAF15, DCAF16, other molecules in the CRL4 E3 ligase family, other molecules in the E3 ligase family, or other molecules in the UPS system, drive the formation of a complex with another substrate or neosubstrate, and thereby cause ubiquitination and degradation of the substrate or neosubstrate by the UPS, acting as molecular glues.
  • Non-limiting examples of agents that can cause targeted protein degradation and can be delivered by a cell -penetrating peptide of the present disclosure may be found in Collins, et al (Biochem J. 2017 Apr 1; 474(7): 1127-1147), Chopra, et al (Drug Discov Today Technol, 2019 Apr; 31 :5-13), and Chamberlain, et al (Nat Chem Biol, 2019 Oct; 15(10):937- 944) each of which is incorporated by reference in its entirety.
  • Molecular glues can be small molecules, but small molecules may not be able to induce formation of ternary complexes between proteins desired for degradation and the E3 ligase complex.
  • a cell-penetrating peptide can deliver an agent that functions as a molecular glue to the cytosol. In some embodiments, a cell-penetrating peptide can deliver an agent that functions as a targeted degradation complex to the cytosol.
  • Targeted degradation complexes may comprise a molecule that binds to a ubiquitin ligase (e.g., cereblon, VHL, DCAF15, DCAF16, other molecules in the CRL4 E3 ligase family, other molecules of the Cul2-Rbxl-EloN/C-VHL E3 ligase, the MDM2 E3 ligase, cIAPl, cIAP, APCZC(CDHl), or other molecules in the E3 ligase family), a linker, and target-binding molecule that binds to a protein targeted for degradation.
  • a ubiquitin ligase e.g., cereblon, VHL, DCAF15, DCAF16, other molecules in the CRL4 E3 ligase family, other molecules of the Cul2-Rbxl-EloN/C-VHL E3 ligase, the MDM2 E3 ligase, cIAPl, cI
  • a targeted degradation complex may comprise a small molecule that does not fit the “rule of five” often used to constrain molecule size and solubility parameters for successful small molecule therapeutics.
  • Protein degradation can also be caused by molecules that bind the protein targeted for degradation and that also contain an IMiD, a Boc3-Arg tag, an adamantyl group, or a carborane as hydrophobic tag that causes binding to the 20S proteosome or to HSP70.
  • the cell-penetrating peptides of this disclosure may act as molecular glues or as PROTACS or otherwise cause targeted protein degradation.
  • the cell-penetrating peptides of this disclosure may serve to deliver active agents that act as molecular glues or targeted degradation complexes or that otherwise drive targeted degradation to the cytosol, thereby allowing those active agents to reach their targets and cause targeted protein degradation.
  • Proteins that can targeted for degradation by a cell-penetrating peptide or a cell-penetrating peptide complex may include TEAD, cold-inducible RNA-binding protein (also referred to as CIRP or CIRBP), androgen receptor, ikaros, aiolos, nuclear receptors, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF8
  • a cellpenetrating peptide complex of the present disclosure may target AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, P-catenin, IKK, TEAD, NF-KB, PKC, AP-1, Jun, Fos, REL or a transcription factor for degradation.
  • a cell -penetrating peptide or an active agent for targeted protein degradation can have one or more lysine residues removed or replaced with other amino acids to prevent labeling of the lysine residues with ubiquitin. By targeting proteins for degradation, they may be reduced or removed from cells, thereby removing functions that contribute to disease.
  • Proteins that can be degraded using the cell -penetrating peptides or cellpenetrating peptide complexes of the present disclosure may be transcription factors, kinases, zinc finger domain containing, may perform a scaffolding function, or may be adaptor proteins.
  • the cell-penetrating peptides of the presence disclosure may themselves be agents that cause a therapeutic effect by binding to or interacting with various molecules within the cytosol, nucleus, or other subcellular, intracellular, or paracellular compartments.
  • a cell-penetrating peptide may promote or disrupt a protein-protein interaction in the subcellular, intracellular, or paracellular compartment.
  • a cell -penetrating peptide may deliver an agent that cause a therapeutic effect to the cytosol, nucleus, or other subcellular compartment.
  • a cell -penetrating peptide including, but not limited to, designed or engineered peptides, recombinant peptides, synthetic peptides, and small cystine-dense peptides (or disulfide-knotted peptides, knottins, knotted peptides, or hitchins), that are capable of penetrating cellular layers (e.g., cell membranes, nuclear envelopes, endosomes, lysosomes, or other subcellular compartments) to access intracellular or intercellular spaces.
  • cellular layers e.g., cell membranes, nuclear envelopes, endosomes, lysosomes, or other subcellular compartments
  • Cell-penetrating peptides can include calcines (e.g., imperatoxin A (IpTxa), maurocalcin (MCa), hadrucalcin (Had), hemicalcin, vejocalcin, intrepicalcin, opicalcin-1, opicalcin-2, or urocalcin), toxins (e.g., chlorotoxin (CTX), huwentoxin IV (HwTx-IV), potassium channel toxin-like Tx677, potassium channel toxin KTx2.2, potassium channel toxin KTxl5.8), CTI, and variants and fragments thereof.
  • calcines e.g., imperatoxin A (IpTxa), maurocalcin (MCa), hadrucalcin (Had), hemicalcin, vejocalcin, intrepicalcin, opicalcin-1, opicalcin-2, or urocalcin
  • toxins e.g
  • a cell-penetrating peptide of the present disclosure may be conjugated to, linked to, or fused with a cargo molecule (e g., a peptide, a cystine-dense peptide, a small molecule, a protein, an RNA, an mRNA, a DNA, an active agent, a detectable agent, a therapeutic agent, or a drug) to generate a cell-penetrating peptide complex.
  • a cargo molecule e e a peptide, a cystine-dense peptide, a small molecule, a protein, an RNA, an mRNA, a DNA, an active agent, a detectable agent, a therapeutic agent, or a drug
  • the cellpenetrating peptide may itself be an active agent.
  • cell-penetrating peptide complexes may be used to deliver cargo molecules across cellular layers (e.g., cell membranes, nuclear envelopes, intercellular spaces, paracellular spaces, endosomal membranes, lysosomal membranes, blood brain barriers, or nanolumen) and into cellular, intercellular, or paracellular compartments or spaces (e.g., cytosols, nuclei, or nanolumen).
  • cellular layers e.g., cell membranes, nuclear envelopes, intercellular spaces, paracellular spaces, endosomal membranes, lysosomal membranes, blood brain barriers, or nanolumen
  • cytosols e.g., cytosols, nuclei, or nanolumen
  • the cell-penetrating peptides and cell-penetrating peptide complexes of the present disclosure may be used in various methods to deliver cargo molecules to intracellular and intercellular targets (e.g., drug targets).
  • a cell-penetrating peptide of the present disclosure may be conjugated to a therapeutic agent (e.g., a small molecule drug, a peptide drug, a protein drug, a biologic drug, a transcription factor, a gene editing agent, or a disruptor of PPIs or a driver of PPIs) that would otherwise be excluded from intracellular spaces, and the resulting cell-penetrating peptide complex may be administered to a subject (e.g., a human subject) to deliver the therapeutic agent to a target of the therapeutic agent (e g., a protein or a nucleic acid) located in a cellular, intercellular, or paracellular compartment or space (e.g., cytoplasm, nucleus, or nanolumen).
  • a therapeutic agent e.g., a small molecule drug, a peptide drug, a protein drug, a biologic drug, a transcription factor, a gene editing agent, or a disruptor of PPIs or a driver of PPIs
  • alanine A, Ala
  • arginine R, Arg
  • asparagine N, Asn
  • aspartic acid D, Asp
  • cysteine C, Cys
  • glutamic acid E, Glu
  • glutamine Q, Gin
  • glycine G, Gly
  • histidine H, His
  • isoleucine I, He
  • leucine L, Leu
  • lysine K, Lys
  • methionine M, Met
  • phenylalanine F, Phe
  • proline P, Pro
  • serine S, Ser
  • threonine T, Thr
  • tryptophan W, Trp
  • tyrosine Y, Tyr
  • valine V, Vai
  • Xaa can indicate any amino acid.
  • X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).
  • a peptide of the present disclosure can comprise a nonnatural amino acid, wherein the non-natural amino acid can be an insertion, appendage, or substitution for another amino acid.
  • a non-natural amino acid may comprise a D-isomer, a homo-amino acid, a B-homo-amino acid, an N-methyl amino acid, an a-methyl amino acid.
  • a non-natural amino acid can comprise citrulline (Cit), hydroxyproline (Hyp), norleucine (Nle), 3-nitrotyrosine, nitroarginine, ornithine (Om), naphtylalanine (Nal), aminobutyric acid (a, Abu), di aminobutyric acid (Dab), methionine sulfoxide, or methionine sulfone.
  • a position can be referred to as “position X,” wherein X is the position number within a sequence not counting, if present, the N-terminal GS.
  • position 5 in SEQ ID NO: 59 refers to the “H” residue at position 5 within the sequence, wherein position 1 does not count the N-terminal “GS,” and is, thus, the first D residue.
  • position 9 in SEQ ID NO: 66 refers to the “A” residue within the sequence at position 9, wherein position 1 does not count the N-terminal “GS,” and is, thus, the first E residue.
  • the N-terminal GS is included in the sequence as a byproduct of proteolytic cleavage during recombinant protein production or a spacer.
  • position 11 in SEQ ID NO: 18 refers to the “K” residue at position 11 within the sequence, wherein position 1 is the very first G residue and there is no N-terminal GS.
  • a position can be referred to as “XY,” wherein X is the single-letter abbreviation for an amino acid and wherein Y is the position number within a sequence not counting, if present, the N-terminal GS.
  • G15 refers to the “G” residue at position 15 within a sequence, not counting, if present, an N-terminal GS.
  • Y23 refers to the “Y” residue at position 23 within a sequence, not counting, if present, an N-terminal GS.
  • E25 refers to the “E” residue at position 25 within a sequence, not counting, if present, an N-terminal GS.
  • a position can be referred to as “XYZ,” wherein X and Z are the single-letter abbreviation for an amino acid and wherein Y is the position number within a sequence not counting, if present, the N-terminal GS.
  • L23A refers to a “L” residue at position 23 within a sequence (not counting, if present, an N- terminal GS), which has been substituted with a “A” residue.
  • E25A refers to an “E” residue at position 25 within a sequence (not counting, if present, an N-terminal GS), which has been substituted with an “A” residue.
  • F27A refers to an “F” residue at position 27 within a sequence (not counting, if present, an N-terminal GS), which has been substituted with an “A” residue.
  • Some embodiments of the disclosure contemplate D-amino acid residues of any standard or non-standard amino acid or analogue thereof.
  • an amino acid sequence is represented as a series of three-letter or one-letter amino acid abbreviations, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy terminal direction, in accordance with standard usage and convention.
  • peptide complex may refer to a peptide that is fused to, recombinantly fused to, linked to, conjugated to, chemically conjugated to, recombinantly expressed with, complexed with, or is otherwise connected to an additional agent (e g., a protein, a peptide, a cystine-dense peptide, a small molecule, an RNA, an mRNA, a DNA, an active agent, macromolecular agent, a detectable agent, a therapeutic agent, or a drug).
  • a peptide complex may be a peptide construct.
  • a peptide complex may be a peptide conjugate.
  • a peptide complex of the present disclosure may comprise a cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254, or a variant or fragment thereof) that is fused, linked, conjugated, recombinantly expressed with, or is otherwise connected to a cargo molecule (e.g., a peptide, a peptide, a cystine-dense peptide, a small molecule, an RNA, an mRNA, a DNA, an active agent, macromolecular agent, a detectable agent, a therapeutic agent, or a drug), and may be referred to herein as a cell-penetrating peptide complex.
  • a cargo molecule e.g., a peptide, a peptide
  • a peptide complex may comprise a linker (e.g., a peptide linker of any one SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485, or a small molecule linker as described herein).
  • a linker e.g., a peptide linker of any one SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485, or a small molecule linker as described herein.
  • a “cargo,” “cargo peptide,” “cargo protein,” or “cargo molecule” may be any protein, peptide, or molecule that is fused, linked, conjugated, recombinantly expressed with, or is otherwise connected to a cell-penetrating peptide.
  • a cargo, cargo peptide, cargo protein, or cargo molecule may be carried across a cellular layer (e.g., a plasma membrane, a vesicular membrane, an endosomal membrane, a nuclear envelope, or a blood brain barrier) by a cell-penetrating peptide of the present disclosure.
  • a cargo, cargo peptide, cargo protein, or cargo molecule may be carried within the vesicles of a cell's secretory system (e.g., within the endoplasmic reticulum, Golgi apparatus, lysosomes, or other subcellular compartment, whether to or from the plasma membrane) or within a vesicle as a structure within or outside a cell, comprising liquid or cytoplasm enclosed by a lipid bilayer.
  • a cell's secretory system e.g., within the endoplasmic reticulum, Golgi apparatus, lysosomes, or other subcellular compartment, whether to or from the plasma membrane
  • a vesicle as a structure within or outside a cell, comprising liquid or cytoplasm enclosed by a lipid bilayer.
  • a cargo peptide, cargo protein, or cargo molecule may be a protein, a peptide, a cystine-dense peptide, a small molecule, an RNA, an mRNA, a DNA, an active agent, macromolecular agent, a detectable agent, a therapeutic agent, or a drug.
  • penetration includes movement of a molecule from the extracellular space to the cytosol, the nucleus, or other subcellular compartments by any means, and may also be described as cytosolic access or cytosolic delivery.
  • Cell penetration may also include endosomal uptake, such as by macropinocytosis, clathrin-mediated endocytosis, calveolae-dependent endocytosis, or other endocytosis, followed by endosomal escape such that the molecule leaves the endosome and enters the cytosol. It is understood that cell penetration is not limited to a molecule solely entering endosomes not further accessing or entering the cytosol.
  • a peptide capable of penetrating a cellular layer e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, a cell membrane, an endosomal membrane, a lysosomal membrane, a nuclear envelope, or other subcellular compartment membrane, a blood brain barrier, or a nanolumen
  • a cellpenetrating peptide may also be referred to herein as a cellpenetrating peptide.
  • a cell -penetrating peptide may enter the cytosol. Alternatively or in addition, a cell-penetrating peptide may enter the nucleus.
  • the cellpenetrating peptides disclosed herein can penetrate or enter target cells, such as cancerous or tumor cells, liver cells, pancreas cells, colon cells, ovarian cells, breast cells, and/or lung cells, or any combination thereof.
  • target cells such as cancerous or tumor cells, liver cells, pancreas cells, colon cells, ovarian cells, breast cells, and/or lung cells, or any combination thereof.
  • cell penetration includes movement of a molecule from the extracellular space to the cytosol, the nucleus, or other subcellular compartments by any means, and may also be described as cytosolic access or cytosolic delivery.
  • Cell penetration may also include endosomal uptake, such as by macropinocytosis, clathrin-mediated endocytosis, calveolae-dependent endocytosis, or other endocytosis, followed by endosomal escape such that the molecule leaves the endosome and enters the cytosol. It is understood that cell penetration is not limited to a molecule solely entering endosomes not further accessing or entering the cytosol.
  • a peptide or a library of peptides is designed in silica without derivation from a naturally occurring knottin scaffold.
  • a peptide or a library of peptides is designed in silica by derivation, grafting relevant or important proteinbinding residues, or conserved residues, in the protein-binding interface, or structural modeling based on a naturally occurring peptide or protein known to bind to a protein or receptor of interest.
  • a library of peptides is screened for the ability to access a cellular compartment, such as the cytoplasm or the nucleus, using a fluorescence-based screening method or a mass spectrometry -based screening method, as described herein.
  • a peptide with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is used as a scaffold or base sequence for further modifications, including addition, deletion, or amino acid substitution.
  • a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 may be modified by performing one or more amino acid substitutions to introduce one or more lysine, arginine, proline, or histidine amino acids.
  • residues GS are added at the N-terminus of a peptide.
  • peptides lack GS at the N-terminus.
  • peptides undergo one or more post-translational modifications.
  • peptides are truncated so that they contain fewer amino acids, or are less than 50, less than 40, less than 30, less than 20, less than 15, less than 12, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, or less than 4 amino acids long.
  • TABLE 1 lists exemplary peptide sequences according to the present disclosure.
  • a cell-penetrating peptide of the present disclosure may comprise one or more positively charged amino acid residues (e.g., lysine or arginine). Positively charged amino acid residues may mediate interactions between the cell-penetrating peptide and negatively charged cellular layers (e.g., cell membranes, nuclear envelopes, intercellular spaces, paracellular spaces, endosomal membranes, lysosomal membranes, or other subcellular compartment membrane, blood brain barriers, or nanolumen).
  • positively charged amino acid residues e.g., lysine or arginine
  • Positively charged amino acid residues may mediate interactions between the cell-penetrating peptide and negatively charged cellular layers (e.g., cell membranes, nuclear envelopes, intercellular spaces, paracellular spaces, endosomal membranes, lysosomal membranes, or other subcellular compartment membrane, blood brain barriers, or nanolumen).
  • a cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positively charged amino acid residues.
  • a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 positively charged amino acid residues.
  • a cell-penetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 positively charged amino acid residues.
  • Positively charged amino acid residues may include lysine residues and arginine residues.
  • a cell-penetrating peptide may comprise at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1. 1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1 .6, at least 1 .7, at least 1.8, at least 1 .9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3.7, at least 3.8, at least 3.9, or at least 4 positively charged amino acid residues per 10 amino acid residues.
  • a cell-penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.
  • no more than 2 no more than 1.9, no more than 1.8, no more than 1.7, no more than 1.6, no more than 1.5, no more than 1.4, no more than 1.3, no more than 1.2, no more than 1.1, or no more than 1 positively charged amino acid residues per 10 amino acid residues.
  • a cell-penetrating peptide may comprise no more than 0.5, no more than 0.6, no more than 0.7, no more than 0.8, no more than 0.9, no more than 1, no more than 1.1, no more than 1.2, no more than 1.3, no more than 1.4, no more than 1.5, no more than 1.6, no more than 1.7, no more than 1.8, no more than 1.9, no more than 2, no more than 2.1, no more than 2.2, no more than 2.3, no more than 2.4, no more than 2.5, no more than 2.6, no more than 2.7, no more than 2.8, no more than 2.9, no more than 3, no more than 3.
  • a cellpenetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 positively charged amino acid residues per 10 amino acid residues.
  • Positively charged amino acid residues may include lysine residues and arginine residues.
  • a cell-penetrating peptide of the present disclosure may comprise one or more arginine amino acid residues.
  • a cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 arginine amino acid residues.
  • a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 arginine amino acid residues.
  • a cell-penetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 arginine amino acid residues.
  • a cell-penetrating peptide may comprise at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1. 1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1 .6, at least 1 .7, at least 1.8, at least 1 .9, at least 2, at least 2. 1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.2, at least 3.4, at least 3.6, at least 3.8, or at least 4 arginine amino acid residues per 10 amino acid residues.
  • a cell -penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.
  • a cell-penetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 arginine amino acid residues per 10 amino acid residues.
  • the cell-penetrating peptide comprises no arginine amino acids.
  • a cell-penetrating peptide of the present disclosure may comprise one or more lysine amino acid residues.
  • a cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 lysine amino acid residues.
  • a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 lysine amino acid residues.
  • a cell-penetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 lysine amino acid residues.
  • the cellpenetrating peptide comprises no lysine amino acid residues.
  • a cell-penetrating peptide may comprise at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1. 1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1 .6, at least 1 .7, at least 1.8, at least 1 .9, at least 2, at least 2. 1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.2, at least 3.4, at least 3.6, at least 3.8, or at least 4 lysine amino acid residues per 10 amino acid residues.
  • a cell -penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.
  • a cellpenetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 lysine amino acid residues per 10 amino acid residues.
  • a cell-penetrating peptide of the present disclosure may comprise one or more negatively charged amino acid residues (e g., aspartic acid or glutamic acid).
  • a cellpenetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 negatively charged amino acid residues.
  • a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 negatively charged amino acid residues.
  • a cellpenetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 negatively charged amino acid residues.
  • Negatively charged amino acid residues may include aspartic acid residues (or aspartate residues) and glutamic acid residues (or glutamate residues).
  • a cell-penetrating peptide may comprise at least 0.03, at least 0.05, at least 0.1, at least 0.2, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least
  • a cell-penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, no more than 2, no more than 1.9, no more than 1.8, no more than 1.7, no more than 1.6, no more than 1.5, no more than 1.4, no more than 1.3, no more than 1.2, no more than 1.1, or no more than 1 negatively charged amino acid residues per 10 amino acid residues.
  • a cell-penetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 negatively charged amino acid residues per 10 amino acid residues.
  • Positively charged amino acid residues may include lysine residues and arginine residues.
  • Negatively charged amino acid residues may include aspartic acid residues (or aspartate residues) and glutamic acid residues (or glutamate residues).
  • a cell-penetrating peptide of the present disclosure may comprise more positively charged amino acid residues than negatively charged amino acid residues.
  • a cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 more positively charged amino acid residues than negatively charged amino acid residues.
  • a cellpenetrating peptide may comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 more positively charged amino acid residues than negatively charged amino acid residues.
  • a cell-penetrating peptide may comprise from 1 to 30, from 1 to 20, from 1 to 10, from 5 to 30, from 5 to 20, from 5 to 10, from 3 to 30, from 3 to 20, from 3 to 10, from 4 to 30, from 4 to 20, from 4 to 10, from 5 to 30, from 5 to 20, or from 5 to 10 more positively charged amino acid residues than negatively charged amino acid residues.
  • a cell-penetrating peptide of the present disclosure may comprise more negatively charged amino acid residues than positively charged amino acid residues.
  • a cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 more negatively charged amino acid residues than positively charged amino acid residues.
  • a cellpenetrating peptide may comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 more negatively charged amino acid residues than positively charged amino acid residues.
  • a cell-penetrating peptide may comprise from 1 to 30, from 1 to 20, from 1 to 10, from 5 to 30, from 5 to 20, from 5 to 10, from 3 to 30, from 3 to 20, from 3 to 10, from 4 to 30, from 4 to 20, from 4 to 10, from 5 to 30, from 5 to 20, or from 5 to 10 more negatively charged amino acid residues than positively charged amino acid residues.
  • a cell-penetrating peptide may comprise a ratio of positively charged amino acid residues to negatively charged amino acid residues that is at least 1.0, at least 1.5, at least 2, at least 2.5, at least 2.75, at least 3, at least 3.5, at least 4, at least 5, at least 6, or at least 7.
  • a cell-penetrating peptide may comprise a ratio of negatively charged amino acid residues to positively charged amino acid residues that is no more than 1.0, no more than 1.5, no more than 2, no more than 2.5, no more than 2.75, no more than 3, no more than 3.5, or no more than 4.
  • Proline amino acid residues may provide structural stability or specific conformations for the cell-penetrating peptides of the present disclosure.
  • proline residues may provide a conformation or stability to short cell-penetrating peptide sequences (e g., cellpenetrating peptides of less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, or less than 4 amino acid residues).
  • a cell-penetrating peptide of the present disclosure e.g., a short cell-penetrating peptide
  • a cell-penetrating peptide may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 proline amino acid residues.
  • Histidine amino acid residues may facilitate membrane penetration of the cellpenetrating peptides of the present disclosure.
  • the side chain of a histidine amino acid may be, on average, neutral at an extracellular pH such as pH 7.4, pH 7.3, pH 7.2, pH 7.1, or pH 7.0.
  • the side chain of a histidine amino acid may, on average, become increasingly positively charged as the pH drops in an early endosome, in a late endosome, or in a lysosome. Histidine residue side chains may contribute more positive charge to the net charge of a peptide as pH decreases, for example as the pH of an endosome goes below pH 6.0.
  • a histidine residue side chain may have a pK a of 6.0, such that, as the pH drops below pH 7.4, the amount of positive charge increases.
  • a histidine residue may develop a positive charge in an endosome or a lysosome, thereby causing a change in the conformation of the molecule or a change in interaction with the endosomal membrane such that the cell-penetrating peptide containing one or more histidine residues disrupts the endosome or otherwise escapes the endosome or lysosome as the pH in the endosome or lysosomes drops below about pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7,
  • a cellpenetrating peptide of the present disclosure may contain a histidine amino acid residue.
  • a cell-penetrating peptide may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 histidine amino acid residues.
  • a short cell-penetrating peptide sequences e.g., any one of SEQ ID NO: 4 - SEQ ID NO: 7, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 21 - SEQ ID NO: 29, SEQ ID NO: 41 - SEQ ID NO: 58, SEQ ID NO: 62 - SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 82 SEQ ID NO: 84, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 214, or
  • the penetration properties of a cell-penetrating peptide increase as the pH decreases.
  • a cell-penetrating peptide of the present disclosure may penetrate membranes, including the endosomal membrane or lysosomal membrane, when the pH is below pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, or pH 4.5.
  • the pH change or the ionic strength change can cause changes in conformation of the cell-penetrating peptide or interaction of the cell-penetrating peptide with the endosomal or lysosomal membrane.
  • CDPs or knotted peptides including engineered, non-naturally occurring CDPs and those found in nature (e g., a target-binding peptide), can be conjugated to, linked to, or fused to the cell-penetrating peptides of the present disclosure, such as those described in TABLE 1, TABLE 2, or TABLE 3, to deliver a target molecule to cellular, intracellular, or paracellular compartment or space.
  • the cell can be a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, bone marrow cell, or any combination thereof.
  • An engineered peptide can be a peptide that is non-naturally occurring, artificial, synthetic, designed, or recombinantly expressed.
  • a cellpenetrating peptide of the present disclosure, or a peptide complex comprising a cell- penetrating enables cell-penetrating, and an additional CDP or knotted peptide that is conjugated to, linked to, or fused to the cell-penetrating peptide can be delivered to the cellular, intracellular, or paracellular compartment or space in a cell associated with a disease or condition.
  • the cell is a cancer cell.
  • Cancers can include breast cancer, liver cancer, colon cancer, brain cancer, leukemia, acute myeloid leukemia (AML), lymphoma, nonHodgkin lymphoma, myeloma, blood-cell-derived cancer, spleen cancer, cancers of the salivary gland, kidney cancer, muscle cancers, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, sarcoma, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, diffuse intrinsic pontine glioma, lung cancer, bone marrow cell cancers, skin cancer, genitourinary cancer, osteosarcoma, muscle-derived sarcoma, melanoma, head and neck cancer, a neuroblastoma, glioblastoma, astrocytoma, glioma, medulloblastom
  • TABLE 2 lists exemplary peptide sequences according to the present disclosure.
  • a peptide can be modified with biotin, for example at a lysine residue (e.g., in SEQ ID NO: 102 - SEQ ID NO: 117).
  • a peptide can be modified with a fluorophore, for example at the N-terminus or the C-terminus of the peptide (e.g., Cy5.5 in SEQ ID NO: 135 - SEQ ID NO: 154).
  • a peptide can contain a non-proteinogenic amino acid, for example an a-aminobutyric acid, abbreviated “a” or “Abu” (e.g., in SEQ ID NO: 118 - SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 140 - SEQ ID NO: 154, SEQ ID NO: 163 - SEQ ID NO: 166, or SEQ ID NO: 168).
  • a a-aminobutyric acid
  • peptides of the present disclosure may comprise one or more cellpenetrating peptides.
  • peptides can comprise at least one or multiple peptides having cell penetration properties (e.g., one or more of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254).
  • a cell-penetrating peptide may comprise one or more Arg residues (e g., SEQ ID NO: 1 - SEQ ID NO: 8, SEQ ID NO: 16 - SEQ ID NO: 41, SEQ ID NO: 43 - SEQ ID NO: 82, SEQ ID NO: 84 - SEQ ID NO: 120, SEQ ID NO: 124 - SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 153 - SEQ ID NO: 194, SEQ ID NO: 197 SEQ ID NO: 209, SEQ ID NO: 212 SEQ ID NO: 235, SEQ ID NO: 237 - SEQ ID NO: 243, SEQ ID NO: 250 - SEQ ID NO: 254, SEQ ID NO: 408 - SEQ ID NO: 431, or SEQ ID NO: 433 - SEQ ID NO: 456).
  • a cell -penetrating peptide of the present disclosure may comprise one or more Lys residues. In some embodiments, a cellpenetrating peptide of the present disclosure may comprise one or more Asp residues. In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more Glu residues. In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more His residues. In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more Pro residues.
  • a cellpenetrating peptide of the present disclosure may comprise one or more Cys residues (e g., SEQ ID NO: 1 - SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 18 - SEQ ID NO: 20, SEQ ID NO: 30 - SEQ ID NO: 40, SEQ ID NO: 59 - SEQ ID NO: 61, SEQ ID NO: 64 - SEQ ID NO: 69, SEQ ID NO: 71 - SEQ ID NO: 81, SEQ ID NO: 85 - SEQ ID NO: 117, SEQ ID NO: 119 - SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 169 - SEQ ID NO: 193, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 202 - SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 211
  • one or more Lys residues may be substituted with Arg residues. In some embodiments, one or more Arg residues may be substituted with Lys residues. In some embodiments, one or more Cys residues may be substituted with Ala residues.
  • a cell-penetrating peptide may be a short cell-penetrating peptide (e.g., SEQ ID NO: 4 - SEQ ID NO: 7, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 21 - SEQ ID NO: 29, SEQ ID NO: 41 - SEQ ID NO: 58, SEQ ID NO: 62 - SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 82 - SEQ ID NO: 84, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 214, or SEQ ID NO: 216).
  • a cell -penetrating peptide may be a fragment of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • a cell-penetrating peptide may be any consecutive fragment of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 SEQ ID NO: 254 that is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids long.
  • a cell -penetrating peptide may comprise a functional fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • the functional fragment may be a consecutive fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • a functional fragment may be a fragment that has cell -penetrating properties.
  • a functional fragment may be capable of penetrating a cellular layer, cell membrane, a nuclear envelope, an endosomal membrane, a lysosomal membrane, other cellular or subcellular membrane, or a blood brain barrier.
  • a functional fragment may be a fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 18 - SEQ ID NO: 20, SEQ ID NO: 30 - SEQ ID NO: 40, SEQ ID NO: 59 - SEQ ID NO: 61, SEQ ID NO: 64 - SEQ ID NO: 69, SEQ ID NO: 71 - SEQ ID NO: 81, SEQ ID NO: 85 - SEQ ID NO: 117, SEQ ID NO: 119 - SEQ ID NO: 162, SEQ ID NO 166,
  • SEQ ID NO: 224 SEQ ID NO: 226, SEQ ID NO: 244, or SEQ ID NO: 247 comprising at least
  • a functional fragment may be a fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 positively charged amino acid residues.
  • a functional fragment may be a fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 8, SEQ ID NO: 16 - SEQ ID NO: 41, SEQ ID NO: 43 - SEQ ID NO: 82, SEQ ID NO: 84 SEQ ID NO: 120, SEQ ID NO: 124 SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 153 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, SEQ ID NO: 197 - SEQ ID NO: 209, SEQ ID NO: 212 - SEQ ID NO: 235, SEQ ID NO: 237 SEQ ID NO: 243, or SEQ ID NO: 250 SEQ ID NO: 254 comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 arginine amino acid residues.
  • a functional fragment may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 lysine amino acid residues. In some embodiments, a functional fragment may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 histidine amino acid residues. In some embodiments, a functional fragment may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 proline amino acid residues. In some embodiments, a functional fragment may comprise an amphipathic a-helix.
  • a short cell-penetrating peptide may have a sequence of any one of GSXDALPHLKL (SEQ ID NO: 334) or XDALPHLKL (SEQ ID NO: 325), wherein X is any amino acid.
  • a short cell -penetrating peptide may have a sequence of any one of GSGXALPHLKL (SEQ ID NO: 335) or GXALPHLKL (SEQ ID NO: 326), wherein X is any amino acid.
  • a short cell -penetrating peptide may have a sequence of any one of GDXLPHLKL (SEQ ID NO: 327) or GSGDXLPHLKL (SEQ ID NO: 336), wherein X is any amino acid.
  • a short cell -penetrating peptide may have a sequence of any one of GDAXPHLKL (SEQ ID NO: 328) or GSGDAXPHLKL (SEQ ID NO: 337), wherein X is any amino acid.
  • a short cell-penetrating peptide may have a sequence of any one of GDALXHLKL (SEQ ID NO: 329) or GSGDALXHLKL (SEQ ID NO: 338), wherein X is any amino acid
  • a short cell -penetrating peptide may have a sequence of any one of GDALPXLKL (SEQ ID NO: 330) or GSGDALPXLKL (SEQ ID NO: 339), wherein X is any amino acid.
  • a short cell-penetrating peptide may have a sequence of any one of GDALPHXKL (SEQ ID NO: 331) or GSGDALPHXKL (SEQ ID NO: 340), wherein X is any amino acid.
  • a short cell-penetrating peptide may have a sequence of any one of GDALPHLXL (SEQ ID NO: 332) or GSGDALPHLXL (SEQ ID NO: 341), wherein X is any amino acid.
  • a short cell -penetrating peptide may have a sequence of any one of GDALPHLKX (SEQ ID NO: 333) or GSGDALPHLKX (SEQ ID NO: 342), wherein X is any amino acid.
  • An amino acid may be a natural amino acid or a non-natural amino acid.
  • Cell-penetrating peptides may be derived from calcins or calcin variants. For example, the peptides may be derived from the calcin variants shown in FIG. 17.
  • a cell-penetrating peptide derived from a calcin may have a sequence of DCLX 1 X 2 LRX 3 CX 4 X 5 X 6 X 7 DCCX 8 RX 9 CX 10 RR, wherein X'-X 1 ' 1 are each independently any amino acid or no amino acid (SEQ ID NO: 343).
  • X ⁇ X 10 in SEQ ID NO: 343 can be deleted, resulting in no amino acid in the position with reference to SEQ ID NO: 343.
  • a cell-penetrating peptide derived from a calcin may have a sequence of DCLX 1 X 2 LRX 3 CX 4 X 5 X 6 X 7 DCCX 8 RX 9 CX 10 RR, wherein X 1 is selected from P or A, X 2 is selected from H or R, X 3 is selected from L or R, X 4 is selected from R or K, X 5 is selected from E, R, or A, X 6 is selected from N or D, X 7 is selected from N or R, X 8 is selected from S or G, X 9 is selected from R or S, and X 10 is selected from R, S, or K (SEQ ID NO: 344).
  • known calcin sequences may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 344.
  • a cell-penetrating peptide derived from a calcin may have a sequence of DCLPHLRLCRX 1 X 2 X 3 DCCSRRCRRRGT X 4 ERRCR, wherein X'-X 4 are each independently any amino acid (SEQ ID NO: 345), or wherein X 1 is E, D, A, R, K, or G, X 2 is N or D or E, X 3 is R, K, N, and X 4 is I, A, P, or G (SEQ ID NO: 346).
  • known calcin sequences may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 345 or SEQ ID NO: 346.
  • a cell-penetrating peptide derived from a calcin may have a sequence of X 1 DCLX 2 X 3 LRX 4 CX 5 X 6 X 7 X 8 DCCX 9 RX 10 CX 11 RRX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 , wherein X 4 -X 21 are each independently any amino acid or no amino acid (SEQ ID NO: 347).
  • a cellpenetrating peptide derived from a calcin may have a sequence of X 1 DX 2 LX 3 X 4 LRX 5 , wherein X 1 any amino acid or no amino acid and X 2 -X 5 are each independently any amino acid (SEQ ID NO: 348), or wherein X 1 is G, A, S, R, or K, X 2 is C, A, G, S, X 3 is P, R, K, A, or G, X 4 is H, R, or K, and X 5 is L, R, or K (SEQ ID NO: 349).
  • known calcin sequences may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 348 or SEQ ID NO: 349.
  • a cell-penetrating peptide derived from a calcin may have a sequence of LX L X 2 LRX 3 , wherein X 4 -X 3 are each independently any amino acid (SEQ ID NO: 350), or wherein X 1 is P, R, K, A, or G, X 2 is H, R, or K, and X 3 is L, R, or K (SEQ ID NO: 351).
  • known calcin sequences may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 350 or SEQ ID NO: 351.
  • a cell-penetrating peptide described herein as a consensus sequence or as variants with respect to percent identity may exclude known cell-penetrating sequences.
  • Additional cell-penetrating tag peptide sequences can include Tat (GRKKRRQRRR; SEQ ID NO: 225), CysTat (CYRKKRRQRRR; SEQ ID NO: 226), S19-TAT (PFVIGAGVLGALGTGIGGIGRKKRRQRRR; SEQ ID NO: 227), R8 (RRRRRRRR; SEQ ID NO: 228), pAntp (RQIKIWFQNRRMKWKK; SEQ ID NO: 229), Pas-TAT (FFLIPKGGRKKRRQRRR; SEQ ID NO: 230), Pas-R8 (FFLIPKGRRRRRRRR; SEQ ID NO: 231), PasFHV (FFLIPKGRRRRNRTRRNRRRVR; SEQ ID NO: 232), Pas-pAntP (FFLIPKGRQIKIWFQNRRMKWKK; SEQ ID NO: 233), F2R4 (FFRRRR; SEQ ID NO: 234), B55 (KAV
  • a cell-penetrating peptide can comprise an Arginine patch (Arg patch), for example, an RRRRRRRR (SEQ ID NO: 228), or a variant or fragment thereof
  • the Arg patch comprises two or more Arg residues, or Argn wherein n is a whole number and can be 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 250).
  • the cell -penetrating peptide can comprise a Tat peptide (Tat proteins are reviewed in Gump et al. TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med. 2007 Oct;13(10):443-8 and Harada et al.
  • the Tat peptide can have a sequence of, for example, YGRKKRRQRRR (SEQ ID NO: 251), GRKKRRQRRR (SEQ ID NO: 225), or any modification, variant, or fragment thereof.
  • the Tat peptide sequence can be GRKKRRQRRRPQ (SEQ ID NO: 252), GRKKRRQRRR (SEQ ID NO: 225), or a fragment or variant thereof.
  • Cell-penetrating peptides may be capable of penetrating and transferring a cargo, either covalently or non-covalently attached to the peptides, into a cell.
  • Such cellpenetrating peptides can be synthesized or derived from known proteins, such as penetratin (RQIKIWFQNRRMKWKKGG; SEQ ID NO: 254), VP22 (MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFV QYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTT APRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKL HFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDL NELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAAS
  • a cell-penetrating peptide can may be derived from maurocaline (GDCLPHLKLCKENKDCCSKKCKRRGTNIEKRCR; SEQ ID NO: 197), imperatoxin (GDCLPHLKRCKADNDCCGKKCKRRGTNAEKRCR; SEQ ID NO: 202), hadrucalcin (SEKDCIKHLQRCRENKDCCSKKCSRRGTNPEKRCR; SEQ ID NO: 200), hemicalcin (GDCLPHLKLCKADKDCCSKKCKRRGTNPEKRCR; SEQ ID NO: 205), opicalcin-1 (GDCLPHLKRCKENNDCCSKKCKRRGTNPEKRCR; SEQ ID NO: 206), opicalcin-2 (GDCLPHLKRCKENNDCCSKKCKRRGANPEKRCR; SEQ ID NO: 207), huwentoxin (ECLEIFKACNPSNDQCCKSSKLVCSRKTRWCKYQIG
  • the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any sequence of SEQ ID NO: 1 - SEQ ID NO: 84. In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any sequence of SEQ ID NO: 85 - SEQ ID NO: 194 or SEQ ID NO: 408 - SEQ ID NO: 457.
  • the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any sequence of SEQ ID NO: 195 - SEQ ID NO: 254. In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a fragment of SEQ ID NO: 1 - SEQ ID NO: 84.
  • the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a fragment of SEQ ID NO: 85 - SEQ ID NO: 194 or SEQ ID NO: 408 - SEQ ID NO: 457. In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a fragment of SEQ ID NO: 195 - SEQ ID NO: 254.
  • a variant may comprise one or more Lys residues replaced with Arg. In some embodiments, a variant may comprise one or more Arg residues replaced with Lys. In some embodiments, a variant may comprise one or more Cys residues replaced with Ala.
  • a fragment may be a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46 at least 47, at least 48, at least 49, at least 50, at least 51
  • a fragment may be a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, no more than 30, no more than 31, no more than 32, no more than 33, no more than 34, no more than 35, no more than 36, no more than 37, no more than 38, no more than 39, no more than 40, no more than 41, no more than
  • the peptide sequence is flanked by additional amino acids.
  • additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
  • nuclear localization signals can be coupled to, conjugated to, linked to, or fused to a cell-penetrating peptide or a cell-penetrating peptide complex described herein to promote nuclear localization.
  • cell-penetrating peptides are conjugated to, linked to, or fused to a nuclear localization signal, such as a four-residue sequence of K-K/R-X-K/R (SEQ ID NO: 352), wherein X can be any amino acid, or a variant thereof.
  • cell-penetrating peptides are conjugated to, linked to, or fused to a nuclear localization signal as described in Lange et al, J Biol Chem. 2007 Feb 23 ;282(8): 5101 - 5, such as PKKKRRV (SEQ ID NO: 353) or KRPAATKKAGQAKKKK (SEQ ID NO: 354)
  • a cell-penetrating peptide described herein is conjugated to, linked to, or fused to a nuclear localization signal comprising KxRy (SEQ ID NO: 355), wherein x and y independently can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, such as KKRR (SEQ ID NO: 356), KKKRR (SEQ ID NO: 357), or KKKK (SEQ ID NO: 358).
  • cell-penetrating moieties can also be linked to, conjugated to, linked to, or fused to the peptides described herein, including, but not limited to, polycations, polyorganic acids, endosomal releasing polymers, poly(2- propylacrylic acid), poly(2-ethylacrylic acid), or any combination thereof.
  • cell penetration can be increased by using high dosage of a peptide described here, such as up to 10 pM, or 10 pM or more of the cell-penetrating peptide.
  • a peptide described here such as up to 10 pM, or 10 pM or more of the cell-penetrating peptide.
  • Up 10 pM, or 10 pM or more cell-penetrating peptide e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • Protein transfection agents can also be used to increase cell penetration of a peptide.
  • direct cytosolic expression of a peptide can be used.
  • physical disruption methods such as electroporation can be used to improve delivery of a peptide into a cell.
  • cell penetrance of a peptide described herein can be improved.
  • the binding interface of a peptide described herein can modified from a cellpenetrating peptide, such as a calcine.
  • calcines can be imperatoxin-A, maurocalcine, hemicalcin, opiclacin 1, opicalcin 2, and hadrucalcin.
  • the cellpenetrating peptide can comprise at least 60%, 70%, 80%, 90%, 95%, or 98% with any one of SEQ ID NO: 1 - SEQ ID NO: 100, SEQ ID NO: 102 - SEQ ID NO: 224.
  • the cell penetrance peptide can be calcines, modified calcines, derivatives of calcines, or fragments thereof, or variants thereof, which can be used to increase cell penetration.
  • Modified calcines, derivatives of calcines, or fragments can be screened for cell penetration activity such as activation of sarcoplasmic reticulum ryanodine receptors, activity on ryanodine-sensitive Ca 2+ channels RyRl, Ryr2, or both, or as a selective agonist of the foregoing.
  • modified calcines can include substitution, addition or reduction of Lysine residues, addition or reduction of Arginine residues, or other charged residues, within a native calcine in order to modify activity and optimize such calcine cell -penetration activity or activity on the RyRl or RyR2 receptors.
  • residues of a cell-penetrating peptide of this disclosure are modified so they do not act on or have reduced activity on RyRl, RyR2, or other receptors.
  • cell-penetrating peptides can penetrate into a target cell or into the nucleus of a target cell.
  • target cells include cancerous cells, tumors, neurons, inflammatory cells, cells of metastases, cancerous stem cells, and other cell types.
  • the target cell can be a human cell, a mammalian cell, a human or mammalian cell line, a cancer cell line, a cell extracted from a subject, in vivo, or in vitro.
  • the peptide can contain only one lysine residue, or no lysine residues. In some instances, some or all of the lysine residues in the peptide are replaced with arginine residues. In some instances, some or all of the methionine residues in the peptide are replaced by leucine or isoleucine. Some or all of the tryptophan residues in the peptide can be replaced by phenylalanine or tyrosine. In some instances, some or all of the asparagine residues in the peptide are replaced by glutamine. In some embodiments, some or all of the aspartic acid residues can be replaced by glutamic acid residues.
  • cysteine residues are replaced by alanine residues. In some embodiments, some or all of the cysteine residues are replaced by serine residues. In some embodiments, some or all of the cysteine residues are replaced by glycine residues.
  • the C-terminal Arg residues of a peptide is modified to another residue such as Ala, Asn, Asp, Gin, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai.
  • the C-terminal Arg residue of a peptide can be modified to He.
  • the C-terminal Arg residue of a peptide can be modified to any non-natural amino acid. This modification can prevent clipping of the C-terminal residue during expression, synthesis, processing, storage, in vitro, or in vivo including during treatment, while still allowing maintenance of a key hydrogen bond.
  • a key hydrogen bond can be the hydrogen bond formed during the initial folding nucleation and is critical for forming the initial hairpin.
  • the N-terminus of the peptide is blocked, such as by an acetyl group.
  • the C-terminus of the peptide is blocked, such as by an amide group.
  • the peptide is modified by methylation on free amines. For example, full methylation can be accomplished through the use of reductive methylation with formaldehyde and sodium cyanoborohydride.
  • GS can be added as the first two N-terminal amino acids, as shown in SEQ ID NO: 59 - SEQ ID NO: 84, SEQ ID NO: 93 - SEQ ID NO: 101, or SEQ ID NO: 210 - SEQ ID NO: 224, or such N-terminal amino acids (GS) can be absent as shown in SEQ ID NO: 1 - SEQ ID NO: 58, SEQ ID NO: 85 - SEQ ID NO: 92, SEQ ID NO: 102 - SEQ ID NO: 209, or SEQ ID NO: 225 - SEQ ID NO: 254, or can be substituted by any other one or two amino acids.
  • GS is used as a linker or used to couple to a linker to make a protein conjugate or fusion.
  • the linker comprises a G x S y (SEQ ID NO: 256) peptide, wherein x and y independently can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the peptide linker comprises (GS)x (SEQ ID NO: 258), wherein x can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the peptide linker comprises GGSSG (SEQ ID NO: 259), GGGGG (SEQ ID NO: 260), GSGSGSGS (SEQ ID NO: 261), GGGGS (SEQ ID NO: 263), GGGS (SEQ ID NO: 264), or a variant or fragment thereof or any number of repeats and combinations thereof.
  • KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx
  • EPKSSDKTHT (SEQ ID NO: 267) from human IgG3
  • the peptide linker comprises GGGSGGSGGGS (SEQ ID NO: 292) or a variant or fragment thereof or any number of repeats and combinations thereof. It is understood that any of the foregoing linkers or a variant or fragment thereof can be used with any number of repeats or any combinations thereof. It is also understood that other peptide linkers in the art or a variant or fragment thereof can be used with any number of repeats or any combinations thereof.
  • the linker between the cell -penetrating peptides in the protein conjugate or fusion within the selective depletion complex is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least
  • the C-terminal Arg residues of a peptide is modified to another residue such as Ala, Asn, Asp, Gin, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai.
  • the C-terminal Arg residue of a peptide can be modified to He.
  • the C-terminal Arg residue of a peptide can be modified to any non-natural amino acid. This modification can prevent clipping of the C-terminal residue during expression, synthesis, processing, storage, in vitro, or in vivo including during treatment, while still allowing maintenance of a key hydrogen bond.
  • a key hydrogen bond can be the hydrogen bond formed during the initial folding nucleation and is critical for forming the initial hairpin.
  • the peptide comprises the sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • a peptide can be a fragment comprising a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is at least 3, at least 4, at least 5, at least 6, at least 7, at least, 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46 at least 47, at least 48, at least 49, at least 50, at least 51
  • the peptide sequence is flanked by additional amino acids.
  • additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
  • a peptide can be a fragment comprising a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is not greater than 3, not greater than 4, not greater than 5, not greater than 6, not greater than 7, not greater than, 8, not greater than 9, not greater than 10, not greater than 11, not greater than 12, not greater than 13, not greater than 14, not greater than 15, not greater than 16, not greater than 17, not greater than 18, not greater than 19, not greater than 20, not greater than 21, not greater than 22, not greater than 23, not greater than 24, not greater than 25, not greater than 26, not greater than 27, not greater than 28, not greater than 29, not greater than 30, not greater than 31, not greater than 32, not greater than 33, not greater than 34, not greater than 35, not greater than 36, not greater than 37, not greater than 38, not greater than 39,
  • the peptide sequence is flanked by additional amino acids.
  • additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
  • peptide sequences comprise primarily beta-sheets and/or alphahelix structures.
  • cell-penetrating peptides of the present disclosure are small, compact mini-proteins stabilized by intra-chain disulfide bonds (mediated by cysteines) and a well-packed hydrophobic core.
  • cell-penetrating peptides e.g., SEQ ID NO: 1 SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • structures comprising helical bundles with at least one disulfide bridge between each of the alpha helices, thereby stabilizing the peptides.
  • a cell-penetrating peptide can be amphipathic, comprising a hydrophilic region and a hydrophobic region, which can facilitate interactions with cell membranes and insertion dynamics.
  • a cell-penetrating peptide can comprise an amphipathic a- helix, which can facilitate interactions with cellular membranes.
  • the amphipathicity of a cell-penetrating peptide is pH dependent and is present to a higher degree at endosomal or lysosomal pHs than at extracellular pH.
  • cell-penetrating peptides contain one or more positively charged residues or carry a net positive charge.
  • a cell-penetrating peptide is cyclized peptide or a stapled peptide.
  • peptides can be conjugated to, linked to, or fused to a cargo peptide or cargo molecule to deliver the cargo peptide or cargo molecule to a target of interest in a cell.
  • peptides can be conjugated to, linked to, or fused to a molecule that extends half-life or modifies the pharmacodynamic and/or pharmacokinetic properties of the peptides, or any combination thereof.
  • a peptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positively charged residues, such as Arg or Lys, or any combination thereof.
  • one or more lysine residues in the peptide are replaced with arginine residues.
  • one or more arginine residues in the peptide are replaced with lysine residues.
  • 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more Arg or Lys residues are solvent exposed on a peptide.
  • the peptides of the present disclosure can further comprise neutral amino acid residues.
  • the peptide has 35 or fewer neutral amino acid residues.
  • the peptide has 81 or fewer neutral amino acid residues, 70 or fewer neutral amino acid residues, 60 or fewer neutral amino acid residues, 50 or fewer neutral amino acid residues, 40 or fewer neutral amino acid residues, 36 or fewer neutral amino acid residues, 33 or fewer neutral amino acid residues, 30 or fewer neutral amino acid residues, 25 or fewer neutral amino acid residues, or 10 or fewer neutral amino acid residues.
  • the peptides of the present disclosure can further comprise negative amino acid residues.
  • the peptide has 6 or fewer negative amino acid residues, 5 or fewer negative amino acid residues, 4 or fewer negative amino acid residues, 3 or fewer negative amino acid residues, 2 or fewer negative amino acid residues, or 1 or fewer negative amino acid residues.
  • negative amino acid residues can be selected from any negatively charged amino acid residues, in some embodiments, the negative amino acid residues are either E, or D, or a combination of both E and D.
  • a peptide comprises no Cys or disulfides. In some cases, a peptide comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more Cys or disulfides. In other embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Ser residues.
  • 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Thr residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Ala, Gly, or Met residues.
  • the NMR solution or x-ray crystallography structures of related structural homologs can be used to inform mutational strategies that may improve the folding, stability, manufacturability, while maintaining a particular biological function. They can be used to predict the 3D pharmacophore of a group of structurally homologous scaffolds, as wells as to predict possible graft regions of related proteins to create chimeras with improved properties. For example, this strategy was used to identify critical amino acid positions and loops that may be used to design peptides with improved cell-penetrating properties, high expression, high stability in vivo, or any combination thereof.
  • a cell-penetrating peptide capable of penetrating a cellular layer comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with with a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, as provided in TABLE 1, or a fragment thereof.
  • a cell-penetrating peptide capable of penetrating a cellular layer comprises a sequence with no more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a peptide of any one of SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, as provided in TABLE 2, or a fragment thereof.
  • a cell -penetrating peptide capable of penetrating a cellular layer comprises a sequence with no more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a peptide of any one of SEQ ID NO: 195 — SEQ ID NO: 254, as provided in TABLE 3, or a fragment thereof.
  • Two or more peptides can share a degree of sequence identity or homology and share similar properties in vivo.
  • a peptide can share a degree of sequence identity or homology with any one of the peptides of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
  • one or more peptides of the disclosure can have up to about 20% pairwise sequence identity or homology, up to about 25% pairwise sequence identity or homology, up to about 30% pairwise sequence identity or homology, up to about 35% pairwise sequence identity or homology, up to about 40% pairwise sequence identity or homology, up to about 45% pairwise sequence identity or homology, up to about 50% pairwise sequence identity or homology, up to about 55% pairwise sequence identity or homology, up to about 60% pairwise sequence identity or homology, up to about 65% pairwise sequence identity or homology, up to about 70% pairwise sequence identity or homology, up to about 75% pairwise sequence identity or homology, up to about 80% pairwise sequence identity or homology, up to about 85% pairwise sequence identity or homology, up to about 90% pairwise sequence identity or homology, up to about 95% pairwise sequence identity or homology, up to about 96% pairwise sequence identity or homology, up to about 97% pairwise sequence identity or homology, up to about 98% pairwise sequence identity or homology,
  • one or more peptides of the disclosure can have at least about 20% pairwise sequence identity or homology, at least about 25% pairwise sequence identity or homology, at least about 30% pairwise sequence identity or homology, at least about 35% pairwise sequence identity or homology, at least about 40% pairwise sequence identity or homology, at least about 45% pairwise sequence identity or homology, at least about 50% pairwise sequence identity or homology, at least about 55% pairwise sequence identity or homology, at least about 60% pairwise sequence identity or homology, at least about 65% pairwise sequence identity or homology, at least about 70% pairwise sequence identity or homology, at least about 75% pairwise sequence identity or homology, at least about 80% pairwise sequence identity or homology, at least about 85% pairwise sequence identity or homology, at least about 90% pairwise sequence identity or homology, at least about 95% pairwise sequence identity or homology, at least about 96% pairwise sequence identity or homology, at least about 97% pairwise sequence identity or homology, at least about 98% pairwise sequence identity or homology,
  • Various methods and software programs can be used to determine the homology between two or more peptides, such as NCBI BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method or algorithm. Pairwise sequence alignment is used to identify regions of similarity that may indicate functional, structural and/or evolutionary relationships between two biological sequences (protein or nucleic acid) By contrast, multiple sequence alignment (MSA) is the alignment of three or more biological sequences. From the output of .MSA applications, homology can be inferred and the evolutionary relationship between the sequences assessed.
  • MSA multiple sequence alignment
  • sequence homology and “sequence identity” and “percent (%) sequence identity” and “percent (%) sequence homology” have been used interchangeably to mean the sequence relatedness or variation, as appropriate, to a reference polynucleotide or amino acid sequence.
  • the peptide is any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or a functional fragment thereof.
  • the peptide of the disclosure further comprises a peptide with 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99%, 97%, 95%, 90%, 85%, or 80% sequence identity or homology to any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or fragment thereof.
  • the peptide can be a peptide that is homologous to any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or a functional fragment thereof.
  • homologous is used herein to denote peptides having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity or homology to a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or a functional fragment thereof.
  • variant nucleic acid molecules that encode a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408
  • SEQ ID NO: 457 or SEQ ID NO: 195 - SEQ ID NO: 254 can be identified by either a determination of the sequence identity or homology of the encoded peptide amino acid sequence with the amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254, or by a nucleic acid hybridization assay.
  • SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 can be characterized as nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule having the nucleotide sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1 x-0.2xSSC with 0.1% SDS at 50-65° C., and (2) that encode a peptide having at least 70%, at least 80%, at least 90*Eo, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least
  • a peptide is modified to increase homology to a human protein sequence.
  • a peptide is modified to increase resistance to degradation.
  • a peptide is modified to reduce an affinity of the peptide for a human leukocyte antigen complex, a major histocompatibility complex, or both.
  • peptides that have cell-penetrating capabilities can be aligned where amino acids that are the same or similar are aligned.
  • Positional amino acid variants at one position in a given peptide may be placed in the same position in a different aligned peptide to create a new peptide.
  • fragments of one peptide that is penetrant may identify fragments in aligned peptides that may also be cell penetrant.
  • Amino acids of like charge can be varied, such as changing Lys to Arg and vice versa, and changing Asp to Glu and vice versa.
  • Cysteine residues may be replaced in peptide fragments or where unpaired with other Cys residues to improve manufacturability, shelf-life stability, purity, in vivo stability, in vivo safety, and in vivo activity.
  • one or more cysteine residues may be mutated to alanine.
  • one or more cysteine residues may be mutated to any other amino acid, or to glycine or aminobutyric acid.
  • Histidine residues may be added to facilitate endosomal escape.
  • Proline residues may be added to facilitate endosomal escape.
  • An alignment may be generated using R language and an “MSA” software package, which codes for R language specific for multiple alignments (Bodenhofer, U et al.
  • peptides of the family of sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO 22 - SEQ ID NO: 29, SEQ ID NO: 44 - SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, or SEQ ID NO: 68 may retain essential properties such as structure, function, peptide folding, biodistribution, or stability.
  • peptides comprising a segment corresponding to SEQ ID NO: 350 or SEQ ID NO: 351 may have structure, function, peptide folding, biodistribution, binding, accumulation, retention, or stability favorable for cell penetration.
  • a positively charged region for example as seen in residues 30-33 of SEQ ID NO: 197 or SEQ ID NO: 202 or residues 32-35 of SEQ ID NO: 200, may improve cell-penetrating properties of a peptide (e.g., a cystine-dense peptide).
  • Other conserved regions within sequences of the present disclosure can be identified.
  • an alignment may be generated using R language and an “msa” software package, which codes for R language specific for multiple alignments (Bodenhofer, U et al.
  • cell-penetrating peptides, cargo peptides, or cell -penetrating peptide complexes of the present disclosure comprise one or more Cys, or one or more disulfide bonds.
  • the cell-penetrating peptides are derived from cystine-dense peptides (CDPs), knotted peptides, or hitchins.
  • the cargo peptides are derived from cystine-dense peptides (CDPs), knotted peptides, or hitchins.
  • peptide is considered to be interchangeable with the terms “knotted peptide”, “cystine- dense peptide”, “CDP”, and “hitchin”. (See e.g., Correnti et al. Screening, large-scale production, and structure-based classification for cystine-dense peptides. Nat Struct Mol Biol. 2018 Mar; 25(3): 270-278).
  • the peptides of the present disclosure are cystine-dense peptides (CDPs), related to knotted peptides or hitchin-derived peptides or knottin-derived peptides.
  • CDPs cystine-dense peptides
  • the cellpenetrating peptides can be cystine-dense peptides (CDPs).
  • Hitchins can be a subclass of CDPs wherein six cysteine residues form disulfide bonds according to the connectivity [1-4], 2-5, 3-6 indicating that the first cysteine residue forms a disulfide bond with the fourth residue, the second with the fifth, and the third cysteine residue with the sixth.
  • the brackets in this nomenclature indicate cysteine residues form the knotting disulfide bond.
  • Knotting can be a subclass of CDPs wherein six cysteine residues form disulfide bonds according to the connectivity 1-4, 2-5, [3-6], Knotting are a class of peptides, usually ranging from about 20 to about 80 amino acids in length that are often folded into a compact structure. Knottins are typically assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks and can contain beta strands and other secondary structures.
  • the peptides described herein can be derived from knotted peptides.
  • the amino acid sequences of peptides as disclosed herein can comprise a plurality of cysteine residues. In some cases, at least six cysteine residues of the plurality of cysteine residues present within the amino acid sequence of a peptide participate in the formation of disulfide bonds. In some cases, all cysteine residues of the plurality of cysteine residues present within the amino acid sequence of a peptide participate in the formation of disulfide bonds.
  • the term “knotted peptide” can be used interchangeably with the terms “cystine-dense peptide”, “CDP”, or “peptide”.
  • CDPs comprise alternatives to existing biologies, primarily antibodies, which can bypass some of the liabilities of the immunoglobulin scaffold, including poor tissue permeability, immunogenicity, and long serum half-life that can become problematic if toxicities arise.
  • Peptides of the present disclosure in the 20-80 amino acid range represent medically relevant therapeutics that are mid-sized, with many of the favorable binding specificity and affinity characteristics of antibodies but with improved cell penetration, stability, reduced immunogenicity, and simpler manufacturing methods.
  • the intramolecular disulfide architecture of CDPs provides particularly high stability metrics, reducing fragmentation and immunogenicity, while their smaller size could improve tissue penetration or cell penetration and facilitate tunable serum half-life.
  • peptides representing candidate peptides that can serve as vehicles for delivering target molecules to cellular, intracellular, and paracellular compartments or spaces.
  • cell-penetrating peptides can be engineered peptides.
  • An engineered peptide can be a peptide that is non-naturally occurring, artificial, isolated, synthetic, designed, or recombinantly expressed.
  • the cell-penetrating peptides of the present disclosure comprise one or more properties of CDPs, knotted peptides, or hitchins, such as stability, resistance to proteolysis, resistance to heat, resistance to denaturation, resistance to reducing conditions, and/or ability to cross the blood brain barrier.
  • cell-penetrating peptides as described herein contain no disulfides or Cys (e g., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 21 - SEQ ID NO: 29, SEQ ID NO: 41 - SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 70, SEQ ID NO: 82 - SEQ ID NO: 84, SEQ ID NO: 188, SEQ ID NO: 163 - SEQ ID NO: 165, SEQ ID NO: 168, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 198 - SEQ ID NO: 201, SEQ ID NO: 210, SEQ ID NO: 213, SEQ ID NO: 225, SEQ ID NO: 227 - SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 246, or S
  • cell-penetrating peptides may comprise one or more Cys, or one or more disulfide bond (e g., SEQ ID NO: 1 - SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 18 - SEQ ID NO: 20, SEQ ID NO: 30 - SEQ ID NO: 40, SEQ ID NO: 59 - SEQ ID NO: 61, SEQ ID NO: 64 - SEQ ID NO: 69, SEQ ID NO: 71 - SEQ ID NO: 81, SEQ ID NO: 85 - SEQ ID NO: 117, SEQ ID NO: 119 SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 169 - SEQ ID NO: 193, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 202 - SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO:
  • the cell-penetrating peptides are derived from knotted or knottin peptides.
  • such knotted peptides can penetrate a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen).
  • a peptide that penetrates a cellular layer comprises one or more properties of some knottin peptides, such as stability, resistance to heat, resistance to denaturation, resistance to proteolysis, and/or resistance to reducing conditions.
  • knotted peptides can be conjugated to, linked to, or fused to cellpenetrating peptides, such as those described in TABLE 1, TABLE 2, or TABLE 3, to deliver the knotted peptide to a target cell, such as a cancer cell, a pancreatic cell, liver cell, colon cell, a smooth muscle cell, ovarian cell, breast cell, lung cell, brain cell, skin cell, ocular cell, blood cell, lymph cell, immune system cell, reproductive cell, reproductive organ cell, prostate cell, fibroblast, kidney cell, adenocarcinoma cell, glioma stem cell, tumor cell, or any combination thereof.
  • a target cell such as a cancer cell, a pancreatic cell, liver cell, colon cell, a smooth muscle cell, ovarian cell, breast cell, lung cell, brain cell, skin cell, ocular cell, blood cell, lymph cell, immune system cell, reproductive cell, reproductive organ cell, prostate cell, fibroblast, kidney cell, adenocarcinoma cell, gliom
  • knotted peptides may perform a function inside a target cell, such a cancer cell, such as a breast cancer, liver cancer, colon cancer, brain cancer, pancreatic cancer, lung cancer, leukemia, lymphoma, myeloma, skin cancer, fibroblastic cancer, kidney cell cancer, adenocarcinoma cell, glioma stem cell, or tumor cell.
  • a knotted peptide may promote or inhibit a protein-protein interaction within a target cell, or a knotted peptide may promote or inhibit transcription of a target gene.
  • knotted peptides are conjugated to, linked to, or fused to cell-penetrating peptides that are capable of delivering the knotted peptide across a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) to target cells in the central nervous system.
  • cell-penetrating peptides can be used to deliver a cytotoxic agent to the target cell.
  • a knotted cargo peptide may be conjugated to a knotted cell-penetrating peptide to form a diknottin (e g., SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 316, or SEQ ID NO: 317).
  • a diknottin e g., SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 316, or SEQ ID NO: 317.
  • Knottins are a class of peptides, usually ranging from about 11 to about 81 amino acids in length that are often folded into a compact structure. Knottins are typically assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks and may contain beta strands, alpha helices, and other secondary structures. The presence of the disulfide bonds gives knottins remarkable environmental stability, allowing them to withstand extremes of temperature and pH and to resist the proteolytic enzymes of the blood stream. The presence of a disulfide knot may provide resistance to reduction by reducing agents.
  • knottins also allows them to bind to targets without paying the “entropic penalty” that a floppy peptide accrues upon binding a target.
  • binding is adversely affected by the loss of entropy that occurs when a peptide binds a target to form a complex. Therefore, “entropic penalty” is the adverse effect on binding, and the greater the entropic loss that occurs upon this binding, the greater the “entropic penalty.”
  • unbound molecules that are flexible lose more entropy when forming a complex than molecules that are rigidly structured, because of the loss of flexibility when bound up in a complex.
  • rigidity in the unbound molecule also generally increases specificity by limiting the number of complexes that molecule can form.
  • knotted peptides can bind targets with antibody -like affinity, or with nanomolar or picomolar affinity.
  • a wider examination of the sequence structure and sequence identity or homology of knottins reveals that they have arisen by convergent evolution in all kinds of animals and plants. In animals, they are often found in venoms, for example, the venoms of spiders and scorpions and have been implicated in the modulation of ion channels.
  • the knottin proteins of plants can inhibit the proteolytic enzymes of animals or have antimicrobial activity, suggesting that knottins can function in the native defense of plants.
  • the present disclosure provides peptides that comprise or are derived from these knotted peptides (or knottins).
  • the term “knotted peptide” is considered to be interchangeable with the terms “knottin.”
  • Knotted peptides of the present disclosure comprise cysteine amino acid residues.
  • the peptide has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 cysteine amino acid residues.
  • the peptide has at least 8 cysteine amino acid residues.
  • the peptide has at least 10 cysteine amino acid residues, at least 12 cysteine amino acid residues, at least 14 cysteine amino acid residues or at least 16 cysteine amino acid residues.
  • a knotted peptide can comprise disulfide bridges.
  • a knotted peptide can be a peptide wherein 5% or more of the residues are cysteines forming intramolecular disulfide bonds.
  • a disulfide-linked peptide can be a drug scaffold.
  • the disulfide bridges form a knot.
  • a disulfide bridge can be formed between cysteine residues, for example, between cysteines 1 and 4, 2 and 5, or, 3 and 6. In some cases, one disulfide bridge passes through a loop formed by the other two disulfide bridges, for example, to form the knot. In other cases, the disulfide bridges can be formed between any two cysteine residues.
  • the present disclosure further includes peptide scaffolds that, e g., can be used as a starting point for generating additional peptides.
  • these scaffolds can be derived from a variety of knotted peptides (or knottins).
  • knotted peptides are assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks, and optionally contain beta strands and other secondary structures such as an alpha helix.
  • knotted peptides include, in some embodiments, small disulfide-rich proteins characterized by a disulfide through disulfide knot.
  • This knot can be, for example, obtained when one disulfide bridge crosses the macrocycle formed by two other disulfides and the interconnecting backbone.
  • the knotted peptides can include growth factor cysteine knots or inhibitor cysteine knots.
  • Other possible peptide structures include peptide having two parallel helices linked by two disulfide bridges without p- sheets (e g., hefutoxin).
  • a knotted peptide can comprise at least one amino acid residue in an L configuration.
  • a knotted peptide can comprise at least one amino acid residue in a D configuration.
  • a knotted peptide is 15-40 amino acid residues long.
  • a knotted peptide is 11-57 amino acid residues long.
  • a knotted peptide is 11-81 amino acid residues long.
  • a knotted peptide is at least 20 amino acid residues long.
  • Knotted peptides or peptides can be derived from a class of proteins known to be present or associated with toxins or venoms.
  • the peptide can be derived from toxins or venoms associated with scorpions or spiders.
  • the peptide can be derived from venoms and toxins of spiders and scorpions of various genus and species.
  • the peptide can be derived from a venom or toxin of the Leiurus quinquestriatus hebraeus, Buthus occitanus tunetanus, Hottentotta judaicus, Mesobuthus eupeus, Buthus occitanus Israelis, Hadrurus gertschi, Androctonus australis, Centruroides noxius, Heteroflowers laoticus, Opistophthalmus carinatus, Haplopelma schmidti, Isometrus maculatus, Haplopelma huwenum, Haplopelma hainanum, Haplopelma schmidti, Agelenopsis aperta, Haydronyche
  • a peptide of the present disclosure can comprise a sequence having cysteine residues at one or more of corresponding positions 11, 12, 13, 14, 19, 20, 21, 22, 36, 38, 39, 41, for example with reference to SEQ ID NO: 295.
  • a peptide comprises Cys at corresponding positions 11, 12, 19, 20, 36, 39, or any combination thereof.
  • a peptide can comprise a sequence having a cysteine residue at corresponding position 11.
  • a peptide can comprise a sequence having a cysteine residue at corresponding position 12.
  • a peptide can comprise a sequence having a cysteine residue at corresponding position 13. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 14. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 19. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 20. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 21. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 22.
  • a peptide can comprise a sequence having a cysteine residue at corresponding position 36. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 38. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 39. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 41.
  • the first cysteine residue in the sequence can be disulfide bonded with the 4th cysteine residue in the sequence
  • the 2nd cysteine residue in the sequence can be disulfide bonded to the 5th cysteine residue in the sequence
  • the 3rd cysteine residue in the sequence can be disulfide bonded to the 6th cysteine residue in the sequence.
  • a peptide can comprise one disulfide bridge that passes through a ring formed by two other disulfide bridges, also known as a “two-and-through” structure system.
  • the peptides disclosed herein can have one or more cysteines mutated to serine.
  • peptides of the present disclosure comprise at least one cysteine residue. In some embodiments, peptides of the present disclosure comprise at least two cysteine residues. In some embodiments, peptides of the present disclosure comprise at least three cysteine residues. In some embodiments, peptides of the present disclosure comprise at least four cysteine residues. In some embodiments, peptides of the present disclosure comprise at least five cysteine residues. In some embodiments, peptides of the present disclosure comprise at least six cysteine residues.
  • peptides of the present disclosure comprise at least ten cysteine residues. In some embodiments, a peptide of the present disclosure comprises six cysteine residues. In some embodiments, a peptide of the present disclosure comprises seven cysteine residues. In some embodiments, a peptide of the present disclosure comprises eight cysteine residues. In some embodiments, a peptide of the present disclosure comprises an even number of cysteine residues.
  • a peptide of the present disclosure comprises an amino acid sequence having cysteine residues at one or more positions, for example with reference to SEQ ID NO: 295.
  • the one or more cysteine residues are located at any one of the corresponding amino acid positions 6, 10, 20, 34, 44, 48, or any combination thereof.
  • the one or more cysteine (C) residues participate in disulfide bonds with various pairing patterns (e.g., C10-C20).
  • the corresponding pairing patterns are C6-C48, C10-C44, and C20-C34.
  • the peptides as described herein comprise at least one, at least two, or at least three disulfide bonds. In some embodiments, at least one, at least two, or at least three disulfide bonds are arranged according to the corresponding C6-C48, C10-C44, and C20-C34 pairing patterns, or a combination thereof. In some embodiments, peptides as described herein comprise three disulfide bonds with the corresponding pairing patterns C6-C48, C10-C44, and C20-C34.
  • a peptide (e.g., a cell-penetrating peptide or a cell-penetrating peptide complex) comprises a sequence having a cysteine residue at corresponding position 6. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 10. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 20. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 34. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 44.
  • a peptide comprises a sequence having a cysteine residue at corresponding position 50.
  • the first cysteine residue in the sequence is disulfide bonded with the last cysteine residue in the sequence.
  • the second cysteine residue in the sequence is disulfide bonded with the second to the last cysteine residue in the sequence.
  • the third cysteine residue in the sequence is disulfide bonded with the third to the last cysteine residue in the sequence and so forth.
  • the first cysteine residue in the sequence is disulfide bonded with the 6th cysteine residue in the sequence
  • the 2nd cysteine residue in the sequence is disulfide bonded to the 5th cysteine residue in the sequence
  • the 3rd cysteine residue in the sequence is disulfide bonded to the 4th cysteine residue in the sequence.
  • a peptide can comprise one disulfide bridge that passes through a ring formed by two other disulfide bridges, also known as a “two-and-through” structure system.
  • the peptides disclosed herein have one or more cysteines mutated to serine.
  • a peptide of the present disclosure has an even number of cysteine residues.
  • a peptide of the present disclosure comprises no cysteine residues.
  • a peptide (e.g., a cell-penetrating peptide or a cell-penetrating peptide complex) comprises no cysteine or disulfides.
  • a peptide comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more cysteine or disulfides.
  • 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more cysteine residues have been replaced with serine residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more cysteine residues have been replaced with threonine residues.
  • a peptide (e.g., a cell-penetrating peptide or a cell-penetrating peptide complex) comprises no Cys or disulfides.
  • a peptide comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more Cys or disulfides.
  • 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Ser residues.
  • 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Thr residues.
  • one or more or all of the methionine residues in the peptide are replaced by leucine or isoleucine.
  • one or more or all of the tryptophan residues in the peptide are replaced by phenylalanine or tyrosine.
  • one or more or all of the asparagine residues in the peptide are replaced by glutamine.
  • the N-terminus of the peptide is blocked, such as by an acetyl group.
  • the C-terminus of the peptide is blocked, such as by an amide group.
  • the peptide is modified by methylation on free amines.
  • Percent sequence identity or homology is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (Id.). The sequence identity or homology is then calculated as: ([Total number of identical matches]/[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).
  • FASTA similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of sequence identity or homology shared by an amino acid sequence of a peptide disclosed herein and the amino acid sequence of a peptide variant
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat'lAcad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
  • the ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are “trimmed” to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch- Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, Siam J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions.
  • FASTA can also be used to determine the sequence identity or homology of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as described above.
  • ⁇ amino acids that are a “conservative amino acid substitution” are illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat'lAcad. Sci.
  • the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention.
  • conservative amino acid substitution preferably refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that can be more or less tolerant of change and maintain the overall tertiary structure of the molecule.
  • Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity or homology and computer analysis using available software (e.g., the Insight II.RTM. viewer and homology modeling tools; MSI, San Diego, Calif), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, G.J., Current Opin. Struct. Biol. 5'312-6 (1995) and Cordes, M.H. et al., Current Opin. Struct. Biol. 6:3-10 (1996)).
  • modifications to molecules or identifying specific fragments determination of structure can typically be accompanied by evaluating activity of modified molecules.
  • peptides can have a net charge, for example, of -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, or +5.
  • the net charge is zero, the peptide can be uncharged or zwitterionic.
  • the peptide contains one or more disulfide bonds and has a positive net charge at physiological pH where the net charge can be +0.5 or less than +0.5, +1 or less than +1, +1.5 or less than +1.5, +2 or less than +2, +2.5 or less than +2.5, +3 or less than +3, +3.5 or less than +3.5, +4 or less than +4, +4.5 or less than +4.5, +5 or less than +5, +5.5 or less than +5.5, +6 or less than +6, +6.5 or less than +6.5, +7 or less than +7, +7.5 or less than +7.5, +8 or less than +8, +8.5 or less than +8.5, +9 or less than +9.5, +10 or less than +10.
  • the peptide has a negative net charge at physiological pH where the net charge can be -0.5 or less than -0.5, -1 or less than -1, -1.5 or less than -1.5, -2 or less than -2, -2.5 or less than -2.5, -3 or less than -3, -3.5 or less than -3.5, -4 or less than -4, -4.5 or less than -4.5, -5 or less than -5, -5.5 or less than -5.5, -6 or less than -6, -6.5 or less than -6.5, -7 or less than -7, - 7.5 or less than -7.5, -8 or less than -8, -8.5 or less than -8.5, -9 or less than -9.5, -10 or less than -10.
  • peptides of the present disclosure can have an isoelectric point (pl) value from 3 and 10. In other embodiments, peptides of the present disclosure can have a pl value from 4.3 and 8.9. In some embodiments, peptides of the present disclosure can have a pl value from 3-4. In some embodiments, peptides of the present disclosure can have a pl value from 3-5. In some embodiments, peptides of the present disclosure can have a pl value from 3-6. In some embodiments, peptides of the present disclosure can have a pl value from 3-7. In some embodiments, peptides of the present disclosure can have a pl value from 3-8.
  • pl isoelectric point
  • peptides of the present disclosure can have a pl value from 3-9. In some embodiments, peptides of the present disclosure can have a pl value from 4-5. In some embodiments, peptides of the present disclosure can have a pl value from 4-6. In some embodiments, peptides of the present disclosure can have a pl value from 4-7. In some embodiments, peptides of the present disclosure can have a pl value from 4-8. In some embodiments, peptides of the present disclosure can have a pl value from 4-9. In some embodiments, peptides of the present disclosure can have a pl value from 4-10 In some embodiments, peptides of the present disclosure can have a pl value from 5-6.
  • peptides of the present disclosure can have a pl value from 5-7. In some embodiments, peptides of the present disclosure can have a pl value from 5-8. In some embodiments, peptides of the present disclosure can have a pl value from 5-9. In some embodiments, peptides of the present disclosure can have a pl value from 5-10. In some embodiments, peptides of the present disclosure can have a pl value from 6-7. In some embodiments, peptides of the present disclosure can have a pl value from 6-8. In some embodiments, peptides of the present disclosure can have a pl value from 6-9. In some embodiments, peptides of the present disclosure can have a pl value from 6-10.
  • peptides of the present disclosure can have a pl value from 7-8. In some embodiments, peptides of the present disclosure can have a pl value from 7-9. In some embodiments, peptides of the present disclosure can have a pl value from 7-10. In some embodiments, peptides of the present disclosure can have a pl value from 8-9. In some embodiments, peptides of the present disclosure can have a pl value from 8-10. In some embodiments, peptides of the present disclosure can have a pl value from 9-10.
  • the engineering of one or more mutations within a peptide yields a peptide with an altered isoelectric point, charge, surface charge, or rheology at physiological pH.
  • Such engineering of a mutation to a peptide derived from a scorpion or spider can change the net charge of the peptide, for example, by decreasing the net charge by 1, 2, 3, 4, or 5, or by increasing the net charge by 1, 2, 3, 4, or 5.
  • the engineered mutation can facilitate the ability of the peptide to penetrate a cell, an endosome, or the nucleus.
  • Suitable amino acid modifications for improving the rheology and potency of a peptide can include conservative or non-conservative mutations.
  • a peptide can comprise at most 1 amino acid mutation, at most 2 amino acid mutations, at most 3 amino acid mutations, at most 4 amino acid mutations, at most 5 amino acid mutations, at most 6 amino acid mutations, at most 7 amino acid mutations, at most 8 amino acid mutations, at most 9 amino acid mutations, at most 10 amino acid mutations, or another suitable number as compared to the sequence of the venom or toxin component that the peptide is derived from.
  • a peptide, or a functional fragment thereof comprises at least 1 amino acid mutation, at least 2 amino acid mutations, at least 3 amino acid mutations, at least 4 amino acid mutations, at least 5 amino acid mutations, at least 6 amino acid mutations, at least 7 amino acid mutations, at least 8 amino acid mutations, at least 9 amino acid mutations, at least 10 amino acid mutations, or another suitable number as compared to the sequence of the venom or toxin component that the peptide is derived from.
  • mutations can be engineered within a peptide to provide a peptide that has a desired charge or stability at physiological pH.
  • the NMR solution structures, the x-ray crystal structures, as well as the primary structure sequence alignment of related structural homologs or in silico design can be used to inform mutational strategies that can improve the folding, stability, and/or manufacturability, while maintaining a particular biological function.
  • the general strategy for producing homologs or in silico designed peptides or proteins can include identification of a charged surface patch or conserved residues of a protein, mutation of critical amino acid positions and loops, and testing of sequences. This strategy can be used to design peptides with improved properties or to correct deleterious mutations that complicate folding and manufacturability.
  • the present disclosure also encompasses multimers of the various peptides described herein.
  • multimers include dimers, trimers, tetramers, pentamers, hexamers, heptamers, and so on.
  • a multimer may be a homomer formed from a plurality of identical subunits or a heteromer formed from a plurality of different subunits.
  • a peptide of the present disclosure is arranged in a multimeric structure with at least one other peptide, or two, three, four, five, six, seven, eight, nine, ten, or more other peptides.
  • the peptides of a multimeric structure each have the same sequence.
  • the present disclosure further includes peptide scaffolds that, e.g., can be used as a starting point for generating additional peptides.
  • these scaffolds can be derived from a variety of knotted peptides or knottins.
  • Suitable peptides for scaffolds can include, but are not limited to, CDPS, chlorotoxin, brazzein, circulin, stecrisp, hanatoxin, midkine, hefutoxin, potato carboxypeptidase inhibitor, bubble protein, attractin, a-GI, a-GID, p- PIIIA, co-MVIIA, ®-CVID, %-MrIA, p-TIA, conantokin G, conantokin G, conantokin G, conantakin G, GsMTx4, margatoxin, shK, toxin K, chymotrypsin inhibitor (CTI), and EGF epiregulin core.
  • the peptide sequence is flanked by additional amino acids.
  • One or more additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
  • a cell-penetrating peptide sequence (e.g., a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) can be fused or linked to a cargo molecule, such a small molecule or peptide.
  • a cell-penetrating peptide may be appended to either the N- terminus or the C-terminus of a peptide (e.g., a cargo peptide, an active agent peptide, a therapeutic peptide, or a detectable peptide).
  • the cell -penetrating peptide can be appended to the N-terminus of a peptide (e.g., a cargo peptide, an active agent peptide, a therapeutic peptide, or a detectable peptide) following an N-terminal GS dipeptide and preceding, for example, a GGGS (SEQ ID NO: 257) linker.
  • a peptide e.g., a cargo peptide, an active agent peptide, a therapeutic peptide, or a detectable peptide
  • cellpenetrating peptide sequence e.g., a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • a peptide e.g., a cargo peptide, an active agent peptide, a therapeutic peptide, or a detectable peptide
  • a peptide linker such as G x S y (SEQ ID NO: 256) peptide linker, wherein x and y can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the peptide linker comprises (GS)x (SEQ ID NO: 258), wherein x can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the peptide linker comprises GGSSG (SEQ ID NO: 259), GGGGG (SEQ ID NO: 260), GSGSGSGS (SEQ ID NO: 261), GSGG (SEQ ID NO: 262), GGGGS (SEQ ID NO: 263), GGGS (SEQ ID NO: 264), GGS (SEQ ID NO: 265), GGGSGGGSGGGS (SEQ ID NO: 255), or a variant or fragment thereof.
  • KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx
  • EPKSSDKTHT (SEQ ID NO: 267) from human IgG3
  • the cell-penetrating peptide can be appended to the peptide at any amino acid residue.
  • the cell-penetrating peptide can be appended to the peptide at any amino acid residue without interfering with an activity of the peptide (e.g., a therapeutic activity, an enzymatic activity, a binding activity, or a transcription factor activity).
  • the tag peptide is appended via conjugation, linking, or fusion techniques. In some embodiments, the tag peptide is placed between two other peptides with active functions. In other embodiments, the cell-penetrating peptide can be appended to the peptide at any amino acid residue. In further embodiments, the cell-penetrating peptide can be appended to the peptide at any amino acid residue without interfering with peptide activity (e.g., therapeutic activity, enzymatic activity, binding activity, or transcription factor activity). In some embodiments, the cell-penetrating peptide is appended via conjugation, linking, or fusion techniques. Examples of cell -penetrating peptide complexes that may improve cell penetration of the cargo peptide are provided in TABLE 4.
  • a cargo molecule is conjugated to, linked to, or fused to one or more cell-penetrating peptides (e.g., a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) for delivery of a cargo molecule across a cellular layer or into the cytoplasm or nucleus of a cell.
  • Conjugation, linking, or fusion can be direct or with a spacer in between (chemical or peptide-based).
  • a spacer can be any peptide linker.
  • a spacer can be GGGSGGGSGGGS (SEQ ID NO: 255), KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx, EPKSSDKTHT (SEQ ID NO: 267) from human IgG3 or any variant or fragment thereof.
  • a cell-penetrating peptide may be linked to a cargo peptide via a peptide linker (e.g., any of SEQ ID NO: 255 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485).
  • a cell-penetrating peptide may be linked to a cargo molecule via a small molecule linker (e.g., a linker provided in TABLE 11).
  • a cargo molecule can be delivered carried across a cellular layer (e g., a plasma membrane, a vesicular membrane, an endosomal membrane, a nuclear envelope, or a blood brain barrier) to a cellular, intracellular, or paracellular space (e.g., a cytosol, a nucleus, or a nanolumen) when linked, fused, conjugated, or otherwise connected to a cell -penetrating peptide of the present disclosure.
  • a cellular layer e.g., a plasma membrane, a vesicular membrane, an endosomal membrane, a nuclear envelope, or a blood brain barrier
  • a cellular, intracellular, or paracellular space e.g., a cytosol, a nucleus, or a nanolumen
  • a cargo molecule can be an active agent (e.g., a therapeutic agent or a detectable agent).
  • a cargo molecule can be a cystine-dense peptide, an anti-cancer agent, a transcription factor binding agent, an inhibitor of protein-protein interactions, a promoter of protein-protein interactions, a transcription factor, an RNA, a Cas enzyme or other CRISPR component, a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, a protein (e.g., a protein that inhibits proteinprotein interactions), an oligonucleotide, or an immunomodulating agent.
  • an active agent e.g., a therapeutic agent or a detectable agent.
  • a cargo molecule can be a cystine-dense peptide, an anti-cancer agent, a transcription factor binding agent, an inhibitor of protein-protein interactions,
  • a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254, or a fragment thereof can be conjugated, fused, linked, or otherwise connected to an active agent comprising a cystine- dense peptide, an anti-cancer agent, a transcription factor binding agent, an inhibitor of proteinprotein interactions, a promoter of protein-protein interactions, a transcription factor, an RNA, a Cas enzyme or other CRISPR component, a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, a protein (e g., a protein that inhibits proteinprotein interactions), an oligonucleotide, or an immunomodulating agent for delivery
  • one or more cell-penetrating peptides can be fused, linked, or conjugated to a cargo peptide or cargo molecule for improved cell penetration.
  • a cargo molecule may be a target-binding molecule that binds a target of interest, such as a target peptide.
  • the cargo molecule inhibits the target upon binding. For example, biding of the cargo molecule may inhibit formation of protein-protein or protein-nucleic acid interactions with the target. In another example, binding of the cargo molecule may inhibit a conformation change or enzymatic activity of the target molecule.
  • target molecules include TEAD, coldinducible RNA-binding protein, androgen receptor, ikaros, aiolos, a nuclear receptor, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, tau, AKT, mTOR, STAT3, RAS, RAF
  • a cargo molecule that binds a target may comprise a sequence of any one of SEQ ID NO: 293 - SEQ ID NO: 298 or SEQ ID NO: 364 - SEQ ID NO: 407.
  • a cargo molecule that binds a target may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NO: 293 - SEQ ID NO: 298 or SEQ ID NO: 364 - SEQ ID NO: 407. Examples of cargo-binding molecules are provided in TABLE 5.
  • a cargo molecule may comprise a small molecule (e.g., a small molecule ligand) that binds a target.
  • a cargo molecule may comprise a small molecule that binds a ubiquitin ligase.
  • cargo molecules that binds ubiquitin ligase include thalidomide, pomalidomide, lenalidomide, methyl bestatin, bestatin, nutlin-3, and VHL ligand 1, as well as other immunomodulatory drugs (IMiDs).
  • a cargo molecule such as a cargo molecule of a targeted degradation complex, may comprise a target-binding peptide (e.g., a peptide that binds TEAD, cold-inducible RNA-binding protein, androgen receptor, ikaros, aiolos, nuclear receptors, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WI
  • a peptide complex of the present disclosure may be delivered across a cellular layer of a cell by way of the cell-penetrating peptide.
  • the peptide complex may be delivered to a cellular compartment (e g., the cytosol or nucleus) of the cell.
  • the peptide complex may be delivered to an intercellular compartment (e.g., a nanolumen, intercellular space, or paracellular space) across the cellular layer.
  • At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 22%, at least about 25%, at least about 27%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of the peptide complex is delivered across the cellular layer.
  • the cell-penetrating peptides described herein are attached to another molecule (e.g., a cargo molecule), such as a cystine-dense peptide (CDP) that provides a functional capability.
  • a cell-penetrating peptide attached to a CDP may have a sequence of any one of SEQ ID NO: 299 - SEQ ID NO: 308 or SEQ ID NO: 312 - SEQ ID NO: 321.
  • cell-penetrating peptides can direct the CDP into the cell.
  • cell-penetrating peptides can direct the CDP into the nucleus.
  • cell-penetrating peptides can direct the CDP cross the blood brain barrier. In further embodiments, cell-penetrating peptides can direct the CDP into a nanolumen between cells (e.g., cancer cells). In some embodiments, the CDP has a therapeutic effect inside the cell. In further embodiments, cell-penetrating peptides can direct the CDP into a nanolumen between cells (e.g., cancer cells).
  • cell-penetrating peptides can direct the CDP into or across a cellular space or compartment (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, or other subcellular compartment membrane, a blood brain barrier, or a nanolumen).
  • a cellular space or compartment e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, or other subcellular compartment membrane, a blood brain barrier, or a nanolumen.
  • the CDP may bind to a cytosolic, nuclear, intracellular, or paracellular protein and modulate an activity or a protein-protein interaction of the protein.
  • the CDP may bind to and modulate a transcription factor, cereblon, VHL, DCAF15, DCAF16, other molecules in the CRL4 E3 ligase family, other molecules in the E3 ligase family, or other molecules in the UPS system.
  • the CDP may bind to target molecule, such as a target peptide.
  • Binding of the CDP to the target molecule may inhibit the target molecule (e g., inhibit a conformational change, inhibit an enzymatic activity, inhibit ligand binding, inhibit a protein-protein interaction, or inhibit a protein-nucleic acid interaction of the target).
  • binding of the CDP may recruit additional agents to the target, such as a ubiquitin ligase.
  • the CDP is a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a protein (e.g., a protein that inhibits protein-protein interactions), or a proteolysis targeting chimera.
  • the cell-penetrating peptides or peptide complexes of the present disclosure can be conjugated to, linked to, or fused with an active agent (e.g., a cargo molecule, a therapeutic agent, a drug, a biologic, or a peptide).
  • an active agent e.g., a cargo molecule, a therapeutic agent, a drug, a biologic, or a peptide.
  • the cell-penetrating peptide may be linked to more than one active agent.
  • the cell-penetrating peptide may be linked to a cargo peptide (e.g., a targetbinding peptide) and an additional active agent (e.g., a target-binding ligand, a therapeutic agent, a drug, a biologic, or a peptide).
  • a cargo peptide e.g., a targetbinding peptide
  • an additional active agent e.g., a target-binding ligand, a therapeutic agent, a drug, a biologic, or a peptide.
  • the cell-penetrating peptide may deliver the active agent into a cellular compartment (e.g., across a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, a blood brain barrier, or a nanolumen).
  • Peptides according to the present disclosure can be conjugated to, linked to, or fused to an agent for use in the treatment of a disease or a condition (e.g., cancer, neurodegeneration, over-expression or under-expression of a gene, inflammation, or protein over-expression or accumulation).
  • a disease or a condition e.g., cancer, neurodegeneration, over-expression or under-expression of a gene, inflammation, or protein over-expression or accumulation.
  • the peptides described herein are fused to another molecule, such as an active agent that provides an additional functional capability.
  • An active agent may be a small molecule active agent, a peptide active agent, or a nucleic acid active agent.
  • a cell-penetrating peptide may be conjugated to a small molecule therapeutic agent.
  • the cell-penetrating peptides of the present disclosure may be fused to an active agent that may otherwise be excluded from a cell or a cellular compartment to facilitate absorption or permeation of the active agent.
  • a cell-penetrating peptide of the present disclosure may facilitate cytosolic delivery of an active agent with multiple hydrogen bond donors or hydrogen bond acceptors, a large molecular weight, or a high partition coefficient, which may lead to poor cytosolic delivery of the active agent in the absence of the cell -penetrating peptide.
  • a cell-penetrating peptide may be conjugated to an active agent comprising at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors. In some embodiments, the cell-penetrating peptide may be conjugated to an active agent comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors.
  • the cell-penetrating peptide may be conjugated to an active agent comprising a molecular weight of at least 200 Da, at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, at least 1000 Da, at least 1500 Da, at least 2000 Da, at least 3000 Da, at least 4000 Da, at least 5000 Da, at least 6000 Da, at least 7000 Da, at least 8000 Da, at least 9000 Da, or at least 10,000 Da.
  • an active agent comprising a molecular weight of at least 200 Da, at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, at least 1000 Da, at least 1500 Da, at least 2000 Da, at least 3000 Da, at least 4000 Da, at least 5000 Da, at least 6000 Da, at least 7000 Da, at least 8000 Da, at least 9000 Da, or at least 10,000 Da.
  • the cell-penetrating peptide may be conjugated to an active agent comprising a molecular weight of up to 1000 Da, up to 2000, up to 3000 Da, up to 4000 Da, up to 5000 Da, up to 6000 Da, up to 7000 Da, up to 8000 Da, up to 9000 Da, up to 10,000 Da, up to 12,000 Da, up to 15,000 Da, up to 20,000 Da, or up to 25,000 Da.
  • the cell-penetrating peptide may be conjugated to an active agent comprising a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0.
  • the partition coefficient may be a measure of a ratio of the solubility of the agent in a hydrophobic solvent (e.g., octanol) relative to water.
  • a peptide can be fused with an active agent through expression of a vector containing the sequence of the peptide with the sequence of the active agent.
  • the sequence of the peptide and the sequence of the active agent can be expressed from the same Open Reading Frame (ORF).
  • ORF Open Reading Frame
  • the sequence of the peptide and the sequence of the active agent can comprise a contiguous sequence.
  • the peptide and the active agent can each retain similar functional capabilities in the fusion peptide compared with their functional capabilities when expressed separately.
  • examples of active agents can include other peptides.
  • the peptides described herein are attached to another molecule, such as an active agent that provides a functional capability.
  • cell-penetrating peptides can direct the active agent into the cell.
  • cell-penetrating peptides can direct the active agent into the nucleus.
  • cell-penetrating peptides can direct the active agent across the blood brain barrier.
  • cell-penetrating peptides can direct the active agent into a nanolumen between cells (e.g., cancer cells).
  • cell-penetrating peptides can direct the active agent into or across a cellular space or compartment (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen).
  • the active agent has a therapeutic effect inside the cell.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 active agents can be linked to a peptide.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell-penetrating peptides of this disclosure can be linked to an active agent.
  • Multiple active agents can be attached by methods such as conjugating to multiple lysine residues and/or the N-terminus, or by linking the multiple active agents to a scaffold, such as a polymer or dendrimer and then attaching that agent-scaffold to the peptide (such as described in Yurkovetskiy, A. V., Cancer Res 75(16): 3365-72 (2015)) or by recombinant fusion.
  • a scaffold such as a polymer or dendrimer
  • active agents include but are not limited to: a peptide, an oligopeptide, a polypeptide, a peptidomimetic, a polynucleotide, a polyribonucleotide, a cysteine-dense peptide, an affibody, an avimer, an adnectin, a B-hairpin, a stapled peptide, a kunitz domain, a nanofttin, a fynomer, a bicycle peptide, a Cas protein, a transcription factor, a DNA, a cDNA, a ssDNA, a RNA, a dsRNA, a micro RNA, a guide RNA, a U1 adaptor, a crRNA, a tracrRNA, an oligonucleotide, an antibody, a single chain variable fragment (scFv), a nanobody, an antibody fragment, an aptamer, a cytokine, an
  • the peptide is covalently or non-covalently linked to an active agent, e.g., directly or via a linker.
  • cytotoxic molecules that can be used include auristatins, MMAE, MMAF, dolostatin, auristatin F, monomethylaurstatin D, DM1, DM4, maytansinoids, maytansine, calicheamicins, N-acetyl-y-calicheamicin, pyrrolobenzodiazepines, PBD dimers, doxorubicin, vinca alkaloids (4-deacetylvinblastine), duocarmycins, cyclic octapeptide analogs of mushroom amatoxins, epothilones, and anthracy lines, CC-1065, taxanes, paclitaxel, cabazitaxel, docetaxel, SN-38, irinotecan, vincristine, vinblastine, platinum compounds,
  • the peptides or fusion peptides of the present disclosure can also be conjugated to, linked to, or fused to other moieties that can serve other roles, such as providing an affinity handle (e.g., biotin) for retrieval of the peptides from tissues or fluids.
  • an affinity handle e.g., biotin
  • peptides or fusion peptides of the present disclosure can also be conjugated to, linked to, or fused to biotin.
  • biotin can also act as an affinity handle for retrieval of peptides or fusion peptides from tissues or other locations.
  • fluorescent biotin conjugates that can act both as a detectable label and an affinity handle can be used.
  • Non limiting examples of commercially available fluorescent biotin conjugates include Atto 425- Biotin, Atto 488-Biotin, Atto 520-Biotin, Atto-550 Biotin, Atto 565-Biotin, Atto 590-Biotin, Atto 610-Biotin, Atto 620-Biotin, Atto 655-Biotin, Atto 680-Biotin, Atto 700-Biotin, Atto 725- Biotin, Atto 740-Biotin, fluorescein biotin, biotin-4-fluorescein, biotin-(5-fluorescein) conjugate, and biotin-B-phycoerythrin, ALEXA FLUOR 488 biocytin, ALEXA FLUOR 546, ALEXA FLUOR 549, lucifer yellow cadaverine biotin-X, Lucifer yellow biocytin, Oregon green 488 biocytin, biotin-rhodamine
  • the conjugates could include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels.
  • the peptide-active agent fusions described herein can be attached to another molecule.
  • the peptide sequence also can be attached to another active agent (e.g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar, etc.).
  • the peptide can be fused with, or covalently or non-covalently linked to an active agent.
  • the conjugates can include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels.
  • the peptide described herein can also be attached to another molecule.
  • the peptide sequence also can be attached to another active agent (e.g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding agents, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar).
  • another active agent e.g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding agents, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar.
  • the peptide can be conjugated to, linked to, or fused with, or covalently or non- covalently linked to an active agent.
  • a peptide sequence derived from a toxin or venom knottin protein can be present on, conjugated to, linked to, or fused with a particular peptide.
  • a peptide can be incorporated into a biomolecule by various techniques.
  • a peptide can be incorporated by a chemical transformation, such as the formation of a covalent bond, such as an amide bond.
  • a peptide can be incorporated, for example, by solid phase or solution phase peptide synthesis.
  • a peptide can be incorporated by preparing a nucleic acid sequence encoding the biomolecule, wherein the nucleic acid sequence includes a subsequence that encodes the peptide.
  • the subsequence can be in addition to the sequence that encodes the biomolecule or can substitute for a subsequence of the sequence that encodes the biomolecule.
  • Active agents that may be delivered using the cell -penetrating peptides of the present disclosure include a RIG-I ligand, or ligands targeting a related receptor, such as melanoma differentiation-associated protein 5 (MDA5) or toll-like receptor 3 (TLR3).
  • Ligands for RIG-I, MDA5, and TLR3 that may be complexed with a cell -penetrating peptide include abnormal double stranded RNA (dsRNA) comprising a 5’ diphosphate or, in the case of RIG-I, a 5’ triphosphate, such as a dsRNA associated with viral infection.
  • dsRNA abnormal double stranded RNA
  • Activation of RIG-I and MDA5 using RIG-I or MDA5 ligand can be effective at promoting antitumor immunity and apoptosis.
  • RIG-I, MDA5, or TLR3 ligands that may be complexed with a cell-penetrating peptide of the present disclosure include dsRNA, 5’ diphosphate dsRNA, 5’ triphosphate dsRNA, double stranded hairpin RNA, a benzobisthiazole compound, and polyinosinic:polycytidylic acid. Delivery of a RIG-I, MDA5, or TLR3 ligand across a cellular layer may promote anti -tumor or anti-viral activity in a subject, thereby treating a cancer or viral infection in a subject.
  • RIG-I is active intracellularly
  • the present disclosure provides a peptide-RIG-I ligand complex in which the peptide can be capable of cell penetration such that the RIG-I ligand can access the cytoplasm of the target cell.
  • RIG-I ligands by themselves may be able to activate the RIG-I helicase when in contact with it, but when applied to cells in vivo or in vitro, may access the cytoplasm at only low levels and thus not be able to access and activate RIG-I.
  • RIG-I ligands may require formulation with transfection reagents or other components to access the cytoplasm, which may not be feasible for human cancer therapy, due to toxicity, stability, safety issues, or inability to apply the formulation systemically (such as by intravenous or subcutaneous administration) and deliver sufficient amounts of active agent to the tumor.
  • RIG-I can be activated in vivo for anti-cancer therapy.
  • the RIG-I ligand in the peptide- RIG-I complex can access the cytoplasm of the target cell via cleavage of the peptide- RIG-I complex in the endosome or after exit from the endosome into the cytosol and dissociated RIG-I ligand therefrom can access the cytoplasm, or via any other mechanism as described herein.
  • the RIG-I ligand can also optionally access the cytoplasm and RIG-I without cleavage. Cleavage can also occur in the cytosol.
  • the peptide- RIG-I ligand complex can be designed such that the peptide is distal from the end of the RIG-I ligand that activates the helicase, and as such the peptide-RIG-I ligand complex may be active without cleavage.
  • the interaction between RIG-I ligands and the RIG-I helicase, such as shown in the crystal structure in Devarker et al., Proc Natl Acad Sci, 113(3): 596-601, 2016, can be analyzed to design peptide-RIG-I ligand complexes.
  • the peptides of this disclosure can be located a number of base pairs away from the 5’ triphosphate end of the RIG-I ligand, such as 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs away from the 5 ’ triphosphate end. In some embodiments, the peptide of this disclosure is conjugated 7-20 base pairs away from the 5’ triphosphate end end. In some embodiments, the peptide of this disclosure is conjugated more than 20 base pairs away from the 5’ triphosphate end. In some embodiments, the RIG-I ligand can be chemically conjugated to the peptide.
  • the peptide and RIG-I ligand can be linked with a cleavable linker, such that the linker can be cleaved selectively once intracellular, such as in the endosome or cytosol, thereby releasing the RIG-I ligand adequately in high concentrations within a cell in order to target intracellular RIG-I.
  • the peptide and RIG-I ligand can be linked such that the RIG-I is inactive or blocked from binding to its receptor by the peptide until the peptide is removed, thereby reducing exposure of noncancerous tissues to the RIG-I ligand.
  • the linker can be a disulfide bond. In other embodiments, the linker can be acid labile.
  • the linker can be enzymatically cleavable, such that it is cleaved by enzymes in the endosomal- lysosomal pathway, or within the cytosol.
  • the peptide-RIG-I ligand complex can be co-formulated.
  • the peptide-RIG-I ligand complex can be formulated in a delivery vehicle, such as a liposome.
  • the peptide-RIG-I ligand complex comprises a RIG-I ligand that can be encapsulated in a liposome, which can be further coated with a peptide of the present disclosure.
  • the peptide-RIG-I ligand complex can be linked by a stable linker and is active as a complex.
  • the linker can comprise additional functions as peptides or chemical structures that enhance endosomal escape, endosomal uptake, tissue biodistribution to the tumor, or cell penetration. Cell penetrating or endosomal escape peptide sequences can be added to the linker or to the other end of the peptide.
  • Receptor-specificity of an RNA ligand-cell-penetrating peptide complex may be achieved by selective delivery of the complex to a compartment where the receptor is found (e.g., the cytoplasm or an endosome), by using a receptor-specific ligand, or a combination thereof.
  • RIG-I and MDA5 are both RNA helicases present in the cytoplasm, while various tolllike receptors (TLRs) may be found in the endosome.
  • ligand recognized by two or more receptors found in different cellular compartments may be complexed with a cell-penetrating peptide of the present disclosure to specifically deliver the ligand to a desired cellular compartment (e.g., the cytosol or an endosome) to specifically activate one of the receptors.
  • a desired cellular compartment e.g., the cytosol or an endosome
  • delivery of polyinosinic: poly cytidylic acid to the cytoplasm may specifically activate MDA5, which is present in the cytoplasm, rather than a toll-like receptor found in endosomes (e g., TLR3, TLR7, TLR8, or TLR10).
  • an RNA ligand can be conjugated to a peptide of this disclosure to form a peptide complex that enters the cytoplasm and activates a cytoplasmic receptor (e g., RIG-I or MDA5).
  • a cytoplasmic receptor e g., RIG-I or MDA5
  • complexing the ligand to a cell-penetrating peptide capable of endosomal delivery may enable activation of an endosomal RNA-sensing receptor (e.g., an RNA-sensing TLR).
  • RIG-I and MDA5 each have a C-terminal domain involved in ligand specificity and two N-terminal CARD domains, which enable mitochondrial antiviral -signaling protein (MAVS)- mediated signal transduction, and both recognize the internal RNA duplex structure.
  • MAVS mitochondrial antiviral -signaling protein
  • RIG-I can also recognize the 5’ end of dsRNA.
  • Ligand binding of RIG-I or MDA5 may lead to activation of the MAVS -dependent signaling pathway, stimulating production of proinflammatory substances, including Type I interferons that contribute to antiviral and antitumor immunity, and are distinct from the gene expression induced by TLR3 activation.
  • Activation of RIG-I or MDA5 can additionally lead to the activation of the inflammasome resulting in changes to the tumor microenvironment that promotes antitumor immunity, such as secretion of IL-1, IL- 18, and damage-associated molecular patterns (DAMPs).
  • DAMPs damage-associated molecular patterns
  • RIG-I and MDA5 both signal through mitochondrial antiviral-signaling (MAVS) proteins, which initiates signaling via IRF3/7 and NFKB factors. MAVS is also important for initiation of tumor cell apoptosis, via RIG-I-like receptor (RLR) activation, which leads to immunogenic cell death (ICD). Additionally, RIG-I can utilize multiple interferon regulatory factors (IRF). Thus, engagement of RIG-I or MDA5 with a RIG-I-specific ligand can activate anti-viral immune mechanisms, which can have therapeutic effects against tumors. In some embodiments, engagement of RIG-I or MDA5 with a RIG-I ligand can induce direct immunogenic cell death (ICD) of tumor cells, but not normal cells.
  • ICD interferon regulatory factors
  • a RIG-I or MDA5 ligand can stimulate DC activation including inflammasome activity.
  • a RIG-I ligand can induce tumor cells to produce IFN and CXCL10 via the IRF3 pathway.
  • a RIG-I or mDA5 ligand can induce tumor regression in a subject, such as a human, non-human primate, or any other animal.
  • a RIG-I ligand can additionally inhibit a Thl7 and Treg responses.
  • cell penetrating peptide complexes of this disclosure can target MDA5, a related cytoplasmic sensor for dsRNA, which shares many of the same functions as RIG-I.
  • a RIG-I ligand may comprise a short dsRNA.
  • the short dsRNA may have a length of from 5 base pairs to 60 base pairs, from 5 base pairs to 10 base pairs, from 7 base pairs to 10 base pairs, from 11 base pairs to 18 base pairs, from 14 base pairs to 120 base pairs, from 5 base pairs to 15 base pairs, from 15 base pairs to 25 base pairs, from 25 base pairs to 40 base pairs, from 40 base pairs to 60 base pairs, from 60 base pairs to 80 base pairs, from 80 base pairs to 100 base pairs, from 100 base pairs to 120 base pairs, from 120 base pairs to 140 base pairs, from 140 base pairs to 160 base pairs, for from 19 base pairs to 60 base pairs.
  • a short dsRNA may comprise a length of from 19 base pairs to 60 base pairs and at least one 5’ triphosphate or 5’ diphosphate.
  • the short dsRNA may further comprise an uncapped 5’ A or G nucleotide, a 5’ triphosphate on a blunt end, a 1 nucleotide 5’ overlap, a 1 nucleotide overlap at the 5 ’ end with the triphosphate, or combinations thereof.
  • at least one 5 ’ triphosphate or 5 ’ diphosphate is located on the 5 ’ end of the sense strand of the dsRNA.
  • the short dsRNA may comprise a mismatch, for example a mismatch that is 8 or more base pairs from the 5’ end of the dsRNA.
  • a dsRNA RIG-I ligand may be complexed with a cellpenetrating peptide of the present disclosure via an RNA modification.
  • the RNA modification may be positioned 8 or more base pairs from the 5 ’ end of the dsRNA.
  • Additional types of dsRNA that may function as RIG-I ligands may include dsRNA hairpin RNA (e.g., in which some ribosides are paired with a partner within the same hairpin), or any other short dsRNA comprising a 5’ triphosphate.
  • a dsRNA described herein may be a based paired region within a longer single RNA sequence or may a base paired region of two separate RNA strands.
  • Double stranded RNA RIG-I ligands can be made by a variety of techniques that are used to combine the sense and antisense strands of the RNAs into a double stranded form.
  • the sense and antisense strands of the dsRNA can be separately transcribed or synthesized and combined into dsRNA structures using a variety of recombinant or synthetic techniques.
  • the sense and antisense strands of the dsRNA can be transcribed or synthesized in a single RNA that contains a loop structure (hairpin) that is optionally later cleaved by an RNAse to obtain the dsRNA.
  • a RIG-I ligand may comprise two RNA strands complexed together as a double strand, comprising a 5’ diphosphate or a 5’ triphosphate group on one or both strands, or a single RNA strand complexed together in a hairpin that is double stranded at one or more locations in the molecule.
  • the double strand may extend throughout the sequence or there may be regions of mismatch, and there may be one or more locations of hairpin self-association within one or both strands.
  • the ends of the double strand may be at the same blunt location, or one or the other end may overhang. Hairpins and other structures within the RNA complex can be more immunogenic and activate the RIG-I pathway at higher levels.
  • MDA5 ligands may also exhibit all the above structural variations but may also contain no or one 5’ phosphate.
  • RNA backbone or bases of a RIG-I ligand can be modified to improve in vitro and in vivo stability (e.g., serum stability, manufacturability, shelf stability) or other properties of the molecule including base pairing affinity and immune system activation.
  • Pyrimidines can be 2'- fluoro-modified, which can increase stability to nucleases as well as increase immune system activation.
  • the RNA backbone can be phosphorothioate-substituted (where the non-bridging oxygen is replaced with sulfur), which can increase resistance to nuclease digestion as well as altering the biodistribution and tissue retention and increasing the pharmacokinetics such as by increasing protein binding, and can also induce more immune stimulation.
  • Methyl phosphonate modification of an RNA can also be used.
  • 2’-Omethyl and 2’-F RNA bases can be used, which can protect against base hydrolysis and nucleases and increase the melting temperature of duplexes.
  • the modification can also comprise a bridged nucleic acid, a morpholino nucleic acid, a PNA, an LNA, an ethyl cEt nucleic acid. Bridged, Locked, and other similar forms of Bridged Nucleic Acids (BNA, LNA, cEt) where any chemical bridge such as an N-0 linkage between the 2' oxygen and 4' carbons in ribose can be incorporated to increase resistance to exo- and endonucleases and enhance biostability.
  • BNA, LNA, cEt Bridged Nucleic Acids
  • RNA backbone or base modifications can be placed anywhere in the RNA sequence, at one, multiple, or all base locations.
  • the modifications may be distal from the end of the dsRNA complex that contains the 5’ triphosphate and interacts with the helicase.
  • the RNA backbone or base modifications may enhance, decrease, or have no effect on the level of RIG-I activation by the peptide-RIG-I ligand complex.
  • phosophorothioate nucleic acids may be used at the 2-3 terminal nucleic acids of one or both sequences.
  • 2’F modified nucleic acids may be used at least at 5%, at least at 10%, at least at 25%, at least at 50%, at least at 75%, or at up to 100% of internal positions.
  • 2’F modified nucleic acids may be used at from 2 to 4 positions, at all internal positions, or at all positions.
  • the RIG-I ligand may have additional modified nucleotides or bases present within the sequence, such as to allow chemical modification such as conjugation to a linker and peptide or conjugation to an additional delivery agent such as a lipid, cholesterol, or hydrocarbon chain.
  • a cell -penetrating peptide of the present disclosure may be complexed with an oligonucleotide.
  • the oligonucleotide may function as an active agent, a therapeutic agent, a detectable agent, or a combination thereof.
  • a cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) may be complexed with an oligonucleotide to form a peptide oligonucleotide complex, also referred to as a peptidenucleotide agent conjugate, a peptide oligonucleotide complex, or a peptide target-binding agent complex, may comprise a peptide complexed with a nucleotide (e g., an oligonucleotide).
  • a nucleotide e g., an oligonucleotide
  • the peptide of the peptide oligonucleotide complex may comprise a cell-penetrating peptide, as described herein (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254).
  • a cellpenetrating peptide of a peptide oligonucleotide complex may facilitate cell penetration of the peptide oligonucleotide complex.
  • a peptide oligonucleotide complex comprising a cell-penetrating peptide may cross a cellular membrane, enabling delivery of the peptide oligonucleotide complex to a cytoplasm or a nucleus of the cell and interaction between the nucleotide of the peptide oligonucleotide complex and various cytosolic or nuclear components (e.g., genomic DNA, an ORF, mRNA, pre-mRNA, or DNA).
  • cytosolic or nuclear components e.g., genomic DNA, an ORF, mRNA, pre-mRNA, or DNA.
  • the nucleotide of the peptide oligonucleotide complex may be a target-binding agent comprising single stranded DNA, single stranded RNA, double stranded DNA, double stranded RNA, or a combination thereof.
  • the term “nucleotide” may refer to an oligonucleotide or polynucleotide molecule or to a single nucleotide base.
  • a nucleotide of a peptide complex may comprise a DNA or RNA oligonucleotide.
  • the nucleotide may be a small interfering RNA (siRNA), a micro RNA (miRNA, or miR), an anti-miR, an antisense RNA, an antisense oligonucleotide (ASO), a complementary RNA, a complementary DNA, an interfering RNA, a small nuclear RNA (snRNA), a spliceosomal RNA, an inhibitory RNA, a nuclear RNA, an oligonucleotide complementary to a natural antisense transcript (NAT), an aptamer, a gapmer, a splice blocker ASO, or a U1 adapter.
  • siRNA small interfering RNA
  • miRNA micro RNA
  • ASO anti-miR
  • ASO antisense oligonucleotide
  • a complementary RNA a complementary DNA
  • an interfering RNA a small nuclear RNA (snRNA), a spliceosomal RNA
  • a nucleotide of the peptide oligonucleotide complex may comprise a sequence of any one of any one of SEQ ID NO: 488 - SEQ ID NO: 573 or a sequence complementary to a portion of any sequence provided in SEQ ID NO: 574 - SEQ ID NO: 611 or an open reading frame listed in TABLE 6.
  • the nucleotide may be an siRNA that inhibits translation of a target mRNA by promoting degradation of the target mRNA.
  • the nucleotide may be an miRNA that inhibits translation of a target mRNA by promoting cleavage or destabilization of the target mRNA.
  • the nucleotide may be an aptamer that binds to a target protein, thereby inhibiting protein-protein interactions with the target protein, inhibiting enzymatic activity of the target protein, or activating the target protein.
  • oligonucleotide complexes examples include an aptamer, a gapmer, an anti-miR, an siRNA, a splice blocker ASO, and a U1 adapter.
  • the peptide portion of the peptide oligonucleotide complex (e.g., a CDP of a CDP- oligonucleotide complex) can be used to guide the nucleotide sequence (e g., an oligonucleotide of a CDP- oligonucleotide complex) to a specific tissue, target, or cell, or to deliver the oligonucleotide to the cytosol or nucleus of the cell, or to cause endosomal escape of the oligonucleotide.
  • a CDP of a CDP- oligonucleotide complex can be used to guide the nucleotide sequence (e g., an oligonucleotide of a CDP- oligonucleotide complex) to a specific tissue, target, or cell, or to deliver the oligonucleotide to the cytosol or nucleus of the cell, or to cause endosomal escape of the
  • the peptide oligonucleotide complexes of the present disclosure may include nucleotide and nucleotide variants within the peptide oligonucleotide complex wherein the nucleotide portion is targeted to specific target molecule for modulation.
  • Modulation of a target molecule may comprise degradation, inhibiting translation, decreasing expression, increasing expression, enhancing a binding interaction (e.g., a protein-protein interaction), or inhibiting a binding interaction (e.g., a protein-protein interaction).
  • nucleotide sequences that may be used in the nucleotide portion of the peptide oligonucleotide complex, such as those targeting or complementary to nucleotides (e.g., DNA or RNA molecules) listed in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or to nucleotides (e.g., DNA or RNA molecules) encoding the proteins listed in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or otherwise described herein.
  • nucleotide sequences that may be used in the nucleotide portion of the peptide oligonucleotide complex include SEQ ID NO: 488 - SEQ ID NO: 573.
  • nucleic acid sequences, variants, and properties of the nucleic acids that are used in the nucleic acid portion of the peptide oligonucleotide complex may be referred to as nucleic acids of the present disclosure, nucleotides of the present disclosure, or like terminology.
  • nucleic acids or nucleotides are described in the context of the peptide oligonucleotide complexes disclosed, such as a nucleotide sequence comprising single stranded (ssDNA, ssRNA), double stranded (dsDNA, dsRNA), or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter within the peptide oligonucleotide complex, with the accorded alterations, functions and uses described.
  • sDNA single stranded
  • the nucleotide sequence (e.g., a target binding agent capable of binding a target molecule) is single stranded (ssDNA, ssRNA), double stranded (dsDNA, dsRNA), or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or Ul Adapter.
  • ssDNA, ssRNA double stranded
  • dsDNA double stranded
  • dsRNA double stranded
  • a combination of single and double stranded for example with a mis
  • Peptides according to the present disclosure can be conjugated to, linked to, or fused to such nucleotide sequences to make a peptide oligonucleotide complex.
  • other active agents e.g., small molecule, protein, or peptide active agents
  • active agents as described herein can be conjugated to, linked to, complexed with, or fused to such nucleotide sequences, peptides or peptide oligonucleotide complex to form peptide oligonucleotide complex conjugates.
  • a nucleotide (e.g., a nucleotide of a peptide oligonucleotide complex) may be fully or partially reverse complementary to all or a portion of a target molecule (e.g., a target DNA or RNA sequence).
  • a target molecule expresses or encodes a protein (e.g., an mRNA encoding a protein associated with a disease).
  • a nucleotide may be fully or partially reverse complementary to a portion of an open reading frame encoding a gene or protein of interest.
  • a nucleotide may be reverse complementary to any portion of an RNA or open reading frame encoding a transcript or protein of interest.
  • a target molecule may comprise a fragment of any of the sequences provided in TABLE 6 along any portion of its length.
  • a target molecule may comprise a fragment of any of the sequences provided in SEQ ID NO: 574 - SEQ ID NO: 611.
  • a target molecule may comprise a sequence with one or more T residues replaced with U or one or more U residues replaced with T. TABLE 6 - Examples of Open Reading Frame Reference Sequences
  • a number of technologies can be used to generate therapeutically active nucleotide sequences for use in peptide oligonucleotide complexes that include the cell-penetrating peptides (e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) disclosed herein.
  • cell-penetrating peptides e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • a nucleotide of a peptide oligonucleotide complex may bind to a target molecule (e.g., a target DNA, RNA, or protein) and modulate an activity of the target molecule.
  • a target molecule e.g., a target DNA, RNA, or protein
  • the nucleotide may function as a target-binding agent, also referred to as a targeted agent.
  • nucleotides that may function as target-binding agents include nucleotide antisense RNAs, complementary RNAs, inhibitory RNAs, interfering RNAs, nuclear RNAs, antisense oligonucleotides, microRNAs, oligonucleotides complementary to natural antisense transcripts, small interfering RNAs, small nuclear RNAs, aptamers, gapmers, anti-miRs, splice blocker antisense oligonucleotides, and Ul adapters.
  • Nucleotides may enter into cells through complexation with a cell-penetrating peptide to form a cell-penetrating peptide oligonucleotide complex.
  • the cell-penetrating peptide oligonucleotide complex may be delivered into a cytoplasm or nucleus of a cell.
  • the oligonucleotide, the peptide oligonucleotide complex, or any fragment thereof may enter the cytosol and may enter the nucleus.
  • oligonucleotides upon entry into the nucleus, can bind directly to mRNA structures and prevent the maturation (e g., capping or splicing) of the targeted sequence, modulate alternative splicing of a targeted sequence, and recruit RNaseHl to induce cleavage of a targeted sequence.
  • oligonucleotides in the cytoplasm can bind directly to the target mRNA and sterically block the ribosomal subunits from attaching and/or running along the mRNA transcript during translation hence resulting in lack of translation of the target sequence.
  • oligonucleotides can also be designed to directly bind to microRNA (miRNA) sequences or natural antisense transcripts (NATs) sequences, each of the foregoing thereby prohibiting miRNAs and NATs from inhibiting their own specific RNA targets, which ultimately leads to reduced degradation or increased translation of one or more sequences themselves targeted by the miRNA or NAT.
  • miRNA microRNA
  • NATs natural antisense transcripts
  • siRNA (which may be targeted to a specific sequence and regulate expression of the target sequence) may alternatively be used to bind and regulate a targeted sequence in the cytoplasm, engaging an RNA-induced silencing complex (RISC), which is a multiprotein complex that incorporates one strand of a small interfering RNA (siRNA) or micro RNA (miRNA), using the siRNA or miRNA as a template for recognizing complementary mRNA of the targeted sequence. When it finds a complementary strand, its RNase domain cleaves the targeted sequence.
  • RISC RNA-induced silencing complex
  • siRNA small interfering RNA
  • miRNA micro RNA
  • an aptamer e.g., a nucleotide that modulates a specific protein or other target
  • An aptamer (e.g., extracellular or intracellular) may function by directly binding and modulating activity of a protein target, for example by forming aptamer-protein interactions rather than through base pairing or hybridization interactions.
  • ASO antisense oligonucleotides
  • nt nucleotides
  • the first ASOs are sometimes called “Gapmers” because they have a central region with DNA-based-sugar nucleotides that are often (but not always) flanked by non-DNA-sugar nucleotides with greater resistance to nucleases.
  • the DNA region at least 4 nt in length but typically >6, causes a DNA/RNA hybrid that engages RNase H endonuclease to cleave the target RNA.
  • a DNA region of a gapmer may comprise from about 4 to about 30, from about 4 to about 25, from about 4 to about 20, from about 4 to about 15, from about 4 to about 10, from about 6 to about 30, from about 6 to about 25, from about 6 to about 20, from about 6 to about 15, or from about 6 to about 10 nucleotide residues.
  • a non-DNA region of a gapmer may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues 3’ of the DNA region.
  • a non-DNA region of a gapmer may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues 5’ of the DNA region. Examples of gapmers are provided in TABLE 7.
  • the second conventional ASO simply serves to bind to the target transcript, but not induce RNase degradation, so no DNA-based-sugars are used. Instead, binding is designed to disrupt processing into mature mRNA.
  • One such activity relies on binding to the mRNA at or near splice sites to drive particular splice isoforms in the target RNA, resulting in modulating target RNA by disrupting mRNA splicing and resulting in exon skipping. These are commonly called “splice blocking” or “splice blocker” ASOs amongst other known names.
  • DMD distrophin
  • Duchenne Muscular Dystrophy patients One example is eteplirsen, designed to alter splicing of DMD (dystrophin) gene in Duchenne Muscular Dystrophy patients, correcting a mutation that would otherwise create a truncated and nonfunctional dystrophin by splicing out the mutant exons and creating a different truncated (but functional) protein to appear.
  • DMD distrophin
  • siRNA molecules which specifically interact with the canonical RNAi pathway (the RISC complex) to drive cleavage or steric blocking of hybridized transcripts; cleavage-vs-blocking depends on whether the match is perfect (cleavage) or imperfect but still stable (blocking).
  • Length is typically a double-stranded RNA where the overlapping region is 19-22 and each strand has two extra nt at their 3’ ends.
  • Chemistry is largely RNA-based-sugars, with some DNA-based sugars at the 3’ overhangs.
  • Clinical examples include patisiran (targets TTR) and givosiran (targets ALAS ).
  • an overlapping region of a siRNA may comprise from about 10 to about 40, from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 10 to about 22, from about 10 to about 21, from about 10 to about 20, from about 15 to about 40, from about 15 to about 35, from about 15 to about 30, from about 15 to about 25, from about 15 to about 22, from about 15 to about 21, from about 15 to about 20, from about 17 to about 40, from about 17 to about 35, from about 17 to about 30, from about 17 to about 25, from about 17 to about 22, from about 17 to about 21, from about 17 to about 20, from about 18 to about 40, from about 18 to about 35, from about 18 to about 30, from about 18 to about 25, from about 18 to about 22, from about 18 to about 21, from about 18 to about 20, from about 19 to about 40, from about 19 to about 35, from about 19 to about 30, from about 19 to about 25, from about 19 to about 22, from about 19 to about 21, or from about 19 to about 20 nucleotide residues.
  • an overhang region may be selected from about 19 to about
  • Anti-miRs may function as steric blockers designed against miRNAs that would block a RISC complex loaded with a specific disease-associated miRNA without being subject to cleavage by the RISC complex RNase subunit.
  • One clinical example is miravirsen, a 15-base oligo with a mixture of DNA and LNA sugars that targets miR- 122 in hepatitis C patients.
  • An anti-miR nucleotide may be of sufficient length to anneal specifically and stably to the target miR, but the length of the sequence may vary.
  • an anti-miR may have a length of up to about 21 nt, corresponding to the maximum size loaded into RISC.
  • an anti-miR nucleotide may comprise from about 10 to about 25, from about 10 to about 23, from about 10 to about 21, from about 10 to about 20, from about 10 to about 19, from about 10 to about 18, from about 13 to about 25, from about 13 to about 23, from about 13 to about 21, from about 13 to about 20, from about 13 to about 19, from about 13 to about 18, from about 15 to about 25, from about 15 to about 23, from about 15 to about 21, from about 15 to about 20, from about 15 to about 19, from about 15 to about 18, from about 16 to about 25, from about 16 to about 23, from about 16 to about 21, from about 16 to about 20, from about 16 to about 19, or from about 16 to about 18 nucleotide residues.
  • U1 adapters which have two parts. One anneals to the Ul-snRNA of the Ul-snRNP complex, and the other binds to the target RNA, bringing the Ul-snRNP to the polyA site and inhibiting polyadenylation; absence of a polyA tail causes the mRNA to be degraded.
  • the Ul-binding region is at least 10 nt but up to 19 nt.
  • Target binding region can be from about 15 nt to about 25 nt. Chemistry in early studies made heavy use of LNA and 2’-O- Methyl sugars.
  • a U1 binding region may comprise from about 10 to about 25, from about 10 to about 23, from about 10 to about 21, from about 10 to about 20, from about 10 to about 19, from about 10 to about 18, from about 13 to about 25, from about 13 to about 23, from about 13 to about 21, from about 13 to about 20, from about 13 to about 19, from about 13 to about 18, from about 15 to about 25, from about 15 to about 23, from about 15 to about 21, from about 15 to about 20, from about 15 to about 19, from about 15 to about 18, from about 16 to about 25, from about 16 to about 23, from about 16 to about 21, from about 16 to about 20, from about 16 to about 19, or from about 16 to about 18 nucleotide residues.
  • a target binding region may comprise from about 10 to about 40, from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 10 to about 22, from about 10 to about 21, from about 10 to about 20, from about 15 to about 40, from about 15 to about 35, from about 15 to about 30, from about 15 to about 25, from about 15 to about 22, from about 15 to about 21, from about 15 to about 20, from about 17 to about 40, from about 17 to about 35, from about 17 to about 30, from about 17 to about 25, from about 17 to about 22, from about 17 to about 21, from about 17 to about 20, from about 18 to about 40, from about 18 to about 35, from about 18 to about 30, from about 18 to about 25, from about 18 to about 22, from about 18 to about 21, from about 18 to about 20, from about 19 to about 40, from about 19 to about 35, from about 19 to about 30, from about 19 to about 25, from about 19 to about 22, from about 19 to about 21, or from about 19 to about 20 nucleotide residues.
  • An exemplary nucleic acid sequence contains a U1 adapter for modulating BCL2 mRNA that is highly active against BCL2 can include: 5 ’GCCGUACAGUUCCACAAAGGGCCAGGUzL4GG4 U-3 ’ (SEQ ID NO: 504), wherein the underlined portion (GCCGUACAGUUCCACAAAGG (SEQ ID NO: 573)) corresponds to the BCL2 recognition sequence and the italicized portion (GCCAGGUAAGUAU (SEQ ID NO: 492)) corresponds to the U1 recognition sequence.
  • Another example of a nucleotide of the present disclosure is an aptamer.
  • Aptamers disrupt target activity using a mechanism that differs from other nucleotides described herein that form base pairing interactions with a target nucleotide.
  • Aptamers are nucleic acids that form secondary structures (e.g., where a single strand of nucleic acid base-pairs with itself upon folding, creating loops in various locations).
  • Aptamers may be screened for interaction with target proteins.
  • Aptamers may have varied nucleotide chemistry and may include a mixture of conventional RNA and/or DNA sugars and modified sugars (e.g., 2’-O-Methyl (2’-O-Me) RNA or 2’-Fluoro (2’-F) RNA sugars).
  • pegaptanib a VEGF-binding aptamer
  • pegaptanib has a mixture of 2’-O-Methyl (2’-O-Me) RNA and 2’-Fluoro (2’-F) RNA sugars and regular RNA and DNA sugars.
  • An aptamer sequence may be long enough to form a stable secondary structure (e g., through intramolecular base pairing), but the length may vary.
  • an aptamer sequence may comprise from about 20 nt to about 40 nt. For example, experiments that identified pegaptanib used oligos of 20-40 nt in length.
  • an aptamer may comprise from about 15 to about 60, from about 15 to about 50, from about 15 to about 40, from about 15 to about 35, from about 15 to about 30, from about 20 to about 60, from about 20 to about 50, from about 20 to about 40, from about 20 to about 35, from about 20 to about 30, from about 25 to about 60, from about 25 to about 50, from about 25 to about 40, from about 25 to about 35, or from about 25 to about 30 nucleotide residues.
  • Nucleotides may be designed for use in the peptide nucleotide complexes of the present disclosure.
  • nucleotides that modify processing, translation, or other RNA functions e.g., a gapmer, splice blocker, siRNA, anti-miR, or U1 adapter
  • RNA functions e.g., a gapmer, splice blocker, siRNA, anti-miR, or U1 adapter
  • any length of a nucleotide (nt) can be used within the foregoing ranges; (b) cross-species homology (e g., by targeting highly-conserved motifs) is often a desirable feature but is not necessary for activity or clinical development; (c) avoidance of common SNPs in humans unless that SNP is involved in disease pathology (e.g., an allele-specific oligo) is often a desirable feature but is not necessary for activity or clinical development; (d) gene specificity (they have minimal homology to other sequences; for example, a sequence may have 3 or more mismatches to every other sequence), (e) avoid predicted secondary structures in both the oligo and the target region (there are software tools available to screen in silico for such secondary structure formation); (f) higher G/C content may be preferable, as G/C-rich sequences (e.g.
  • CCAC, TCCC, GCCA may be helpful for increasing affinity of the nucleotide to its target, whereas A/T-rich sequences (e.g. TAA) or runs of 4+ G (GGGG) may exhibit low or result in structural (G-quadruplex) formation.
  • An oligonucleotide sequence can be 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity or match to the target sequence. In some situations, an oligonucleotide with 100% complementarity will result in the target RNA being degraded. In some situations, an oligonucleotide that is less than 100% complementarity may not lead to degradation of the target RNA but may prevent translation and production of the encoded protein.
  • gapmers have one or more of the following properties: (a) 12-30 nt in length. It is understood that any length of a nucleotide (nt) can be used within the foregoing range, (b) target sites are anywhere in the pre-mRNA, including UTRs, exons, or introns (c) central DNA region: minimum of 4 contiguous DNA nucleotides, often 10 or more are used. No artificial substitutions at 2’ site (e.g.
  • flanking region can be DNA- or RNA-based-sugars. 2’ substitutions such as 2’-0-ME or 2’-0-M0E are tolerated.
  • LNA Linked nucleic acids
  • morpholino phosphorodiamidate morpholino oligo
  • Backbone can be natural phosphodiester (PO) or nonnatural phosphorothioate (PS) linkages.
  • a clinical example is fomivirsen, a 21 nt gapmer wherein the whole oligo is PS-backbone DNA.
  • Another example is mipomersen, a 20 nt gapmer wherein the entire backbone is PS linkages, and the central region uses DNA sugar flanked by 2’-0-M0E modified RNA.
  • all C bases are 5-methyl-C, though this is not a strict requirement for engagement of RNase Hl.
  • thiophosphorodiamidate chemistries may be used.
  • steric blockers have one or more of the following properties; (a) as the molecule does not need to engage RNase H or any other enzyme, backbone and sugar chemistry can be more varied, (g) target sites for the nucleotide are complementary to one or more splice sites in the target RNA.
  • a clinical example is eteplirsen, a 30 nt splice blocking ASO wherein whole oligonucleotide uses morpholino (Phosphorodiamidate morpholino oligo) chemistry.
  • nusinersen an 18 nt ASO, whose backbone is entirely PS linked and uses 2’-0-M0E RNA chemistry.
  • siRNA have one or more of the following properties: (a) can be between 15 and 25 nt in length (between 13 to 23 nt overlap respectively), or up to 25 nt (23 nt overlap) per strand, but 21 nt (19 nt overlap) is common.
  • any length of a nucleotide (nt) overlap can be used within the foregoing ranges; (b) complements a sequence typically but not exclusively of 21 -nt length in the target mRNA that typically but not exclusively begins with “AA” (c) target sites are ideally found in the mature spliced mRNA as the RISC complex for RNA cleavage is primarily cytosolic; (d) preferably but not exclusively avoids sequences within 100 nt of the mRNA start site, as the transcript at start site is more likely to be occupied by RNA polymerase, (e) successful siRNA constructs typically have more G/C at 5’ end of sense strand, more A/T at 3’ end of sense strand, and are roughly 30-60% in G/C content.
  • anti-miR have one or more of the following properties: (a) a perfect match to target sequence (specifically the 5 ’ end of the guide strand of the miRNA); (b) length can vary and can even be greater than the length of the mature guide strand. Screening for effective anti-miR constructs may begin with the shortest sequence that achieves specificity (no off-target homology) and increase length from there to empirically determine ideal minimal length for strong miRNA inhibition; (c) 2’ sugar modifications (2’-O- Me, 2’-0-M0E, 2’-F) and LNA sugars are commonly used. Sugars can be a mixture.
  • a clinical example of an anti-miR is miravirsen, which uses a mixture of DNA and LNA sugars
  • PS linkages in backbone are common. PS linkages may reduce affinity, but sugar modifications may increase affinity.
  • aptamers have one or more of the following properties: (a) length of aptamers can vary widely, as there is no biological complex (e.g., RISC) they interact with to function. Although composed of nucleic acids, they are more protein-like in function (e.g., bind to a target protein, etc.). The minimum length may be determined empirically to maintain sufficient stability of intra-strand hybridization to fold into a secondary structure, the upper limit on size is limited only by pharmacology, as longer sequences have a higher risk of engaging inflammatory pathways.
  • RISC biological complex
  • the minimum length may be determined empirically to maintain sufficient stability of intra-strand hybridization to fold into a secondary structure, the upper limit on size is limited only by pharmacology, as longer sequences have a higher risk of engaging inflammatory pathways.
  • Aptamer screening typically begins with libraries of 20-40 nt in length (not including flanking regions required for library amplification during screening); (b) as they form interactions via secondary structure rather than base pairing interactions, there are few limitations for their base patterns, since secondary structures are not only desirable but essential to their function. Design may be empirical for each target; (c) selection is typically via Systematic Evolution of Ligands by Exponential Enrichment (SELEX): random or semirandom sequences between primer-binding flanking regions are exposed to a target of interest on a solid substrate. The pooled oligonucleotide mixture is rinsed from the substrate, leaving only sequences that interact with the target remaining, and then binding sequences are eluted and amplified by PCR.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • sugar modifications commonly used include 2 ’-fluoro (2’-F), 2’-0-M0E, and 2’-O-Me, though other chemistries including (but not limited to) LNA and unlocked- nucleic-acids (UNA) are also possible;
  • backbones are typically PO or PS, but other linkages such as methylphosphonate are possible.
  • a clinical aptamer example, pegaptanib is entirely PO backbone, but others in development use other linkages,
  • aptamer termini are typically capped with unnatural nucleotide chemistries (e.g.
  • aptamers can be much more creative with chemical modifications of the bases themselves; these can include bases designed to induce covalent bonds with target proteins to permanently disable them; (h) such modifications are tested after selection of an active, high affinity aptamer, as unmodified bases are required for nucleic acid amplification during SELEX (i) if the target protein is extracellular, less considerations are necessary than for cell penetration capabilities.
  • PK pharmacokinetic
  • other general design considerations aimed at enhancing pharmacokinetic (PK) properties of the nucleotide, peptide, or peptide oligonucleotide complex include one or more of the following properties: (a) building in conjugation to moieties that reduce clearance or increase cellular uptake including cholesterol or other lipids, diacylglycerol, GalNAc, palmitoyl, PEG, an RGD motif, cell penetrating peptides or moieties (e g., a cellpenetrating peptide or cell penetrating peptide as described herein). Adding cholesterol to the peptide oligonucleotide complex can improve biodistribution to the target tissue, increase cellular uptake by endocytosis, and alter the serum pharmacokinetics.
  • the therapeutic activity and molecular method of the peptide oligonucleotide complex may depend on which target molecule (e.g., a DNA or RNA) that the nucleic acid complements, or in the case of an aptamer, which target molecule (e.g., protein or other macromolecule) it binds.
  • Target choice can fall into one or more non-mutually exclusive categories such as tissuetarget-based or disease-selective.
  • Known targets have known mRNA and genomic sequences that can be used to design a variety of complementary nucleic acids for use in the peptide nucleotide complexes described herein depending on the activity (e.g., gene regulation, protein degradation, reduction of cancer cell activators) desired.
  • tissue-targeting may comprise selecting targets acting in the tissues where a cell-penetrating peptide portion of the peptide oligonucleotide complex would preferentially access.
  • Targets for the peptide oligonucleotide complexes can include oncogenes, for example by designing the nucleic acid portion of the complex to target overexpressed genes or those for which the tumor is lacking a redundant ortholog (i.e., normal cells function by using X or Y, tumors do not express Y, so X is targeted).
  • disease-selective targeting can be used to treat conditions where the transcript is selectively found in the diseased tissue, and preferentially accumulate there, to improve safety and reduce off-target effects.
  • the target-binding agent e.g., a nucleotide of a peptide oligonucleotide complex
  • the target-binding agent may be capable of binding the targets described in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or to nucleotides (e.g., DNA or RNA molecules) encoding the proteins listed in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or otherwise described herein.
  • nucleotide sequences that may be used in the nucleotide portion of the peptide oligonucleotide complex include SEQ ID NO: 488 - SEQ ID NO: 573.
  • any oligonucleotide may be used that is complementary to a portion of the target DNA or RNA molecule.
  • target binding agent may comprise a nucleotide sequence is single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter.
  • ssDNA, ssRNA double stranded
  • dsDNA double stranded
  • Such oligos may be about 5 to 30 nt in length, 10 to 25 nt in length, 15 to 25 nt in length, 19 to 23 nt in length, or at least 10 nt in length, at least 15 nt in length, at least 20 nt in length, at least 25 nt in length, or at least 30 nt in length, at least 50 nt in length, at least 100 nucleotides in length across any portion of the target RNA.
  • sequences to which such oligonucleotides may bind include SEQ ID NO: 574 - SEQ ID NO: 611, or any genomic or ORF sequence referenced in TABLE 6.
  • RNA target described herein including for any of the targets or molecules encoding the targets described in TABLE 12, and SEQ ID NO: 574 - SEQ ID NO: 611, or any genomic or ORF sequence referenced in TABLE 6 such target binding agent of any nt length is described.
  • a nucleotide binds to the target molecule with a melting temperature of not less than 37 °C and not more than 99 °C. In some embodiments, a nucleotide binds to the target molecule with a melting temperature of not less than 40 °C and not more than 85 °C, not less than 40 °C and not more than 65 °C, not less than 40 °C and not more than 55 °C, not less than 50 °C and not more than 85 °C, not less than 60 °C and not more than 85 °C, or not less than 55 °C and not more than 65 °C.
  • a nucleotide binds the target molecule with an affinity of not more than 500 nM, not more than 100 nM, not more than 50 nM, not more than 10 nM, not more than 1 nM, not more than 500 pM, not more than 400 pM, not more than 300 pM, not more than 200 pM, or not more than 100 pM.
  • a nucleotide binds the target molecule with an affinity of not more than 500 nM and not less than 100 pM, not more than 100 nM and not less than 200 pM, not more than 50 nM and not less than 300 pM, not more than 10 nM and not less than 400 pM, or not more than 1 nM and not less than 500 pM.
  • a nucleotide comprises at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 488 - SEQ ID NO: 573.
  • a nucleotide comprises a sequence of any one of SEQ ID NO: 488 - SEQ ID NO: 573, any one of SEQ ID NO: 488 - SEQ ID NO: 573 wherein U is replaced with T, or any one of SEQ ID NO: 488 - SEQ ID NO: 573 wherein T is replaced with U.
  • a nucleotide comprises no more than 1, 2, 3, 4, or 5 base changes relative to a sequence of any one of SEQ ID NO: 488 - SEQ ID NO: 573.
  • a nucleotide is at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% reverse complementary to the target molecule. In some embodiments, a nucleotide is 100% reverse complementary to the target molecule. In some embodiments, a nucleotide comprises no more than 1, 2, 3, 4, or 5 base pair mismatches upon binding to the target molecule. In some embodiments, a nucleotide comprises at least 1, 2, 3, 4, or 5 base pair mismatches upon binding to the target molecule.
  • a nucleotide may modulate an activity of a target molecule.
  • modulating the activity of the target molecule comprises reducing expression of the target molecule, increasing the expression of the target molecule, reducing translation of the target molecule, degrading the target molecule, reducing a level of the target molecule, modifying the processing of the target molecule, modifying the splicing of the target molecule, inhibiting processing of the target molecule, reducing a level of a protein encoded by the target molecule, or blocking an interaction with the target molecule.
  • the expression of the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%.
  • the translation of the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99 9%.
  • the expression of the target molecule is reduced by a factor of at least 2, 4, 8, 10, 15, 16, 20, 32, 50, 64, 100, 128, 200, 256, 500, 512, or 1000.
  • the translation of the target molecule is reduced by a factor of at least 2, 4, 8, 10, 15, 16, 20, 32, 50, 64, 100, 128, 200, 256, 500, 512, or 1000. In some embodiments, at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the target molecule is degraded. In some embodiments, the level of the protein encoded by the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%.
  • modifying the splicing of the target molecule increases a level of a protein encoded by the target molecule by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%.
  • a peptide oligonucleotide complex of the present disclosure may comprise a nucleotide complexed with a peptide (e.g., a cell-penetrating peptide).
  • the nucleotide may comprise single stranded DNA, single stranded RNA, double stranded DNA, double stranded RNA, or combinations thereof.
  • a nucleotide of a peptide oligonucleotide complex may be non-naturally occurring, also referred to as an “engineered nucleotide”.
  • a nucleotide may comprise a naturally occurring sequence.
  • a nucleotide may be exogenously expressed, enzymatically synthesized in vitro, or chemically synthesized.
  • a nucleotide may be expressed in a bacterial, yeast, or mammalian cell line and purified for use in a peptide oligonucleotide complex of the present disclosure.
  • a nucleotide may be enzymatically synthesized in vitro using an RNA or DNA polymerase.
  • a nucleotide may be chemically synthesized on a solid support using protected nucleotides.
  • phosphoramidite synthesis One example of a chemical synthesis method that may be used to prepare a nucleotide for use in a peptide oligonucleotide complex of the present disclosure is phosphoramidite synthesis. Briefly, single nucleotide residues may be sequentially added from 3’ to 5’ to the growing nucleotide chain by repeating the steps of de-blocking (detrityl ati on), coupling, capping, and oxidation. Phosphoramidite synthesis may be performed on a solid support such as controlled pore glass (CPG) or macroporous polystyrene (MPPS). Similarly, thiophosphorodiamidate may be used.
  • CPG controlled pore glass
  • MPPS macroporous polystyrene
  • thiophosphorodiamidate may be used.
  • a nucleotide of a peptide oligonucleotide complex may bind to a target molecule (e.g., a target DNA, a target RNA, or a target protein).
  • a target molecule e.g., a target DNA, a target RNA, or a target protein
  • binding of the oligonucleotide to the target molecule may alter an activity of the target molecule.
  • binding of an oligonucleotide e.g., an siRNA, an miRNA, a gapmer, or a U1 adaptor
  • binding of a nucleotide to a target DNA may increase or decrease expression of a gene encoded by the target DNA.
  • binding of a nucleotide to an RNA may increase or decrease expression of a gene encoded by the target DNA.
  • binding of an oligonucleotide (e.g., an aptamer) to a target protein may increase or decrease activity (e.g., an enzymatic activity or a binding activity) of the target protein.
  • the target molecule may be associated with a disease or condition and increasing or decreasing the activity of the target molecule may treat the disease or condition.
  • a sequence of the oligonucleotide of a peptide oligonucleotide complex may be selected for its ability to bind to or modulate the activity of a target molecule.
  • an oligonucleotide may be reverse complementary to a target DNA or RNA molecule.
  • an siRNA oligonucleotide may be reverse complementary to a target RNA molecule.
  • am oligonucleotide may be partially reverse complementary (e.g., comprising one or more mis-matched base pairs) to a target DNA or RNA molecule.
  • an siRNA oligonucleotide may comprise a base mismatch relative to a target RNA molecule.
  • a sequence of the oligonucleotide may be selected for its annealing temperature relative to a target DNA or RNA molecule.
  • a preferred annealing temperature may be achieved by selecting the length of the nucleotide, the degree of complementarity of the nucleotide to the target molecule, the chemistry of the nucleotides, or any combination thereof.
  • Nucleotide sequence parameters e.g., complementarity, annealing temperature, melting temperature, base mismatches, and binding affinity
  • an oligonucleotide may adopt a secondary structure that binds to a target DNA, RNA, or protein molecule.
  • an aptamer may adopt a secondary structure to bind to a target protein.
  • the aptamer sequence may be selected to adopt a secondary structure that binds to a target protein.
  • Nucleotide secondary structure may be predicted using any available software, such as RNAfold and the like.
  • a nucleotide sequence may be determined experimentally by selecting for the ability to bind to a target molecule. For example, a nucleotide library may be contacted to a target molecule, and sequences that bind to the target molecule may be identified.
  • a nucleotide comprises a G/C content of not less than 20% and not more than 80%. In some embodiments, a nucleotide comprises a G/C content of not less than 30% and not more than 65%. In some embodiments, the nucleotide comprises a G/C content of not less than 20%, not less than 25%, not less than 30%, not less than 35%, not less than 40%, not less than 45%, or not less than 50%. In some embodiments, the nucleotide comprises a G/C content of not more than 80%, not more than 75%, not more than 70%, not more than 65%, or not more than 50%.
  • a nucleotide comprises an A/T content or A/U content of not less than 20% and not more than 80%. In some embodiments, a nucleotide comprises an A/T content or A/U content of not less than 30% and not more than 65%. In some embodiments, the nucleotide comprises a A/U (or A/T, or combination of A/U and A/T) content of not less than 20%, not less than 25%, not less than 30%, not less than 35%, not less than 40%, not less than 45%, or not less than 50%.
  • the nucleotide comprises a A/U content (or A/T, or combination of A/U and A/T) of not more than 80%, not more than 75%, not more than 70%, not more than 65%, or not more than 50%.
  • a nucleotide has a length of no more than 1000 nt, 600 nt, 200 nt, 100 nt, 60 nt, 56 nt, 52 nt, 50 nt, 48 nt, 46 nt, 44 nt, 22 nt, 40 nt, 38 nt, 36, nt, 34 nt, 32 nt, 30 nt, or 24 nt.
  • a nucleotide has a length of from 24 to 100 nt, from 24 to 60 nt, from 24 to 50 nt, or from 36 to 50 nt. In some embodiments, a nucleotide has a length of about 42 nt.
  • a nucleotide has a length of no more than 500 nt, 300 nt, 100 nt, 50 nt, 30 nt, 28 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt, 19 nt, 18, nt, 17 nt, 16 nt, 15 nt, or 12 nt.
  • a nucleotide has a length of from 12 to 50 nt, from 12 to 30 nt, from 12 to 25 nt, from 18 to 25 nt, from 18 to 25 nt, from 19 to 23 nt, or from 20 to 22 nt. In some embodiments, a nucleotide has a length of about 21 nt.
  • a peptide oligonucleotide complex of the present disclosure may be further conjugated, linked, or fused to an active agent in addition to the nucleotide active agent (e.g., a target-binding agent capable of binding a target molecule).
  • an active agent e.g., a target-binding agent capable of binding a target molecule.
  • Such additional active agent may be complexed, fused, linked or conjugated to one or more of the peptide, nucleotide, or linker within the peptide oligonucleotide complex.
  • the active agent may be directly or indirectly linked to the peptide of the peptide oligonucleotide complex or the nucleotide of the peptide oligonucleotide complex.
  • a peptide nucleic acid complex further comprising an additional active agent may be referred to as a peptide-active agent conjugate or a peptide construct.
  • the peptide oligonucleotide complexes of the present disclosure can also be used to deliver another active agent.
  • Peptides according to the present disclosure can be conjugated to, linked to, or fused to an agent for use in the treatment of tumors and cancers or other diseases.
  • the peptides described herein are fused or conjugated to another molecule, such as an active agent that provides an additional functional capability.
  • a peptide or nucleotide can be fused with an active agent through expression of a vector containing the sequence of the peptide with the sequence of the active agent.
  • the sequence of the peptide and the sequence of the active agent can be expressed from the same Open Reading Frame (ORF).
  • ORF Open Reading Frame
  • sequence of the peptide and the sequence of the active agent can comprise a contiguous sequence.
  • the peptide and the active agent can each retain similar functional capabilities in the peptide construct compared with their functional capabilities when expressed separately.
  • examples of active agents can include other peptides.
  • the peptides or nucleotides described herein are attached to another molecule, such as an active agent that provides a functional capability.
  • the active agent may be any active agent (e.g., therapeutic agent, detectable agent, or binding moiety) described herein.
  • the peptide or nucleotide is covalently or non-covalently linked to an active agent, e.g., directly or via a linker. Exemplary linkers suitable for use with the embodiments herein are discussed in further detail below.
  • a peptide e.g., a cell-penetrating peptide of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • peptide complex e.g., comprising a cell-penetrating peptide and a cargo molecule
  • the cell-penetrating peptide may deliver the agent into a cellular space or compartment or across a cellular layer (e g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen).
  • a cellular layer e g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen.
  • the cell-penetrating peptides or peptide complexes of the present disclosure may be fused to a detectable agent that may otherwise be excluded from a cell or a cellular compartment to facilitate absorption or permeation of the detectable agent.
  • a cell -penetrating peptide of the present disclosure may facilitate absorption or permeation of a detectable agent with multiple hydrogen bond donors or hydrogen bond acceptors, a large molecular weight, or a high partition coefficient, which may lead to poor absorption or permeation of the detectable agent in the absence of the cell -penetrating peptide.
  • a cell -penetrating peptide may be conjugated to a detectable agent comprising at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors. In some embodiments, the cell-penetrating peptide may be conjugated to a detectable agent comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors. In some embodiments, the cell-penetrating peptide may be conjugated to a detectable agent comprising a molecular weight of at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, or at least 1000 Da.
  • the cell-penetrating peptide may be conjugated to a detectable agent comprising a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0.
  • the partition coefficient may be a measure of a ratio of the solubility of the agent in a hydrophobic solvent (e.g., octanol) relative to water.
  • a peptide is conjugated to, linked to, or fused with detectable agents, such as a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metalcontaining nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a metal, a radioisotope, a dye, radionuclide chelator, or another suitable material that can be used in imaging.
  • detectable agents such as a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metalcontaining nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a metal, a radioisotope, a dye, radionuclide chelator, or another suitable material that can be used in imaging.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 detectable agents can be linked to a peptide.
  • the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium.
  • the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium.
  • the radioisotope is actinium-225 or lead-212.
  • the near-infrared dyes are not easily quenched by biological tissues and fluids.
  • the fluorophore is a fluorescent agent emitting electromagnetic radiation at a wavelength between 650 nm and 4000 nm, such emissions being used to detect such agent.
  • fluorescent dyes that could be used as a conjugating molecule in the present disclosure include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, or indocyanine green (ICG).
  • near infrared dyes often include cyanine dyes (e.g., Cy7, Cy5.5, and Cy5).
  • Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4', 5'-dichloro-2',7' - dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethyl- rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.),
  • radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters.
  • the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium.
  • the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium.
  • the radioisotope is actinium-225 or lead-212.
  • fluorescent biotin conjugates that can act both as a detectable label and an affinity handle can be used.
  • fluorescent biotin conjugates include Atto 425-Biotin, Atto 488-Biotin, Atto 520-Biotin, Atto- 550 Biotin, Atto 565-Biotin, Atto 590-Biotin, Atto 610-Biotin, Atto 620-Biotin, Atto 655-Biotin, Atto 680-Biotin, Atto 700-Biotin, Atto 725-Biotin, Atto 740-Biotin, fluorescein biotin, biotin-4- fluorescein, biotin-(5-fluorescein) conjugate, and biotin-B-phycoerythrin, ALEXA FLUOR 488 biocytin, ALEXA FLUOR 546, ALEXA FLUOR 549, lucifer yellow cadaverine bio
  • the conjugates could include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels.
  • the peptide-active agent fusions described herein can be attached to another molecule.
  • the peptide sequence also can be attached to another active agent (e.g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar, etc.).
  • the peptide can be fused with, or covalently or non-covalently linked to an active agent.
  • the conjugates can include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels.
  • the peptide described herein can also be attached to another molecule.
  • the peptide sequence also can be attached to another active agent (e g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding agents, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar).
  • another active agent e g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding agents, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar.
  • the peptide can be conjugated to, linked to, or fused with, or covalently or non-covalently linked to an active agent.
  • Peptides can be conjugated to, linked to, or fused to a radiosensitizer or photosensitizer.
  • radiosensitizers include but are not limited to: ABT-263, ABT-199, WEHL539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic acid base derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine).
  • photosensitizers can include but are not limited to: fluorescent molecules or beads that generate heat when illuminated, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metalloporphyrins, metallophthalocyanines, angelicins, chalcogenapyrrillium dyes, chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives, naphthalimides, nile blue derivatives, a
  • this approach can allow for highly specific targeting of diseased cells (e.g., cancer cells) using both a therapeutic agent (e.g., drug) and electromagnetic energy (e.g., radiation or light) concurrently.
  • a therapeutic agent e.g., drug
  • electromagnetic energy e.g., radiation or light
  • the peptide is conjugated to, linked to, fused with, or covalently or non-covalently linked to the agent, e g., directly or via a linker.
  • Exemplary linkers suitable for use with the embodiments herein are discussed in further detail below.
  • a cell-penetrating peptide conjugated to, linked to, or fused with a detectable agent may be used in a method of imaging a cell or tissue.
  • a cell-penetrating peptide complex comprising a cell-penetrating peptide and a detectable agent may be contacted to a cell or tissue, and the cell or tissue may be imaged.
  • the cell or tissue may be imaged to detect the presence, absence, location, intensity, distribution of the detectable agent, or combinations thereof.
  • imaging may comprise diagnostic imaging, for example to detect the presence or absence of a disease state.
  • a peptide e.g., a cell-penetrating peptide, a cargo peptide, or a cell-penetrating peptide complex
  • the peptide can be chemically modified one or more of a variety of ways.
  • the peptide can be mutated to add function, delete function, or modify the in vivo behavior.
  • cell -penetrating peptides of the presenting disclosure may be chemically modified with a molecule that would increase the serum half-life of the peptide when administered to a subject.
  • one or more loops between the disulfide linkages of a knotted cellpenetrating peptide can be modified or replaced to include active elements from other peptides (such as described in Moore and Cochran, Methods in Enzymology, 503, p. 223-251, 2012).
  • loops from cell-penetrating peptides may be grafted into peptide active agents.
  • Amino acids can also be mutated, such as to increase half-life, decrease immunogenicity, modify, add or delete binding behavior in vivo, add new targeting function, modify surface charge and hydrophobicity, or allow conjugation sites.
  • N-methylation is one example of methylation that can occur in a peptide of the disclosure.
  • the peptide is modified by methylation on free amines.
  • full methylation may be accomplished through the use of reductive methylation with formaldehyde and sodium cyanoborohydride.
  • a chemical modification can, for instance, extend the half-life of a peptide or change the biodistribution or pharmacokinetic profile.
  • a chemical modification can comprise a polymer, a poly ether, polyethylene glycol, a biopolymer, a polyamino acid, a fatty acid, a dendrimer, an Fc region, a simple saturated carbon chain such as palmitate or myristolate, an albumin binder, or albumin.
  • a polyamino acid can include, for example, a poly amino acid sequence with repeated single amino acids (e.g., poly glycine), and a poly amino acid sequence with mixed poly amino acid sequences (e.g, gly-ala-gly-ala; SEQ ID NO: 614) that may or may not follow a pattern, or any combination of the foregoing.
  • a poly amino acid sequence with repeated single amino acids e.g., poly glycine
  • a poly amino acid sequence with mixed poly amino acid sequences e.gly-ala-gly-ala; SEQ ID NO: 614
  • the peptides of the present disclosure can be modified such that the modification increases the stability and/or the half-life of the peptides.
  • the attachment of a hydrophobic moiety, such as to the N-terminus, the C-terminus, or an internal amino acid, can be used to extend half-life of a peptide of the present disclosure.
  • the peptides can also be modified to increase or decrease the gut permeability or cellular permeability of the peptide.
  • the peptide of the present disclosure can include post-translational modifications (e g., methylation and/or amidation and/or glycosylation), which can affect, e.g., serum half-life.
  • simple carbon chains can be conjugated to, linked to, the fusion proteins or peptides.
  • the simple carbon chains can render the fusion proteins or peptides easily separable from the unconjugated material.
  • methods that can be used to separate the fusion proteins or peptides from the unconjugated material include, but are not limited to, solvent extraction and reverse phase chromatography.
  • Lipophilic moieties can extend half-life through reversible binding to serum albumin.
  • Conjugated moieties can, e.g., be lipophilic moieties that extend half-life of the peptides through reversible binding to serum albumin.
  • the lipophilic moiety can be cholesterol or a cholesterol derivative including cholestenes, cholestanes, cholestadienes and oxysterols.
  • the peptides can be conjugated to, linked to, myristic acid (tetradecanoic acid) or a derivative thereof.
  • the peptides of the present disclosure can be coupled (e g., conjugated, linked, or fused) to a half-life modifying agent.
  • half-life modifying agents can include, but is not limited to: a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin.
  • PEG polyethylene glycol
  • a hydroxyethyl starch polyvinyl alcohol
  • a water soluble polymer a zwitterionic water soluble polymer
  • a water soluble poly(amino acid) a water soluble poly(amino acid)
  • proline a water soluble polymer of proline
  • alanine and serine a water
  • conjugation of the peptide to a near infrared dye, such as Cy5.5, or to an albumin binder such as Albu-tag can extend serum half-life of any peptide as described herein.
  • immunogenicity is reduced by using minimal non-human protein sequences to extend serum half-life of the peptide.
  • the peptides of the present disclosure can be modified to reduce their immunogenicity.
  • Immunogenicity can limit the utility of a therapeutic peptidic molecule, as described in FDA Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products (2014), which is incorporated by reference in its entirety.
  • a molecule that is immunogenic and cause the formation of, or increased levels of anti-drug antibodies (ADA) can lead to complications such as toxicity, immune incompatibility, or hypersensitivity particularly in the context of human therapeutics. Consequently, it is desirable to limit immunogenicity and ADA in therapeutic applications of peptides.
  • ADA can be neutralizing or binding but not neutralizing.
  • the formation of ADA can reduce the efficacy of a therapeutic biologic molecule, including by causing earlier clearance of or reduced exposure to the therapeutic.
  • ADA can reduce the safety of a therapeutic biologic molecule, such as by causing cytokine release syndrome, infusion reactions, or clearance of endogenous proteins.
  • a therapeutic biologic molecule such as by causing cytokine release syndrome, infusion reactions, or clearance of endogenous proteins.
  • There are approaches to predicting and reducing the immunogenicity of therapeutics as described in Preclinical models immunogenicity prediction of therapeutic proteins (Brinks, 2013), which is incorporated by reference in its entirety. Some peptide sequences are more likely to cause immunogenicity. Sequences may be designed for reduced immunogenicity by screening sequences and variants using experimental and in silico methods and selecting sequences that are less immunogenic. When two or more peptides or proteins are fused together, the point of linkage may create new sequences that could cause immunogenicity.
  • Peptides that are resistant to enzymatic degradation can also have reduced immunogenicity, because in some cases peptides must undergo proteolysis into shorter peptide fragments, such as 9-11 amino acids long, in order to be presented on the MHCII complex to immune cells resulting in immunogenic effects.
  • the peptides of the present disclosure include variants with deduced immunogenicity.
  • the first two N-terminal amino acids (GS) of SEQ ID NO: 59 - SEQ ID NO: 84, SEQ ID NO: 93 - SEQ ID NO: 101, or SEQ ID NO: 210 - SEQ ID NO: 224 serve as a spacer or linker in order to facilitate conjugation or fusion to another molecule, as well as to facilitate cleavage of the peptide from such conjugated to, linked to, or fused molecules.
  • the fusion proteins or peptides of the present disclosure can be conjugated to, linked to, or fused to other moieties that, e.g., can modify or effect changes to the properties of the peptides.
  • a cell -penetrating peptide of the present disclosure may be modified with a tag that further promotes endosomal escape (e.g., an endosomal escape motif).
  • a tag that further promotes endosomal escape e.g., an endosomal escape motif
  • S19 PFVIGAGVLGALGTGIGGI; SEQ ID NO: 359
  • CM18 KWKLFKKIGAVLKVLTTG; SEQ ID NO: 360
  • PAS FTLIPKG; SEQ ID NO: 361), Aureinl .2 (GLFDIIKKIAESF; SEQ ID NO: 362), or B18 (LGLLLRHLRHHSNLLANI; SEQ ID NO: 363)
  • LGLLLRHLRHHSNLLANI LGLLLRHLRHHSNLLANI
  • the cell-penetrating peptides of the present disclosure e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 and complexes thereof penetrate cells with and intracellular concentration to exert a prophylactic or therapeutic effect.
  • 0.01% or more, 0.05% or more, 0.1% or more, 0.2% or more, 0.5% or more, 1% or more, 3% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, up to 100% of a molecule or cellpenetrating peptide can enter the cytosol from the extracellular space or circulation.
  • a cell-penetrating peptide may penetrate a cellular layer such that the cytosolic, nuclear, intracellular, or paracellular concentration of the peptide is at least about 0.1%, at least about 0.2% at least about 0.5%, at least about 1%, at least about 3%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about at least about 100% of the extracellular peptide concentration.
  • the nucleic acid portion of a peptide oligonucleotide complex (e g., an oligonucleotide of a cell-penetrating peptide oligonucleotide complex) contains one or more bases within the nucleic acid molecule that are modified.
  • nucleic acid portion a single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter.
  • ssDNA, ssRNA double stranded
  • dsDNA, dsRNA double stranded
  • a combination of single and double stranded for example with a mismatched sequence, hairpin or other structure
  • an antisense RNA complementary RNA, inhibitory RNA,
  • One or more bases in a given nucleotide sequence may be modified to increase in vivo stability, to increase resistance to enzymes such as nucleases, increase protein binding including to serum proteins, increase in vivo half-life, to modify the tissue biodistribution, or to modify how the immune system responds.
  • the phosphonate, the ribose, or the base may be modified.
  • the modification comprises a phosphorothioate modification, a phosphodiester modification, a thio- phosphoramidate modification, a methyl phosphonate modification, a phosphorodi thioate modification, a methoxypropylphosphonate modification, a 5’-(E)-vinylphosphonate modification, a 5 ’methyl phosphonate modification, an (S)-5’-C-methyl with phosphate modification, a 5 ’-phosphorothioate modification, a peptide nucleic acid (PNA), a 2’-0 methyl modification, a 2’-O-methoxyethyl (2’-O-Me) modification, a 2’-fluoro (2’-F) modification, a 2’-deoxy-2’-fluoro modification, a 2’ arabino-fluor modification, a 2’-O-benyzl modification, a 2’-O-methyl-4-pyridine modification, a locked nucle
  • the oligonucleotide may be comprised entirely of a combination of 2’-O-Me and 2’-F modifications. Diastereomers or one or both stereoisomers may be used. Any of the stabilization chemistries or patterns, including STC, ESC, advanced, ESC, ADI-3, AD5, disclosed in Hu Signal Transduction and Targeted Therapy 2020,5: 101 can be used. Pyrimidines can be 2’-fluoro-modified, which can increase stability to nucleases but can also increase immune system activation.
  • the RNA backbone can be phosphorothioate- substituted (where the non-bridging oxygen is replaced with sulfur), which can increase resistance to nuclease digestion as well as altering the biodistribution and tissue retention and increasing the pharmacokinetics such as by increasing protein binding, but can also induce more immune stimulation.
  • Methyl phosphonate modification of an RNA can also be used.
  • 2’-Omethyl and 2’-F RNA bases can be used, which can protect against base hydrolysis and nucleases and increase the melting temperature of duplexes.
  • Bridged, Locked, and other similar forms of Bridged Nucleic Acids where any chemical bridge such as an N-0 linkage between the 2’ oxygen and 4’ carbons in ribose can be incorporated to increase resistance to exo- and endonucleases and enhance biostability.
  • BNA Bridged Nucleic Acids
  • LNA Low-Nodecyl-N-(2-aminoe)
  • cEt Bridged Nucleic Acids
  • phosophorothioate nucleic acid linkages may be used between the 2-4 terminal nucleic acids of one or both sequences.
  • 2’F modified nucleic acids may be used at least at 2-4 positions, at least 5%, at least 10% at least 25% of internal positions, at least 50%, at least 75%, or up to 100% of internal positions, all internal positions or all positions.
  • one or more of 2’F base, an LNA base, a BNA base, an ENA base, a 2’O-MOE base, a morpholino base, a 2’0Me base, a 5 ’-Me base, a (S)-cEt base or combinations thereof may be used at least at 2-4 positions, at least 5%, at least 10% at least 25% of internal positions, at least 50%, at least 75%, or up to 100% of internal positions, all internal positions or all positions.
  • Modified bases can be used to increase in the in vivo half-life of the oligonucleotide. They can allow the oligonucleotide to remaining intact in the serum, endosome, cytosol, or nucleus, including for days, weeks, or months. This can allow ongoing activity, including if the oligonucleotide is slowly released from the endosome over days, weeks, or months within a given cell (such as described in Brown et al., Nucleic Acids Research, 2020, pl 1827-11844).
  • a nucleotide comprises at least one phosphorothioate linkage.
  • a peptide oligonucleotide complex comprises from 1 to 12 phosphorothioate linkages. In some embodiments, a nucleotide comprises at least one thiophosphoroamidate linkage. In some embodiments, a nucleotide comprises from 1 to 12 thiophosphoroamidate linkages. In some embodiments, a nucleotide comprises at least one modified base. In some embodiments, at least modified base comprises a 2’F base, an LNA base, a BNA base, an ENA base, a 2’O-MOE base, a 5’-Me base, a (S)-cEt base, a 2’OMe base, a morpholino base, or combinations thereof.
  • the cell-penetrating peptides of the presented disclosure can be linked to a cargo peptide (e.g., an active agent peptide, a target-binding peptide, a therapeutic peptide, a detectable peptide, or a cystine-dense peptide) in numerous ways.
  • a cargo peptide e.g., an active agent peptide, a target-binding peptide, a therapeutic peptide, a detectable peptide, or a cystine-dense peptide
  • a cell -penetrating peptide can be conjugated to a cargo peptide via a peptide linker to form a cell-penetrating peptide fusion.
  • a peptide linker does not disturb the independent folding of peptide domains (e.g., a cystine-dense peptide).
  • a peptide linker does not negatively impact manufacturability (synthetic or recombinant) of the peptide complex (e g., the cell -penetrating peptide complex).
  • a peptide linker does not impair post-synthesis chemical alteration (e.g. conjugation of a fluorophore or albumin-binding chemical group) of the peptide fusion.
  • a peptide linker can connect the C-terminus of a first peptide (e.g., a cell-penetrating peptide or a cargo peptide) to the N-terminus of a second peptide (e.g., a cell-penetrating peptide or a cargo peptide).
  • a peptide linker can connect the C-terminus of the second peptide (e.g., a cell-penetrating peptide or a cargo peptide) to the N-terminus of a third peptide (e g., a cell -penetrating peptide or a cargo peptide).
  • a linker e.g., any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485
  • a cell-penetrating peptide e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • a cellpenetrating peptide e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
  • a cellpenetrating peptide fusion e.g., SEQ ID NO: 299 - SEQ ID NO: 3
  • a linker e g., any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485
  • a linker can connect the C-terminus of a cargo peptide to the N- terminus of a cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) to form a cell-penetrating peptide fusion.
  • a cell-penetrating peptide e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 -
  • a peptide linker can comprise a Tau-theraphotoxin-Hsla, also known as DkTx (double-knot toxin), extracted from a native knottin-knottin dimer from Haplopelma schmidti (e.g., SEQ ID NO: 266).
  • the linker can lack structural features that would interfere with dimerizing independently functional CDPs (e.g., a cell-penetrating CDP and a target-binding CDP).
  • a linker can comprise a glycine-serine (Gly-Ser or GS) linker (e.g., SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485).
  • Gly-Ser linkers can have minimal chemical reactivity and can impart flexibility to the linker.
  • Serines can increase the solubility of the linker or the peptide complex, as the hydroxyl on the side chain is hydrophilic.
  • a linker can be derived from a peptide that separates the Fc from the Fv domains in a heavy chain of human immunoglobulin G (e.g., SEQ ID NO: 267).
  • a linker derived from a peptide from the heavy chain of human IgG can comprise a cysteine to serine mutation relative to the native IgG peptide.
  • peptides of the present disclosure can be dimerized using an immunoglobulin heavy chain Fc domain. These Fc domains can be used to dimerize functional domains (e.g., a cell-penetrating peptide and a cargo peptide), either based on antibodies or other otherwise soluble functional domains. In some embodiments, dimerization can be homodimeric via Fc sequences.
  • dimerization can be heterodimeric by mutating the Fc domain to generate a “knob-in-hole” format where one Fc CH3 domain contains novel residues (knob) designed to fit into a cavity (hole) on the other Fc CH3 domain.
  • a first peptide domain e.g., a cell-penetrating peptide
  • a second peptide domain e.g., a TfR-binding peptide or target-binding peptide
  • Knob+knob dimers can be highly energetically unfavorable.
  • a purification tag can be added to the “knob” side to remove hole+hole dimers and select for knob+hole dimers.
  • the peptides of the present disclosure can be linked to another peptide (e.g., a cargo peptide) at the N-terminus or C-terminus.
  • one or more peptides can be linked or fused via a peptide linker (e.g., a peptide linker comprising a sequence of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485).
  • a cell -penetrating peptide can be fused to a cargo peptide via a peptide linker of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485.
  • a peptide linker can have a length of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, or about 50 amino acid residues.
  • a peptide linker can have a length of from about 2 to about 5, from about 2 to about 10, from about 2 to about 20, from about 3 to about 5, from about 3 to about 10, from about 3 to about 15, from about 3 to about 20, from about 3 to about 25, from about 5 to about 10, from about 5 to about 15, from about 5 to about 20, from about 5 to about 25, from about 10 to about 15, from about 10 to about 20, from about 10 to about 25, from about 15 to about 20, from about 15 to about 25, from about 20 to about 25, from about 20 to about 30, from about 20 to about 35, from about 20 to about 40, from about 20 to about 45, from about 20 to about 50, from about 3 to about 50, from about 3 to about 40, from about 3 to about 30, from about 10 to about 40, from about 10 to about 30, from about 50 to about 100, from about 100 to about 200, from about 200 to about 300, from about 300 to about 400, from about 400 to about 500, or from about 500 to about 600 amino acid residues.
  • a first peptide e.g., a cell -penetrating peptide
  • a second peptide e.g., a cargo peptide
  • a flexible linker can provide rotational freedom between the first peptide and the second peptide and can allow the first peptide and the second peptide to perform their respective functions with minimal strain.
  • a peptide linker can have a persistence length of no more than 6 A, no more than 7 A, no more than 8 A, no more than 9 A, no more than 10 A, no more than 12 A, no more than 15 A, no more than 20 A, no more than 25 A, no more than 30 A, no more than 40 A, or no more than 50 A.
  • a peptide linker can have a persistence length of from about 4 A to about 100 A, from about 4 A to about 50 A, from about 4 A to about 20 A, from about 4 A to about 10 A, from about 10 A to about 20 A, from about 20 A to about 30 A, from about 30 A to about 50 A, or from about 50 A to about 100 A.
  • the persistence length of the linker can be a measure of the flexibility of the peptide linker and can be quantified as the peptide length over which correlations in the direction of the tangent are lost.
  • a peptide linker can be selected based on a desired linker length, hydrodynamic radius, chromatographic mobility, posttranslational modification propensity, or combinations thereof.
  • a linker separating two or more functional domains of a peptide complex e.g., separating a cell -penetrating peptide and a cargo peptide
  • a linker separating two or more functional domains of a peptide complex can comprise a small, flexible linker, for example to reduce the hydrodynamic radius of the complex for use in tight spaces like dense-core tumor stroma.
  • a peptide linker can support independent folding of the two or more functional domains and may not inhibit functions of the respective functional domains.
  • a peptide can be appended to the N-terminus of any peptide of the present disclosure following an N-terminal GS dipeptide and preceding, for example, a GGGS (SEQ ID NO: 264) spacer.
  • a peptide e.g., a target-binding peptide
  • the peptide linker comprises (GS)x (SEQ ID NO: 258), wherein x can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the peptide linker comprises GGSSG (SEQ ID NO: 259), GGGGG (SEQ ID NO: 260), GSGSGSGS (SEQ ID NO: 261), GSGG (SEQ ID NO: 262), GGGGS (SEQ ID NO: 263), GGGS (SEQ ID NO: 264), GGS (SEQ ID NO: 265), GGGSGGGSGGGS (SEQ ID NO: 255), or a variant or fragment thereof.
  • KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx
  • EPKSSDKTHT (SEQ ID NO: 267) from human IgG3
  • the peptide linker comprises GGGSGGSGGGS (SEQ ID NO: 292).
  • the peptide linker comprises a linker of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485. Examples of peptide linkers compatible with the cell-penetrating peptides and cell-penetrating peptide fusions of the present disclosure are provided in TABLE 10.
  • any of the foregoing linkers or a variant or fragment thereof can be used with any number of repeats or any combinations thereof. It is also understood that other peptide linkers in the art or a variant or fragment thereof can be used with any number of repeats or any combinations thereof.
  • Peptides according to the present disclosure can be attached to another moiety (e.g., an active agent or an detectable agent), such as a small molecule, a second peptide, a protein, an antibody, an antibody fragment, an aptamer, polypeptide, polynucleotide, a fluorophore, a radioisotope, a radionuclide chelator, a polymer, a biopolymer, a fatty acid, an acyl adduct, a chemical linker, or sugar or other active agent or detectable agent described herein through a linker, or directly in the absence of a linker.
  • an active agent or an detectable agent such as a small molecule, a second peptide, a protein, an antibody, an antibody fragment, an aptamer, polypeptide, polynucleotide, a fluorophore, a radioisotope, a radionuclide chelator, a polymer, a biopol
  • an active agent or a detectable agent can be conjugated to, linked to, or fused to the N-terminus or the C-terminus of a peptide to create an active agent or detectable agent fusion peptide.
  • the link can be made by a peptide fusion via reductive alkylation.
  • a cleavable linker is used for in vivo delivery of the peptide, such as a linker that can be cleaved or degraded upon entry in a cell, endosome, or a nucleus.
  • in vivo delivery of a peptide requires a small linker that does not interfere with penetration of a cell or localization to a nucleus of a cell.
  • a linker can also be used to covalently attach a peptide as described herein to another moiety or molecule having a separate function, such a targeting, cytotoxic, therapeutic, homing, imaging, or diagnostic functions.
  • a peptide can be directly attached to another molecule by a covalent attachment.
  • the peptide is attached to a terminus of the amino acid sequence of a larger polypeptide or peptide molecule, or is attached to a side chain, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, or glutamic acid residue.
  • the attachment can be via an amide bond, an ester bond, an ether bond, a carbamate bond, a carbon-nitrogen bond, a triazole, a macrocycle, an oxime bond, a hydrazone bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond.
  • similar regions of the disclosed peptide(s) itself can be used to link other molecules.
  • an amino acid side chain such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, or glutamic acid residue
  • an amide bond, an ester bond, an ether bond, a carbamate bond, a carbon-nitrogen bond, a triazole, a macrocycle, an oxime bond, a hydrazone bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond, or linker as described herein can be used to link other molecules.
  • Attachment via a linker can involve incorporation of a linker moiety between the other molecule and the peptide.
  • the peptide and the other molecule can both be covalently attached to the linker.
  • the linker can be cleavable, labile, non-cleavable, stable, stable self-immolating, hydrophilic, or hydrophobic.
  • non-cleavable or “stable” (such as used in association with an amide, cyclic, or carbamate linker or as otherwise as described herein) is often used by a skilled artisan to distinguish a relatively stable structure from one that is more labile or “cleavable” (e.g., as used in association with cleavable linkers that may be dissociated or cleaved structurally by enzymes, proteases, self-immolation, pH, reduction, hydrolysis, certain physiologic conditions, or as otherwise described herein).
  • Non- cleavable or “stable” linkers offer stability against cleavage or other dissociation as compared to “cleavable” linkers, and the term is not intended to be considered an absolute non-cleavable or non-dissociative structure under any conditions. Consequently, as used herein, a “non- cleavable” linker is also referred to as a “stable” linker.
  • the linker can have at least two functional groups with one bonded to the peptide, the other bonded to the other molecule, and a linking portion between the two functional groups.
  • Non-limiting examples of the functional groups for attachment can include functional groups capable of forming an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond.
  • Non-limiting examples of functional groups capable of forming such bonds can include amino groups; carboxyl groups; hydroxyl groups; aldehyde groups; azide groups; alkyne and alkene groups; ketones; hydrazides; acid halides such as acid fluorides, chlorides, bromides, and iodides; acid anhydrides, including symmetrical, mixed, and cyclic anhydrides; carbonates; carbonyl functionalities bonded to leaving groups such as cyano, succinimidyl, and JV-hydroxysuccinimidyl; hydroxyl groups; sulfhydryl groups; and molecules possessing, for example, alkyl, alkenyl, alkynyl, allylic, or benzylic leaving groups, such as halides, such
  • Non-limiting examples of the linking portion can include alkylene, alkenylene, alkynylene, polyether, such as polyethylene glycol (PEG), hydroxy carboxylic acids, polyester, polyamide, polyamino acids, polypeptides, cleavable peptides, valine-citrulline, aminobenzylcarbamates, D-amino acids, and polyamine, any of which being unsubstituted or substituted with any number of substituents, such as halogens, hydroxyl groups, sulfhydryl groups, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups,
  • a peptide and drug complexed, conjugated, or fused via a linker is described with the formula Peptide-A-B-C-Drug, wherein the linker is A-B-C.
  • A can be a stable amide link, is an amine on the peptide and the linker and can be achieved via a tetrafluorophenyl (TFP) ester or an NHS ester.
  • B can be (-CH2-) X - or a short PEG (-CEECFEO-jx (x is 1-10), and C can be the ester bond to the hydroxyl or carboxylic acid on the drug.
  • C can refer to the “cleavable” or “stable” part of the linker.
  • A can also be the “cleavable” part.
  • A can be amide, carbamate, thioether via maleimide or bromoacetamide, triazole, oxime, or oxacarboline.
  • the cleaved active agent or drug can retain the chemical structure of the active agent before cleavage or can be modified as a result of cleavage.
  • such active agent can be active while linked to the peptide, remain active after cleavage or become inactivated, be inactive while linked to the peptide, or it can be activated upon cleavage.
  • peptide complexes have stable linkers.
  • a peptide of the disclosure can be expressed recombinantly or chemically synthesized.
  • the peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide linkage or a carbamate linkage.
  • the peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide bond using standard 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or dicylcohexylcarbodiimide (DCC) based chemistry or thionyl chloride or phosphorous chloride-based bioconjugation chemistries.
  • EDC 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • DCC dicylcohexylcarbodiimide
  • a stable linker may or may not be cleaved in buffer over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker may or may not be cleaved in body fluids such as plasma or synovial fluid over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker may or may not be cleaved after exposure to enzymes, reactive oxygen species, other chemicals or enzymes that can be present in cells (e.g., macrophages), cellular compartments (e.g., endosomes and lysosomes), inflamed areas of the body (e g., inflamed joints), tissues or body compartments.
  • a stable linker may be cleaved by unknown mechanisms.
  • a stable linker may or may not be cleaved in vivo but remains an active agent after peptide conjugation.
  • a peptide and drug complexed, conjugated, or fused via a linker can be described with the formula Peptide-A-B-C-Drug, wherein the linker is A-B-C.
  • A can be a stable amide link such as that formed by reacting an amine on the peptide with a linker containing a tetrafluorophenyl (TFP) ester or an NHS ester.
  • A can also be a stable carbamate linker such as that formed by reacting an amine on the peptide with an imidazole carbamate active intermediate formed by reaction of CDI with a hydroxyl on the linker.
  • A can also be a stable secondary amine linkage such as that formed by reductive alkylation of the amine on the peptide with an aldehyde or ketone group on the linker.
  • A can also be a stable thioether linker formed using a maleimide or bromoacetamide in the linker with a thiol in the peptide, a triazole linker, a stable oxime linker, or a oxacarboline linker.
  • B can be (-CH2-) X - or a short PEG (-CH2CH2O-) X (x is 0-20) or other spacers or no spacer.
  • C can be an amide bond formed with an amine or a carboxylic acid on the drug, a thioether formed between a maleimide on the linker and a sulfhydroyl on the drug, a secondary or tertiary amine, a carbamate, or other stable bonds.
  • Any linker chemistry described in “Current ADC Linker Chemistry,” Jain et al., Pharm Res, 2015 DOI 10. 1007/sl 1095-015-1657-7 can be used.
  • the resulting peptide complexes can be administered to a human or animal subcutaneously, intravenously, orally, or injected directly into a joint to treat disease.
  • the peptide is not specifically cleaved from the detectable agent or active agent via a targeted mechanism.
  • the peptide can be degraded by mechanisms such as catabolism, releasing a drug that is modified or not modified form its native form (Antibody-Drug Conjugates: Design, Formulation, and Physicochemical Stability, Singh, Luisi, and Pak. Pharm Res (2015) 32:3541- 3571).
  • the peptide drug conjugate exerts its pharmacological activity while still intact, or while partially or fully degraded, metabolized, or catabolized.
  • peptide complexes can have cleavable linkers.
  • a peptide and drug can be complexed, conjugated, or fused via a linker and can be described with the formula Peptide-A-B-C-Drug, wherein the linker is A-B-C.
  • A can be a stable amide link such as that formed by reacting an amine on the peptide with a linker containing a tetrafluorophenyl (TFP) ester or an NHS ester.
  • TFP tetrafluorophenyl
  • A can also be a stable carbamate linker that is formed by an amine reaction on the peptide with an imidazole carbamate active intermediate formed by reaction of CDI with a hydroxyl on the linker.
  • A can also be a stable secondary amine linkage such as that formed by reductive alkylation of the amine on the peptide with an aldehyde or ketone group on the linker.
  • A can also be a stable thioether linker formed using a maleimide or bromoacetamide in the linker with a thiol in the peptide, a triazole linker, a stable oxime linker, or an oxacarboline linker.
  • B can be (-CH2-) X - or a short PEG (-CEECFLO-jx (x is 0-20) or other spacers or no spacer.
  • C can be an ester bond to the hydroxyl or carboxylic acid on the drug, or a carbonate, hydrazone, or acylhydrazone, designed for hydrolytic cleavage.
  • the hydrolytic rate of cleavage can be varied by varying the local environment around the bond, including carbon length (-CH2-)x, steric hindrance (including adjacent side groups such as methyl, ethyl, cyclic), hydrophilicity or hydrophobicity.
  • peptide complexes can have a linear or cyclic ester linkage, which can include or do not include side chains such as methyl or ethyl groups.
  • a linear ester linkage can be more susceptible to cleavage (such as by hydrolysis, an enzyme such as esterase, or other chemical reaction) than a cyclic ester due to steric hindrance or hydrophobicity /hydrophilicity effects.
  • side chains such as methyl or ethyl groups on the linear ester linkage can optionally make the linkage less susceptible to cleavage than without the side chains.
  • hydrolysis rate can be affected by local pH, such as lower pH in certain compartments of the body or of the cell such as endosomes and lysosomes or diseased tissues.
  • C can also be a pH sensitive group such as a hydrazone or oxime linkage.
  • C can be a disulfide bond designed to be released by reduction, such as by glutathione.
  • (or A-B-C) can be a peptidic linkage design for cleavabe by enzymes.
  • a self-immolating group such as pABC can be included to cause release of a free unmodified drug upon cleavage
  • the linker can be cleaved by enzymes such as esterases, matrix metalloproteinases, cathepsins such as cathepsin B, glucuronidases, a protease, or thrombin.
  • the bond designed for cleavage can be at A, rather than C, and C can be a stable bond or a cleavable bond.
  • An alternative design can be to have stable linkers (such as amide or carbamate) at A and C and have a cleavable linker in B, such as a disulfide bond.
  • the rate of reduction can be modulated by local effects such as steric hindrance from methyl or ethyl groups or modulating hydrophobicity /hydrophilicity.
  • peptide complexes can have an ester carbonyl linkage, a long hydrocarbon linker, or carbamate linker, each of which can include hydrophilic groups, such as alcohols, acids, or ethers, or include a hydrocarbon side chain or other moiety that tunes the rate of cleavage.
  • the rate of hydrolysis can be faster with hydrophilic groups, such as alcohols, acids, or ethers, near an ester carbonyl.
  • hydrophobic groups present as side chains or as a longer hydrocarbon linker can slow the cleavage rate of the ester.
  • cleavage of a carbamate group can also be tuned by hindrance, hydrophobicity, and the like.
  • using a less labile linking group, such as a carbamate rather than an ester can slow the cleavage rate of the linker.
  • a linker can comprise a triazole group, such as any one of the heterocyclic compounds with molecular formula C2H3N3, having a five-membered ring of two carbon atoms and three nitrogen atoms, optionally with a hydrogen atom bonded to N at any position in the ring, such as: r example, a 1, 2, 3-Triazole
  • linkers include linear or non-cyclic linkers such as: ach n is independently 0 to about 1,000; 1 to about 1,000; 0 to about 500; 1 to about 500; 0 to about 250; 1 to about 250; 0 to about 200; 1 to about 200; 0 to about 150; 1 to about 150; 0 to about 100; 1 to about 100; 0 to about 50; 1 to about 50; 0 to about 40; 1 to about 40; 0 to about 30; 1 to about 30; 0 to about 25; 1 to about 25; 0 to about 20; 1 to about 20; 0 to about 15; 1 to about 15; 0 to about 10; 1 to about 10; 0 to about 5; or 1 to about 5.
  • ach n is independently 0 to about 1,000; 1 to about 1,000; 0 to about 500; 1 to about 500; 0 to about 250; 1 to about 250; 0 to about 200; 1 to about 200; 0 to about 150; 1 to about 150; 0 to about 100; 1 to about 100; 0 to about 50;
  • each n is independently 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50.
  • m is 1 to about 1,000; 1 to about 500; 1 to about 250; 1 to about 200; 1 to about 150; 1 to about 100; 1 to about 50; 1 to about 40; 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; or 1 to about 5.
  • m is 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50, or any linker as disclosed in Jain, N., Pharm Res. 32(11): 3526-40 (2015) or Ducry, L., Antibody Drug Conjugates (2013).
  • a linker can comprise a cyclic group, such as an organic nonaromatic or aromatic ring, optionally with 3-10 carbons in the ring, optionally built from a carboxylic acid, optionally be used to form a carbamate linkage.
  • a carbamate linkage can be more resistant to cleavage, such as by hydrolysis, enzymes such as esterases, or other chemical reactions, than an ester linkage.
  • a linker can comprise a cyclic carboxylic acid, for example a cyclic di carboxylic acid, for example one of the following groups: 1,4-cyclohexane dicarboxylic acid, 1,2-cyclohexane dicarboxylic acid, or 1,3 -cyclohexane dicarboxylic acid, 1,1- cyclopentanediacetic acid, stereoisomer thereof.
  • the linker can comprise one of the following groups. used to form an ester linkage.
  • a cyclic ester linkage can be more sterically resistant to cleavage, such as by hydrolysis by water, enzymes such as esterases, or other chemical reactions, than a noncyclic or linear ester linkage.
  • a linker can comprise an aromatic dicarboxylic acid, for example terephthalic acid, isophthalic acid, phthalic acid substituted analog thereof.
  • a linker can comprise a natural or non-natural amino acid, for example
  • a linker can comprise alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Vai); or any plurality or combination thereof.
  • the non-natural amino acid can comprise one or more functional groups, e.g., alkene or alkyne, that can
  • a substituted analog or a stereoisomer is a structural analog of a compound disclosed herein, for which one or more hydrogen atoms of the compound can be substituted by one or more groups of halo (e.g., Cl, F, Br), alkyl (e.g., methyl, ethyl, propyl), alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocycloalkyl, or any combination thereof.
  • a stereoisomer can be an enantiomer, a diastereomer, a cis or trans stereoisomer, a E or Z stereoisomer, or a R or S stereoisomer.
  • linear linkers include;
  • each nl, or n2 or m is independently 0 to about 1,000; 1 to about 1,000; 0 to about 500; 1 to about 500; 0 to about 250; 1 to about 250; 0 to about 200; 1 to about 200; 0 to about 150; 1 to about 150; 0 to about 100; 1 to about 100; 0 to about 50; 1 to about 50; 0 to about 40; 1 to about 40; 0 to about 30; 1 to about 30; 0 to about 25; 1 to about 25; 0 to about 20; 1 to about 20; 0 to about 15; 1 to about 15; 0 to about 10; 1 to about 10; 0 to about 5; or 1 to about 5.
  • each n is independently 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50.
  • m is 1 to about 1,000; 1 to about 500; 1 to about 250; 1 to about 200; 1 to about 150; 1 to about 100; 1 to about 50; 1 to about 40; 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; or 1 to about 5.
  • m is 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50.
  • the linker can comprise a linear dicarboxylic acid, e.g., one of the following groups: succinic acid, 2,3 -dimethylsuccinic acid, glutaric acid, adipic acid, 2,5 -dimethyladipic acid, or a substituted analog or a stereoisomer thereof.
  • the linker can be used to form a carbamate linkage.
  • the carbamate linkage can be more resistant to cleavage, such as by hydrolysis, enzymes such as esterases, or other chemical reactions, than an ester linkage
  • the linker can be used to form a linear ester linkage.
  • the linear ester linkage can be more susceptible to cleavage, such as by hydrolysis, enzymes such as esterases, or other chemical reactions, than a cyclic ester or carbamate linkage.
  • Side chains such as methyl groups on the linear ester linkage can optionally make the linkage less susceptible to cleavage than without the side chains.
  • a linker can be a succinic linker, and a targeting agent (e.g., a single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter) or other active agent or detectable agent can be attached to a peptide via an ester bond or an amide bond with two methylene carbons in between.
  • a linker can be any linker
  • a nucleotide e.g., a single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure
  • an antisense RNA complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or Ul Adapter
  • an active agent, or a detectable agent can be attached to a peptide using any one or more of the linkers shown below in TABLE 11.
  • a peptide, an additional active agent, or a detectable agent can be attached to a
  • an active agent is attached to a linker wherein a nucleophilic functional group (e.g., a hydroxyl group) of the active agent molecule acts as the nucleophile and replaces a leaving group on the linker moiety, thereby attaching it to the linker.
  • a nucleophilic functional group e.g., a hydroxyl group
  • an active agent is attached to a linker wherein a nucleophilic functional group (e.g., thiol group, amine group, etc.) of the linker replaces a leaving group on the active agent, thereby attaching it to the linker.
  • a nucleophilic functional group e.g., thiol group, amine group, etc.
  • Such leaving group may be a primary alcohol to form a thioether bond, thereby attaching it to the linker.
  • a primary alcohol can be converted into a leaving group such as a mesylate, a tosylate, or a nosylate in order to accelerate the nucleophilic substitution reaction.
  • the peptide complexes of the present disclosure can comprise a cargo molecule (e g., a target-binding agent, an active agent, a therapeutic agent, or a detectable agent), a linker, and/or a peptide of the present disclosure.
  • a general connectivity between these three components can be active agent-linker-peptide, such that the linker is attached to both the active agent and the peptide.
  • the peptide is attached to a linker via an amide bond.
  • Amide bonds can be relatively stable (e.g., in vivo) compared to other bonds described herein, such as esters, carbonates, etc.
  • the amide bond between the peptide and the linker may thus provide advantageous properties due to its in vivo stability of the active agent is sought to be cleaved from a peptide-active agent-conjugate without the linker being attached to the active agent after such in vivo cleavage.
  • an active agent is attached to the linker-peptide moiety via linkages such as ester, carbonate, carbamate, etc., wherein the peptide or active agent is attached to the linker via an amide bond. This can allow for selective cleavage of the active agent-linker bond (as opposed to the linker-peptide bond) allowing the active agent to be released without a linker moiety attached to it after cleavage.
  • the linker can be a cleavable or a stable linker.
  • the use of a cleavable linker permits release of the conjugated moiety (e.g., a nucleotide targeting agent, a therapeutic agent, a detectable agent, or a combination thereof) from the peptide, e.g., after targeting to the target tissue or cell or subcellular compartment or after endocytosis.
  • the linker is enzyme cleavable, e.g., a valine-citrulline linker that can be cleavable by cathepsin, or an ester linker that can be cleavable by esterase.
  • the linker contains a selfimmolating portion.
  • the linker includes one or more cleavage sites for a specific protease, such as a cleavage site for matrix metalloproteases (MMPs), thrombin, urokinase-type plasminogen activator, or cathepsin (e.g., cathepsin K).
  • MMPs matrix metalloproteases
  • thrombin thrombin
  • urokinase-type plasminogen activator or cathepsin (e.g., cathepsin K).
  • a peptide-active agent complexes of the present disclosure can comprise one or more, about two or more, about three or more, about five or more, about ten or more, or about 15 or more amino acids that can form an amino acid sequence cleavable by an enzyme.
  • enzymes can include proteinases.
  • a peptide-active agent complex can comprise an amino acid sequence that can be cleaved by a Cathepsin, a Chymotrypsin, an Elastase, a Subtilisin, a Thrombin I, or a Urokinas, or any combination thereof.
  • the cleavable linker can be cleaved, dissociated, or broken by other mechanisms, such as via pH, reduction, or hydrolysis. Hydrolysis can occur directly due to water reaction, or be facilitated by an enzyme, or be facilitated by presence of other chemical species.
  • a hydrolytically labile linker, (amongst other cleavable linkers described herein) can be advantageous in terms of releasing active agents from the peptide.
  • an active agent in a conjugate form with the peptide may not be active, but upon release from the conjugate after targeting to the target tissue or cell or subcellular compartment, the active agent is active.
  • the cleaved active agent may retain the chemical structure of the active agent before cleavage or may be modified.
  • a stable linker may optionally not cleave in buffer over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker may optionally not cleave in body fluids such as plasma or synovial fluid over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker optionally may cleave, such as after exposure to enzymes, reactive oxygen species, other chemicals or enzymes that may be present in cells (such as macrophages), cellular compartments (such as endosomes and lysosomes), inflamed areas of the body (such as inflamed joints), or tissues or body compartments.
  • a stable linker may optionally not cleave in vivo but present an active agent that is still active when conjugated to, linked to, or fused to the peptide.
  • the rate of hydrolysis of the linker can be tuned.
  • the rate of hydrolysis of linkers with unhindered esters may be faster compared to the hydrolysis of linkers with bulky groups next an ester carbonyl.
  • a bulky group can be a methyl group, an ethyl group, a phenyl group, a ring, or an isopropyl group, or any group that provides steric bulk.
  • the rate of hydrolysis can be faster with hydrophilic groups, such as alcohols, acids, or ethers, or near an ester carbonyl.
  • hydrophobic groups present as side chains or by having a longer hydrocarbon linker can slow cleavage of the ester.
  • cleavage of a carbamate group can also be tuned by hindrance, hydrophobicity, and the like.
  • using a less labile linker, such as a carbamate rather than an ester can slow the cleavage rate of the linker.
  • the steric bulk can be provided by the drug itself, such as by ketorolac when conjugated via its carboxylic acid.
  • the rate of hydrolysis of the linker can be tuned according to the residency time of the conjugate in the target tissue or cell or subcellular compartment, according to how quickly the peptide accumulates in the target tissue or cell or subcellular compartment, or according to the desired time frame for exposure to the active agent in the target tissue or cell or subcellular compartment. For example, when a peptide is cleared from the target tissue or cell or subcellular compartment relatively quickly, the linker can be tuned to rapidly hydrolyze. In contrast, for example, when a peptide has a longer residence time in the target tissue or cell or subcellular compartment, a slower hydrolysis rate can allow for extended delivery of an active agent.
  • a linker may be tuned for different cleavage rates for similar cleavage rates in different species.
  • a linker can be a succinic linker, and a drug can be attached to a peptide via an ester bond or an amide bond with two methylene carbons in between.
  • a linker can be any linker with both a hydroxyl group and a carboxylic acid, such as hydroxy hexanoic acid or lactic acid.
  • the linker can release the active agent in an unmodified form.
  • the active agent can be released with chemical modification.
  • catabolism can release the active agent still linked to parts of the linker and/or peptide.
  • peptide complexes have stable linkers.
  • a peptide of the disclosure can be expressed recombinantly or chemically synthesized.
  • the peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide linkage or a carbamate linkage.
  • the peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide bond using standard l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or dicylcohexylcarbodiimide (DCC) based chemistry or thionyl chloride or phosphorous chloride-based bioconjugation chemistries.
  • EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • DCC dicylcohexylcarbodiimide
  • a stable linker may or may not be cleaved in buffer over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker may or may not be cleaved in a cellular, intracellular, or paracellular space over extended periods of time (e.g., hours, days, or weeks).
  • a stable linker may or may not be cleaved after exposure to enzymes, reactive oxygen species, other chemicals or enzymes that can be present in cells (e.g., cancer cells, pancreatic cells, liver cells, colon cells, smooth muscle cells, ovarian cells, breast cells, lung cells, brain cells, skin cells, ocular cells, blood cells, lymph cells, immune system cells, reproductive cells, reproductive organ cells, prostate cells, fibroblasts, kidney cells, adenocarcinoma cells, glioma stem cells, or tumor cells,), cellular, paracellular, or intracellular compartments (e.g., cytosols, nuclei, or nanolumen), cells, tissues or body compartments.
  • a stable linker may be cleaved by unknown mechanisms.
  • a stable linker may or may not be cleaved in viv
  • the linker can be a stable linker or a cleavable linker.
  • the stable linker can slowly release the conjugated moiety by an exchange of the conjugated moiety onto the free thiols on serum albumin.
  • the use of a cleavable linker can permit release of the conjugated moiety (e.g., a therapeutic agent) from the peptide, e.g., after administration to a subject in need thereof.
  • the use of a cleavable linker can permit the release of the conjugated therapeutic from the peptide.
  • the linker is enzyme cleavable, e.g., a valine-citrulline linker.
  • the linker contains a self-immolating portion.
  • the linker includes one or more cleavage sites for a specific protease, such as a cleavage site for matrix metalloproteases (MMPs), thrombin, cathepsins, peptidases, or beta-glucuronidase.
  • MMPs matrix metalloproteases
  • thrombin cleavage site for matrix metalloproteases
  • cathepsins peptidases
  • beta-glucuronidase beta-glucuronidase
  • the linker is cleavable by other mechanisms, such as via pH, reduction, or hydrolysis.
  • the rate of hydrolysis or reduction of the linker can be fine-tuned or modified depending on an application.
  • the rate of hydrolysis of linkers with unhindered esters can be faster compared to the hydrolysis of linkers with bulky groups next to an ester carbonyl.
  • a bulky group can be a methyl group, an ethyl group, a phenyl group, a ring, or an isopropyl group, or any group that provides steric bulk.
  • the steric bulk can be provided by the drug itself, such as by ketorolac when conjugated, linked, or fused via its carboxylic acid.
  • the rate of hydrolysis of the linker can be tuned according to the residency time of the conjugate or fusion in the target location. For example, when a peptide is cleared from a tumor, or the brain, relatively quickly, the linker can be tuned to rapidly hydrolyze. When a peptide has a longer residence time in the target location, a slower hydrolysis rate would allow for extended delivery of an active agent. “Programmed hydrolysis in designing paclitaxel prodrug for nanocarrier assembly” Sci Rep 2015, 5, 12023 Fu et al., provides an example of modified hydrolysis rates. [0325] The rate of hydrolysis of the linker (e.g., a linker of a peptide conjugate) can be measured.
  • Such measurements can include determining free active agent in plasma, serum, or synovial fluid, or other fluid or tissue of a subject in vivo and/or by incubating a linker or a peptide conjugate comprising a linker of the present disclosure with a buffer (e.g., PBS) or blood plasma from a subject (e.g., rat plasma, human plasma, etc.) or synovial fluid or other fluids or tissues ex vivo.
  • the methods for measuring hydrolysis rates can include taking samples during incubation or after administration and determine free active agent, free peptide, or any other parameter indicate of hydrolysis, including also measuring total peptide, total active agent, or conjugated active agent-peptide.
  • a hydrolysis half-life of a linker can differ depending on the plasma or fluid or species or other conditions used to determine such half-life. This can be due to certain enzymes or other compounds present in a certain plasma (e.g., rat plasma). For instance, different fluids (such as plasma or synovial fluid) can contain different amounts of enzymes such as esterases, and these levels of these compounds can also vary depending on species (such as rat versus human) as well as disease state (such as normal versus arthritic).
  • the complexes of the present disclosure can be described as having a modular structure comprising various components, wherein each of the components (e.g., peptide, linker, active agent and/or detectable agent) can be selected dependently or independently of any other component.
  • each of the components e.g., peptide, linker, active agent and/or detectable agent
  • a targeted degradation peptide complex for targeted degradation of a target peptide can comprise a cell-penetrating peptide of the present disclosure (e.g., those having the amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 254), a targetbinding peptide (e.g., any one of SEQ ID NO: 293 - SEQ ID NO: 298 or SEQ ID NO: 364 - SEQ ID NO: 407), a linker (e.g., any linker described in TABLE 10 or TABLE 11, SEQ ID NO: 255 - SEQ ID NO: 292, SEQ ID NO: 458 - SEQ ID NO: 485, or otherwise described), and a ubiquitin ligase-binding agent (e.g., thalidomide, pomalidomide, lenalidomide, methyl bestatin, bestatin, nutlin-3, or VHL ligand 1).
  • the linker for example, can be selected and/or modified to achieve a certain active agent release (e.g., a certain release rate) via a certain mechanism (e.g., via hydrolysis, such as enzyme and/or pH-dependent hydrolysis) at the target site (e.g., in the brain) and/or to minimize systemic exposure to the active agent.
  • a certain active agent release e.g., a certain release rate
  • a certain mechanism e.g., via hydrolysis, such as enzyme and/or pH-dependent hydrolysis
  • any one or more of the components of the conjugate can be modified and/or altered to achieve certain in vivo properties of the conjugate, e.g., pharmacokinetic (e.g., clearance time, bioavailability, uptake and retention in various organs) and/or pharmacodynamic (e.g., target engagement) properties.
  • pharmacokinetic e.g., clearance time, bioavailability, uptake and retention in various organs
  • pharmacodynamic e.
  • the conjugates of the present disclosure can be modulated to prevent, treat, and/or diagnose a variety of diseases and conditions, while reducing side effects (e.g., side effects that occur if such active agents are administered alone (i.e., not conjugated to a peptide)).
  • side effects e.g., side effects that occur if such active agents are administered alone (i.e., not conjugated to a peptide)
  • the non-natural amino acid can comprise one or more functional groups, e.g., alkene or alkyne, that can be used as functional handles.
  • a multiple bond of such functional groups can be used to add one or more molecules to the conjugate.
  • the one or more molecules can be added using various synthetic strategies, some of which may include addition and/or substitution chemistries.
  • an addition reaction using a multiple bond can comprise the use of hydrobromic acid, wherein the bromine can act as a leaving group and thus be substituted with various moieties, e.g., active agents, detectable agents, agents that can modify or alter the pharmacokinetic (e.g., plasma half-life, retention and/or uptake in central nervous system (CNS) or elsewhere) and/or pharmacodynamic (e.g., hydrolysis rate such as an enzymatic hydrolysis rate) properties of the conjugate.
  • various moieties e.g., active agents, detectable agents, agents that can modify or alter the pharmacokinetic (e.g., plasma half-life, retention and/or uptake in central nervous system (CNS) or elsewhere) and/or pharmacodynamic (e.g., hydrolysis rate such as an enzymatic hydrolysis rate) properties of the conjugate.
  • a conjugate as described herein comprises one or more nonnatural amino acid and/or one or more linkers.
  • Such one or more non-natural amino acid and/or one or more linkers can comprise one or more functional groups, e g., alkene or alkyne (e.g., non-terminal alkenes and alkynes), which can be used as functional handles.
  • a multiple bond of such functional groups can be used to add one or more molecules to the conjugate.
  • the one or more molecules can be added using various synthetic strategies, some of which may include addition and/or substitution chemistries, cycloadditions, etc.
  • an addition reaction using a multiple bond can comprise the use of hydrogen bromide (e g., via hydrohalogenation reactions), wherein the bromide substituent, once attached, can act as a leaving group and thus be substituted with various moieties comprising a nucleophilic functional groups, e.g., active agents, detectable agents, agents.
  • a multiple bond can be used as a functional handle in a cycloaddition reaction.
  • Cycloaddition reactions can comprise 1,3-dipolar cycloadditions, [2+2] -cycloadditions (e.g., photocatalyzed), Di els- Alder reactions, Huisgen cycloadditions, nitrone -olefin cycloadditions, etc.
  • Such cycloaddition reactions can be used to attached various functional groups, functional moieties, active agents, detectable agents, and so forth to the conjugate.
  • a 1,3-dipolar cycloaddition reaction can be used to attach a molecule to a conjugate, wherein the molecule comprises a 1,3-dipole that can react with, e.g., an alkyne to form a 5 -membered ring, thereby attaching said molecule to the conjugate.
  • attaching such molecule or agent can modify or alter the pharmacokinetic (e g., plasma halflife, retention and/or uptake in CNS or biodistribution) and/or pharmacodynamic (e.g., hydrolysis rate such as an enzymatic hydrolysis rate) properties of the conjugate.
  • Attaching such molecule or agent can also alter (e.g., increase) the depot effect of a conjugate, or provide functionality for in vivo tracking, e.g., using fluorescence or other types of detectable agents.
  • a conjugate of the present disclosure can comprise a linker comprising one or more of the following groups: substituted analog or a stereoisomer thereof, wherein each nl and n2 is independently a value from 1 to 10.
  • a group can be used as a handle to attach one or more molecules to a conjugate, e.g., to alter the pharmacokinetic (e.g., plasma half-life, retention and/or uptake in central nervous system (CNS) or elsewhere) and/or pharmacodyna via nucleophilic or electrophilic addition followed by nucleophilic substitution mic properties of the conjugate.
  • Functionalization of such a group can occur using one or more multiple bonds (e.g., double bonds, triple bonds, etc.) of the groups.
  • Such functionalization can comprise addition and/or substitution chemistries.
  • a functional group of a linker such as a double bond
  • a single bond e.g., via an addition reaction such as a nucleophilic addition reaction
  • one or both of the carbon atoms of the newly formed single bond can have a leaving group (e.g., a bromine) attached to them.
  • a leaving group e.g., via nucleophilic substitution reaction
  • a specific molecule e.g., an active agent, a detectable agent, etc.
  • a multiple bond can be used as a functional handle in a cycloaddition reaction.
  • Cycloaddition reactions can comprise 1,3 -dipolar cycloadditions, [2+2]-cycloadditions (e.g., photocatalyzed), Diels-Alder reactions, Huisgen cycloadditions, nitrone-olefin cycloadditions, etc.
  • Such cycloaddition reactions can be used to attached various functional groups, functional moieties, active agents, detectable agents, and so forth to the conjugate.
  • a 1,3 -dipolar cycloaddition reaction can be used to attach a molecule to a conjugate, wherein the molecule comprises a 1,3-dipole that can react with, e.g., an alkyne to form a 5-membered ring, thereby attaching said molecule (e.g., active agent, detectable agent, etc.) to the conjugate.
  • molecules may be attached to a conjugate to e.g., modulate the half-life, increase the depot effect, or provide new functionality of a conjugate, such as fluorescence for tracking.
  • a peptide of the present disclosure can be stable in various biological or physiological conditions, such as the pH or reducing environments inside a cell, in the cytosol, in a cell nucleus, lysosome, or endosome.
  • a cell-penetrating peptide of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 may be conjugated to a peptide (e.g., a cystine-dense peptide) that can exhibit resistance to reducing agents, heat, denaturation, proteases, oxidative conditions, or acidic conditions, for example a cell -penetrating peptide complex of SEQ ID NO: 299 - SEQ ID NO: 308 or SEQ ID NO: 312 - SEQ ID NO: 321.
  • a peptide e.g., a cystine-dense peptide
  • biologic molecules such as peptides and proteins
  • GI tract can contain a region of low pH (e.g.
  • protease-rich environment that can degrade peptides and proteins.
  • Proteolytic activity in other areas of the body such as the mouth, eye, lung, intranasal cavity, joint, skin, vaginal tract, mucous membranes, and serum, can also be an obstacle to the delivery of functionally active peptides and polypeptides.
  • the half-life of peptides in serum can be very short, in part due to proteases, such that the peptide can be degraded too quickly to have a lasting therapeutic effect when administering reasonable dosing regimens.
  • proteolytic activity in cellular compartments such as lysosomes and reduction activity in lysosomes and the cytosol can degrade peptides and proteins such that they may be unable to provide a therapeutic function on intracellular targets. Therefore, peptides that are resistant to reducing agents, proteases, and low pH may be able to provide enhanced therapeutic effects or enhance the therapeutic efficacy of co-formulated or conjugated, linked, or fused active agents in vivo.
  • oral delivery of drugs can be desirable in order to target certain areas of the body (e.g., disease in the GI tract such as colon cancer, irritable bowel disorder, infections, metabolic disorders, and constipation) despite the obstacles to the delivery of functionally active peptides and polypeptides presented by this method of administration.
  • oral delivery of drugs can increase compliance by providing a dosage form that is more convenient for patients to take as compared to parenteral delivery.
  • Oral delivery can be useful in treatment regimens that have a large therapeutic window. Therefore, peptides that are resistant to reducing agents, proteases, and low pH can allow for oral delivery of peptides without nullifying their therapeutic function.
  • Cell-penetrating peptides or peptide complexes of this disclosure can contain one or more cysteines, which can participate in disulfide bridges that can be integral to preserving the folded state of the peptide. Exposure of peptides to biological environments with reducing agents can result in unfolding of the peptide and loss of functionality and bioactivity.
  • glutathione GSH
  • a peptide can become reduced during trafficking of a peptide across the gastrointestinal epithelium after oral administration. A peptide can become reduced upon exposure to various parts of the GI tract.
  • the GI tract can be a reducing environment, which can inhibit the ability of therapeutic molecules with disulfide bonds to have optimal therapeutic efficacy, due to reduction of the disulfide bonds.
  • a peptide can also be reduced upon entry into a cell, such as after internalization by endosomes or lysosomes or into the cytosol, or other cellular compartments. Reduction of the disulfide bonds and unfolding of the peptide can lead to loss of functionality or affect key pharmacokinetic parameters such as bioavailability, peak plasma concentration, bioactivity, and half-life. Reduction of the disulfide bonds can also lead to loss of functionality due to increased susceptibility of the peptide to subsequent degradation by proteases, resulting in rapid loss of intact peptide after administration.
  • a peptide that is resistant to reduction can remain intact and can impart a functional activity for a longer period of time in various compartments of the body and in cells, as compared to a peptide that is more readily reduced.
  • the peptides of this disclosure can be analyzed for the characteristic of resistance to reducing agents to identify stable peptides.
  • the peptides of this disclosure can remain intact after being exposed to different molarities of reducing agents such as 0.00001 M - 0.0001 M, 0.0001 M - 0.001 M, 0.001 M - 0.01 M, 0.01 M - 0.05 M, 0.05 M - 0.1 M, or 0.1 M to 0.2 M for 15 minutes or more.
  • the reducing agent used to determine peptide stability can be dithiothreitol (DTT), Tris(2- carboxyethyl)phosphine HC1 (TCEP), 2-Mercaptoethanol, (reduced) glutathione (GSH), or any combination thereof.
  • DTT dithiothreitol
  • TCEP Tris(2- carboxyethyl)phosphine HC1
  • GSH 2-Mercaptoethanol
  • GSH 2-Mercaptoethanol
  • GSH reduced glutathione
  • peptides are completely resistant to GSH reducing conditions and are partially resistant to degradation in DTT reducing conditions.
  • peptides described herein can withstand or
  • proteases also referred to as peptidases or proteinases, are enzymes that can degrade peptides and proteins by breaking bonds between adjacent amino acids. Families of proteases with specificity for targeting specific amino acids can include serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, and asparagine proteases. Additionally, metalloproteases, matrix metalloproteases, elastase, carboxypeptidases, Cytochrome P450 enzymes, and cathepsins can also digest peptides and proteins.
  • Proteases can be present at high concentration in blood, in mucous membranes, lungs, skin, the GI tract, the mouth, nose, eye, and in compartments of the cell. Misregulation of proteases can also be present in various diseases such as rheumatoid arthritis and other immune disorders. Degradation by proteases can reduce bioavailability, biodistribution, half-life, and bioactivity of therapeutic molecules such that they are unable to perform their therapeutic function. In some embodiments, peptides that are resistant to proteases can better provide therapeutic activity at reasonably tolerated concentrations in vivo.
  • peptides of this disclosure can resist degradation by any class of protease.
  • peptides of this disclosure resist degradation by pepsin (which can be found in the stomach), trypsin (which can be found in the duodenum), serum proteases, or any combination thereof.
  • the proteases used to determine peptide stability can be pepsin, trypsin, chymotrypsin, or any combination thereof.
  • peptides of this disclosure can resist degradation by lung proteases (e.g., serine, cysteinyl, and aspartyl proteases, metalloproteases, neutrophil elastase, alpha-1 antitrypsin, secretory leucoprotease inhibitor, and elafin), or any combination thereof.
  • lung proteases e.g., serine, cysteinyl, and aspartyl proteases, metalloproteases, neutrophil elastase, alpha-1 antitrypsin, secretory leucoprotease inhibitor, and elafin
  • Peptide Stability in Acidic Conditions Peptides of this disclosure can be administered in biological environments that are acidic. For example, after oral administration, peptides can experience acidic environmental conditions in the gastric fluids of the stomach and gastrointestinal (GI) tract.
  • the pH of the stomach can range from about 1-4 and the pH of the GI tract ranges from acidic to normal physiological pH descending from the upper GI tract to the colon.
  • the vagina, late endosomes, and lysosomes can also have acidic pH values, such as less than pH 7. These acidic conditions can lead to denaturation of peptides and proteins into unfolded states.
  • the peptides of this disclosure can resist denaturation and degradation in acidic conditions and in buffers, which simulate acidic conditions.
  • peptides of this disclosure can resist denaturation or degradation in buffer with a pH less than 1, a pH less than 2, a pH less than 3, a pH less than 4, a pH less than 5, a pH less than 6, a pH less than 7, or a pH less than 8.
  • peptides of this disclosure remain intact at a pH of 1-3.
  • At least 5%- 10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to a buffer with a pH less than 1, a pH less than 2, a pH less than 3, a pH less than 4, a pH less than 5, a pH less than 6, a pH less than 7, or a pH less than 8.
  • the peptides of this disclosure can be resistant to denaturation or degradation in simulated gastric fluid (pH 1-2).
  • low pH solutions such as simulated gastric fluid can be used to determine peptide stability.
  • the peptides described herein are resistant to degradation in vivo, in the serum of a subject, or inside a cell.
  • the peptides are stable at physiological pH ranges, such as about pH 7, about pH 7.5, between about pH 5 to 7.5, between about 6.5 to 7.5, between about pH 5 to 8, or between about pH 5 to 7.
  • the peptides described herein are stable in acidic conditions, such as less than or equal to about pH 5, less than or equal to about pH 3, or within a range from about 3 to about 5.
  • the peptides are stable in conditions of an endosome or lysosome, or inside a nucleus.
  • Peptide Stability at High Temperatures Peptides of this disclosure can be administered in biological environments with high temperatures. For example, after oral administration, peptides can experience high temperatures in the body. Body temperature can range from 36°C to 40°C. High temperatures can lead to denaturation of peptides and proteins into unfolded states. Unfolding of peptides and proteins can lead to increased susceptibility to subsequent digestion by other enzymes as well as loss of biological activity of the peptide. In some embodiments, a peptide of this disclosure can remain intact at temperatures from 25°C to 100°C. High temperatures can lead to faster degradation of peptides. Stability at a higher temperature can allow for storage of the peptide in tropical environments or areas where access to refrigeration is limited.
  • 5%-100% of the peptide can remain intact after exposure to 25°C for 6 months to 5 years. 5%-100% of a peptide can remain intact after exposure to 70°C for 15 minutes to 1 hour. 5%-100% of a peptide can remain intact after exposure to 100°C for 15 minutes to 1 hour. In other embodiments, at least 5%-10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to 25°C for at least 6 months to 5 years.
  • At least 5%- 10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%- 60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to 70°C for 15 minutes to 1 hour.
  • Various expression vector/host systems can be utilized for the recombinant expression of peptides described herein.
  • Non-limiting examples of such systems include microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a nucleic acid sequence encoding peptides or peptide fusion proteins/ chimeric proteins described herein, yeast transformed with recombinant yeast expression vectors containing the aforementioned nucleic acid sequence, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the aforementioned nucleic acid sequence, plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)), or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the aforementioned nucleic acid sequence, or animal cell systems infected with
  • a host cell can be adapted to express one or more peptides described herein.
  • the host cells can be prokaryotic, eukaryotic, or insect cells.
  • host cells are capable of modulating the expression of the inserted sequences or modifying and processing the gene or protein product in the specific fashion desired. For example, expression from certain promoters can be elevated in the presence of certain inducers (e.g., zinc and cadmium ions for metallothionine promoters).
  • inducers e.g., zinc and cadmium ions for metallothionine promoters.
  • modifications e.g., phosphorylation
  • processing e.g., cleavage
  • Host cells can have characteristic and specific mechanisms for the post-translational processing and modification of a peptide.
  • the host cells used to express the peptides secrete minimal amounts of proteolytic enzymes.
  • organisms can be treated prior to purification to preserve and/or release a target polypeptide.
  • the cells are fixed using a fixing agent.
  • the cells are lysed.
  • the cellular material can be treated in a manner that does not disrupt a significant proportion of cells, but which removes proteins from the surface of the cellular material, and/or from the interstices between cells.
  • cellular material can be soaked in a liquid buffer, or, in the case of plant material, can be subjected to a vacuum, in order to remove proteins located in the intercellular spaces and/or in the plant cell wall.
  • proteins can be extracted from the microorganism culture medium.
  • the peptides can be packed in inclusion bodies. The inclusion bodies can further be separated from the cellular components in the medium. In some embodiments, the cells are not disrupted.
  • a cellular or viral peptide that is presented by a cell or virus can be used for the attachment and/or purification of intact cells or viral particles.
  • peptides can also be synthesized in a cell-free system prior to extraction using a variety of known techniques employed in protein and peptide synthesis.
  • a host cell produces a peptide that has an attachment point for a drug.
  • An attachment point could comprise a lysine residue, an N-terminus, a cysteine residue, a cysteine disulfide bond, a glutamic acid or aspartic acid residue, a C-terminus, or a non-natural amino acid.
  • the peptide could also be produced synthetically, such as by solid-phase peptide synthesis, or solution-phase peptide synthesis. Peptide synthesis can be performed by fluorenylmethyloxycarbonyl (Fmoc) chemistry or by butyloxycarbonyl (Boc) chemistry.
  • the peptide could be folded (formation of disulfide bonds) during synthesis or after synthesis or both.
  • Peptide fragments could be produced synthetically or recombinantly. Peptide fragments can be then be joined together enzymatically or synthetically.
  • the peptides of the present disclosure can be prepared by conventional solid phase chemical synthesis techniques, for example according to the Fmoc solid phase peptide synthesis method (“Fmoc solid phase peptide synthesis, a practical approach,” edited by W. C. Chan and P. D. White, Oxford University Press, 2000).
  • the peptides of the present disclosure can be prepared by solution phase peptide synthesis.
  • the peptides can be folded by one cystine at a time, two at a time, three at a time, four at a time, or all at a time, either in solution or on the resin.
  • the peptide made by manufactured as two or more fragments that are subsequently ligated or joined together.
  • sequences from a peptide library generated from computational design can be synthesized using expression vectors or solid phase or solution phase peptide synthesis methods.
  • cell-penetrating peptides or cell-penetrating peptide fusions can be cloned into a secreted, soluble protein production/expression vector and purified, as per Bannesayake et al., 2011.
  • Purification methods include, but are not limited to, affinity purification columns, ion exchange (cation and/or anion columns), reversed-phase, hydrophobic interaction, and size exchange columns.
  • SDS-PAGE followed by Coomassie staining and reverse phase HPLC can be used to analyze a sample of the purified protein. Protein concentrations were determined by UV spectral absorption and/or amino acid analysis.
  • the peptides of this disclosure can be more stable during manufacturing.
  • peptides of this disclosure can be more stable during recombinant expression and purification, resulting in lower rates of degradation by proteases that are present in the manufacturing process, a higher purity of peptide, a higher yield of peptide, or any combination thereof.
  • the peptides can also be more stable to degradation at high temperatures and low temperatures during manufacturing, storage, and distribution.
  • peptides of this disclosure can be stable at 25°C.
  • peptides of this disclosure can be stable at 70°C or higher than 70°C.
  • peptides of this disclosure can be stable at 100°C or higher than 100°C.
  • Improved cell -penetrating peptides may be identified using various screening methods to select for peptides with cell-penetrating properties.
  • a peptide library e g., a library comprising amino acid substitution variants of a parent peptide
  • a single peptide may be screened at a time using cell -based assays to quantify cytosolic or nuclear access.
  • Such cell-based assays can utilize interactions between a tag protein (e.g., a SNAP -tag, a Halo-tag protein, a split GFP, or a biotin binding protein) and a small molecule substrate (e.g., a BG-GLA-NHS SNAP substrate, a chloroalkane substrate, or a biotin molecule) to identify peptides able to penetrate a cellular layer (e.g., a cell membrane or a nuclear membrane).
  • a tag protein e.g., a SNAP -tag, a Halo-tag protein, a split GFP, or a biotin binding protein
  • a small molecule substrate e.g., a BG-GLA-NHS SNAP substrate, a chloroalkane substrate, or a biotin molecule
  • the labeled peptide library can be incubated with cells expressing the tag protein in a cellular location of interest (e.g., in the cytosol or in the nucleus). Labeled peptides that are able to penetrate the cellular layer and reach the location of interest can bind to the expressed tag protein. Peptides that penetrated the cellular layer can be identified in a number of ways. For example, peptides may be identified by adding small molecule substrate labeled with a fluorescent molecule to the cells.
  • Cells with high concentrations of available tag protein, indicative of low peptide pentration, will bind higher levels of fluorescently labeled substrate, and cells with lower concentrations of available tag protein, indicative of higher peptide penetration, will bind lower levels of fluorescently labeled substrate.
  • Cells can be fluorescently imaged to quantify cellpenetration, where cells contacted with high cell -penetrating peptides have lower fluorescence and cells contacted with low cell-penetrating peptides have higher fluorescence.
  • peptides can be identified by purifying the tag protein (e.g., by immunoprecipitation) along with bound peptides, and the peptides can be identified by mass spectrometry. Identified peptides can penetrate cell membranes.
  • SNAP penetration assay uses many reagents that are commonly referred to herein in a shorthand manner to aid in understanding.
  • SNAP-tag protein is also referred to as “SNAP-tag”.
  • the SNAP-tag can be expressed in a variety of eukaryotic cells (e.g., NIH3T3, HEK293, HeLa, CHO, COS cells and the like).
  • the SNAP-tag is introduced into cells via transfection of a plasmid vector such as for SNAP-tag protein (e.g., pSNAPf) or H2B- tagged SNAP-tag protein (e.g., pSNAPf-H2B) resulting in a cell line expressing the SNAP-tag (e.g., commonly referred to as “NI3T3 pSNAPf’, “HEK293 pSNAPf’, “HeLa pSNAPf’, generically as “pSNAPf cells” or “pSNAPf-H2B cells”, and the like).
  • a plasmid vector such as for SNAP-tag protein (e.g., pSNAPf) or H2B- tagged SNAP-tag protein (e.g., pSNAPf-H2B) resulting in a cell line expressing the SNAP-tag (e.g., commonly referred to as “NI3T3 pSNAPf’,
  • the BG-substrate (BG- GLA-NHS) as used herein is a reactive benzylguanine reagent chemical species used in a chemical reaction that reacts with a test reagent such as a peptide to generate a benzylguanine- containing product, for example a BG-peptide as described below.
  • BG-substrates may be used as a reactive moiety, and one or more reactive benzylguanine reagent chemical agents (e.g., such agents with various properties, fluorescence, radiolabel, for imaging, other measurable aspects) may be created and used to “tag” a test peptide, for example, for use in assessing cell penetration.
  • reactive benzylguanine reagent chemical agents e.g., such agents with various properties, fluorescence, radiolabel, for imaging, other measurable aspects
  • SNAP substrate-tagged peptide or peptide that has been reacted with or “tagged” with a reactive benzylguanine reagent (e.g., BG- GLA-NHS)
  • BG-peptide reactive benzylguanine reagent
  • BG-peptide is typically a test peptide used with cells expressing the SNAP-tag to assess e.g., cytosolic and nuclear cell penetration of the BG-peptide in the SNAP assay.
  • BG-GLA- NHS reactive BG-substrate
  • BG-GLA-OH non-reactive form
  • a “SNAP substrate-tagged fluorophore”, or fluorophore that has been reacted with or “tagged” with a benzylguanine reagent are also referred to as a “BG-fluorophore”.
  • An exemplary BG- fluorophore is the “SNAP-Cell TMR-Star” reagent from New England Biolabs. The BG- fluorophore can diffuse across the cell membrane to bind any unoccupied SNAP -tag in the cell, resulting in fluorescence, and the fluorescence of the cells can be visualized after washing away any free BG-fluorophore that has not bound to unoccupied SNAP -tag.
  • a decrease in fluorescence relative to the PBS control indicates that the SNAP -tag is bound to the BG-peptide (rather than the BG-fluorophore), indicating that the BG-peptide has penetrated the cell.
  • the level of fluorescence is in inverse relationship, wherein adding a cell penetrant BG-fluorophore and then washing away unbound BG- fluorophore, and then detecting fluorescence, the absence of which, relative to the negative control (PBS), is indicative of the SNAP -tag covalently bound to the BG-peptide.
  • SNAP substrate is a general term that can describe a variety of substrates that are used in the SNAPPA as an enzymatic substrate for the SNAP -tag.
  • SNAP substrate can be used to describe whether a reactive benzylguanine- containing reagent (such as a BG-peptide or BG-fluorophore) will react to the SNAP -tag protein in the SNAP assay, serving as a substrate to the SNAP -tag itself. Consequently, it is understood that a “SNAP substrate” is non-limiting and can be an enzymatic substrate for the SNAP -tag in the SNAP assay.
  • each of the BG-GLA-NHS (BG- substrate), and its hydrolyzed form BG-GLA-OH, BG-peptide, and BG-fluorophore can serve as a substrate (SNAP substrate) for the SNAP-tag in the SNAP assay.
  • a cell-based assay to identify cell-penetrating peptides may comprise contacting a library of BG-peptide variants to cells expressing a SNAP-tag in either the cytosol or the nucleus.
  • BG-peptides capable of penetrating the cell membrane may covalently bind to SNAP-tag expressed in the cytosol
  • BG-peptides capable of penetrating the cell membrane and the nuclear envelope may covalently bind to SNAP-tag expressed in the nucleus.
  • peptides that enter the cell may be subsequently identified by contacting the cells with a BG-fluorophore that can permeate the cell membrane and nuclear envelope and bind to any remaining SNAP-tag that is not bound to a BG-peptide.
  • peptides that enter the cell may be identified by identifying which cells have a lower level of fluorescence, indicating a higher level of cell penetration.
  • peptides that enter the cell may be identified by immunoprecipitating the SNAP-tag and any bound BG-peptides and performing mass spectrometry to identify the bound peptides. In some embodiments, these screening methods may be performed as high throughput assays, for example, in microarrays.
  • a valuable aspect of a SNAP penetration assay is that can measure delivery of a peptide to the cytosol or nucleus, rather than solely measuring its uptake into the cell.
  • Other assays exist that merely measure general uptake into the cell, such as by labeling a peptide with a fluorophore and measuring total cellular fluorescence.
  • general uptake assays may measure peptide that is only present in the endosome or lysosome, rather than distinguish peptide that has entered the cytosol.
  • Peptides may be taken up by endocytosis but remain trapped and never enter the cytosol which makes general uptake assays not very effective in identifying cytosolic or nuclear penetrating peptides, as described in Depray, et al (Bioconjug Chem, 2019 Apr 17; 30(4): 1006-1027).
  • the SNAP penetration assay can assess delivery to the cytosol or nucleus. Additionally, SNAP penetration assay may avoid possible false positives from surface-bound peptides, which may result from using fluorophore labels combined with alternative methods such as fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the SNAP penetration assay uses SNAP -tag comprising a 20 kDa mutant of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (SNAP-tag) which reacts specifically and rapidly with benzylguanine (BG) derivatives (BG-substrate) (e.g., such BG-substrate conjugated to a peptide (BG-peptide) as a synthetic probe, test peptide, or substrate for the SNAP-tag in the SNAPPA assay, and similarly such BG-substrate can be attached to a small molecule that is known to enter cells for use as a positive control in SNAPPA), leading to irreversible covalent labeling of the SNAP-tag with the synthetic probe, as described in Keppler, et al (Nat.
  • Unoccupied SNAP-tag binding cites may be detected by subsequently incubating with a BG-fluorophore to covalently bind SNAP -tags not bound by BG-peptides.
  • the labeling reaction is measured by detecting fluorescence, the absence of which, relative to the negative control, is indicative of the SNAP-tag covalently bound to the BG- peptide.
  • the small size of the SNAP-tag and relatively neutral charge may be favorable for the SNAP penetration assay. Formation of a covalent bond during the SNAP reaction may facilitate recovery and identification of bound species, for example using mass spectrometry.
  • Such BG derivatives are also referred to herein as BG-substrate or SNAP substrates.
  • SNAP substrates are chemically inert towards other proteins, avoiding nonspecific labeling in cellular applications.
  • a BG-substrate is conjugated to the peptide being tested for cell penetration, creating a BG-peptide.
  • SNAP-tag is expressed in the cell itself, either without organelle targeting or with nuclear localization by way of histone H2B fusion.
  • BG-GLA resembles the enzyme’s native substrate, an alkylated guanine nucleotide.
  • the enzyme can cause the BG-GLA to be covalently bound to the SNAP protein.
  • species covalently attached to the BG- GLA for example via NHS reaction, are also covalently attached to the SNAP -tag, blocking it from further reactions.
  • the BG-peptide for example generated by mixing BG-GLA-NHS with peptides of interest resulting in the BG-GLA to covalently bond with solvent-accessible primary amines in the peptide, can be added to the cell culture media, and if it is cell penetrant, it can enter the cytosol and become bound to a SNAP protein there.
  • the SNAP -tag in the cells may become saturated, and subsequently-dosed fluorescently-labeled BG-GLA, which can penetrate cells due to its small size, may have no SNAP protein with which to react. If, however, the BG-GLA is attached to a peptide without cell penetration capabilities, then the attached BG-GLA may not come into contact with the SNAP protein upon cell media incubation. This leaves the SNAP protein un-saturated and may react with subsequently-dosed BG- fluorophore, rendering the cells fluorescent.
  • plasmid vector for SNAP -tag pSNAPf
  • H2B-tagged SNAP -tag pSNAPf-H2B
  • Plasmids can be sequence validated and purified by Endofree Maxiprep before transfection into cell lines, such as NIH3T3, HeLa or HEK293 cells using Lipofectamine 2000. Following a period of recovery, selection for positive transfectants can be initiated using geneticin over a period of 2-3 weeks. The pool of stable transfectants can be expanded and banked in liquid nitrogen.
  • SNAP conjugation and purification of SNAP-CDPs can be produced and purified, as described in Banechayake et al., Nucleic Acids Res. 2011 Nov; 39(21): el43.
  • lyophilized peptides can be resuspended in PBS to an approximate 1 mg/mL concentration.
  • SNAP substrate can be attached to reactive amine groups on peptides via NHS-ester reaction.
  • Benzylguanine-NHS (BG-GLA-NHS) can be obtained from a commercial vendor and resuspended at 10 pg/uL in anhydrous DMSO.
  • BG-GLA-NHS One molar equivalent of BG-GLA-NHS can be added to resuspended peptide and reaction can be allowed to proceed with stirring at room temperature for one hour. Samples of starting material and crude reaction mixture can be analyzed by HPLC/MS to assess reaction progress. For peptides with only one reactive amine group, a total of three molar equivalents can be added over the course of three hours. For peptides with multiple reactive amine groups, reactions can be halted when the presence of multi-SNAP substrate-tagged species becomes apparent. A molar reaction or reaction time can be chosen to target a single SNAP substrate to be conjugated per peptide. The reaction can occur with the N-terminus of the peptide. The reaction can occur with one or more lysine residues of a peptide. Additionally, if precipitation is noted, addition of BG- GLA-NHS can be halted.
  • BG-GLA- NHS e.g., BG-GLA-OH
  • SEC size exclusion chromatography
  • BG-peptides can be separated and eluted over 1.5 column volumes. Fractions with notable 280 nm/214 nm/200 nm absorbance can be pooled and concentrated through a 3 kDa molecular weight cut off spin filter. Individual BG-peptides may elute in distinct volumes, often around 1 column volume. BG-peptides can be verified using HPLC/MS BG-peptide concentration can be assessed using the bicinchoninic acid assay.
  • BG-peptides and controls can be diluted in standard cell growth media (DMEM +10% FBS +pen/strep). 2 pM hydrolyzed BG-GLA-NHS (hydrolyzed to BG-GLA-OH) can be used as a positive control for cell penetration, while PBS can serve as a negative control. An additional mock conjugation/purification of BG-GLA-NHS can also serve as a negative control for purification.
  • pSNAPf and pSNAPf-H2B cell lines can be plated on collagen-coated 96-well plates, 20,000 cells/well, and can be allowed to recover in growth media overnight.
  • the plates can be rinsed with PBS and incubated with diluted BG-peptides and controls in growth media for 2 hours at 37° C.
  • the cells can then be incubated with labelling media containing 600 nM SNAP-Cell TMR-Star and 1 pg/mL Hoescht 33342 in growth media for 15 minutes at 37° C.
  • the labelling media may contain a SNAP substrate conjugated to a rhodamine dye, an Alexa fluor, an Atto dye, a cyanine dye, or a fluorescein dye.
  • the labelling media can be removed, and cells can be incubated with fresh growth media for 30 minutes at 37° C as a washout phase for excess SNAP-Cell TMR-Star dye.
  • the media can be replaced with phenol red-free OptiMEM for imaging.
  • Imaging can be performed on a Molecular Devices ImageXpress Nano, utilizing DAPI and Texas Red filters.
  • cells can be segmented using ImageXpress software to generate counts for nuclei and average cytoplasmic or nuclear intensity of SNAP-Cell TMR- Star.
  • the average intensity can then be normalized using the positive control of 2 pM hydrolyzed BG-GLA-NHS (BG-GLA-OH) as 100% cell penetration and PBS as 0% penetration to determine normalized SNAP -tag occupancy.
  • the degree of cell penetration is inversely proportional to the level of fluorescence in the cells.
  • pSNAPf cells can be preincubated with a variety of endocytosis inhibitors to assess mechanisms responsible for uptake of BG-peptides.
  • Macropinocytosis can be inhibited using 50 pM ethylisopropyl amiloride (EIP A), which blocks Na+/H+ exchangers and prevents formation of macropinosomes.
  • EIP A ethylisopropyl amiloride
  • Clathrin-mediated endocytosis can be inhibited using either 20 pM nocodazole, which inhibits microtubule polymerization, 3 pM cytochalasin D, which interferes with actin polymerization, or 80 pM dynasore, an inhibitor of dynamin GTPase.
  • Endosomal acidification or lysosomal maturation can be inhibited using 50 nM bafilomycin A, which inhibits the vacuolar ATPase complex, or 50 pM chloroquine, which diffuses into endosomes as a weak base.
  • pSNAPf cells can be preincubated with the specified concentration of inhibitor in growth media for 1 hour prior to SNAPPA, which can be performed as described herein.
  • cells expressing a labeled SNAP -tag in either the cytoplasm or the nucleus can be cultured in a microplate format.
  • the labeled SNAP -tag is labeled with a green fluorescent protein, a yellow florescent protein, a red fluorescent protein, a blue fluorescent protein, or a cyan fluorescent protein.
  • Candidate cell-penetrating peptides can be tagged with SNAP substrate and purified using the method described herein. Each BG-candidate peptide can be added to the cells expressing labeled SNAP -tag in a well of the microplate.
  • Peptides capable of cell penetration may cross the cell membrane and covalently bind to the labeled SNAP -tag expressed in the cytoplasm or the nucleus. Following incubation, excess BG-candidate peptides that did not enter the cells can be washed off. Cells can be pooled and lysed, and labeled SNAP- tag that reacted with cell penetrant BG-peptides can be purified by immunoprecipitation via the label. Candidate peptides that entered the cell can be immunoprecipitated with the SNAP -tag. Cell -penetrating peptides can be identified by mass spectrometry.
  • SDPR protease resistance testing can be used with sequencing-grade enzymes, including Trypsin and Chymotrypsin. Trypsin and trypsin inhibitor can be used for HPLC analysis.
  • screens for cell penetration can be assessed by Illumina sequencing.
  • Illumina sequencing involves collecting cell pellets (1.5E6, 3 technical replicates) resupended in 50 pL Terra Direct PCRMix (from Clontech) and amplified for 16 cycles using the original cloning primers. Up to four aliquots can be diluted 16- fold into 60 pL Phusion DNA Polymerase reactions and amplified using distinct Illumina primers, containing adaptor sequences for flow cell adherence. Forward primers can include a 6 bp barcode for multiplexing.
  • Illumina HiSeq 2500 in rapid mode can be used to run samples, which Bowtie2 software can be used for mapping, and Excel (Microsoft) and MATLAB (MathWorks) used for data analysis.
  • a pharmaceutical composition of the disclosure can be a combination of any peptide as described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, antioxidants, solubilizers, buffers, osmolytes, salts, surfactants, amino acids, encapsulating agents, bulking agents, cryoprotectants, and/or excipients.
  • the pharmaceutical composition facilitates administration of a peptide described herein to an organism.
  • the pharmaceutical composition comprises factors that extend half-life of the peptide and/or help the peptide to penetrate the target cells.
  • compositions can be administered in therapeutically-effective amounts as pharmaceutical compositions by various forms and routes including, for example, intravenous, subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, optic, nasal, oral, sublingual, inhalation, dermal, intrathecal, intravitreal, intratumoral, intranasal, and topical administration.
  • a pharmaceutical composition can be administered in a local or systemic manner, for example, via injection of the peptide described herein directly into an organ, optionally in a depot.
  • Parenteral injections can be formulated for bolus injection or continuous infusion.
  • the pharmaceutical compositions can be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of a peptide described herein in water-soluble form. Suspensions of peptide-antibody complexes described herein can be prepared as oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension can also contain suitable stabilizers or agents which increase the solubility and/or reduce the aggregation of such peptide-antibody complexes described herein to allow for the preparation of highly concentrated solutions.
  • the cell-penetrating peptides described herein can be used to increase cell penetration of a cargo molecule (e g., a therapeutic agent) and deliver the cargo molecule to a cellular compartment.
  • a cargo molecule e g., a therapeutic agent
  • protein transfection agents, direct cytosolic expression of the peptide, or electrophoration of the peptide can be used to increase cell penetration.
  • other excipients can be formulated with a peptide in order to increase the cell penetration of the peptide, such as those approaches described in “Protein and Peptide Drug Delivery: Oral Approaches” Indian J. Pharm. Sci., Shaji and Patole, v70(3) 269-277, 2008. Any combination of these formulations or approaches can be used to increase cell penetration of a peptide as described herein.
  • the peptide described herein can be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a purified peptide is administered intravenously.
  • a peptide described herein can be administered to a subject, home, target, migrate to, or be directed to cancerous cell, a tumor, or a cell with dysregulated HIPPO pathway.
  • a peptide can be conjugated to, linked to, or fused to another peptide that provides a targeting function to a specific target cell type in the central nervous system or across the blood brain barrier.
  • a peptide of the disclosure can be applied directly to an organ, or an organ tissue or cells, such as brain or brain tissue or cells, during a surgical procedure.
  • the recombinant peptide described herein can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, and ointments.
  • Such pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
  • therapeutically effective amounts of a peptide described herein can be administered in pharmaceutical compositions to a subject suffering from a condition that affects the immune system.
  • the subject is a mammal such as a human or a primate.
  • a therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.
  • a peptide is cloned into a viral or non-viral expression vector.
  • Such expression vector can be packaged in a viral particle, a virion, or a non-viral carrier or delivery mechanism, which is administered to patients in the form of gene therapy.
  • patient cells are extracted and modified to express a cell-penetrating peptide ex vivo before the modified cells are returned back to the patient in the form of a cell-based therapy, such that the modified cells will express the peptide once transplanted back in the patient.
  • compositions can be formulated using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulation can be modified depending upon the route of administration chosen.
  • Pharmaceutical compositions comprising a peptide described herein can be manufactured, for example, by expressing the peptide in a recombinant system, purifying the peptide, lyophilizing the peptide, mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or compression processes.
  • the pharmaceutical compositions can include at least one pharmaceutically acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically acceptable salt form.
  • Methods for the preparation of peptide described herein comprising the compounds described herein include formulating peptide described herein with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid composition.
  • Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.
  • Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.
  • compositions can also include permeation or absorption enhancers (Aungst et al. AAPS J. 14(l):10-8. (2012) and Moroz et al. Adv Drug Deliv Rev 101: 108-21. (2016)).
  • Permeation enhancers can facilitate uptake of molecules from the GI tract into systemic circulation.
  • Permeation enhancers can include salts of medium chain fatty acids, sodium caprate, sodium caprylate, N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC), N-(5- chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), hydrophilic aromatic alcohols such as phenoxyethanol, benzyl alcohol, and phenyl alcohol, chitosan, alkyl glycosides, dodecyl-2-N,N- dimethylamino propionate (DDAIPP), chelators of divalent cations including EDTA, EGTA, and citric acid, sodium alkyl sulfate, sodium salicylate, lecithin-based, or bile salt-derived agents such as deoxy cholates,
  • SNAC N-(8-[2-hydroxybenzoyl]amino)caprylic acid
  • 5-CNAC N-(5- chlorosalicyloyl)-8-a
  • compositions can also include protease inhibitors including soybean trypsin inhibitor, aprotinin, sodium glycocholate, camostat mesilate, vacitracin, or cyclopentadecalactone, Use of Peptides in Treatments
  • the cell-penetrating peptides described herein may be conjugated to, linked to, or fused with a therapeutic agent for use in a method of treatment.
  • the cell-penetrating peptides or cell-penetrating peptide complexes may be used to deliver a therapeutic agent that may otherwise be excluded from a cellular compartment across a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) an into a target cell. Delivery of the therapeutic agent using the cell -penetrating peptides described herein may increase access to a target (e.g., a target protein or nucleotide sequence) by the therapeutic agent.
  • a target e.g., a target protein or nucleotide sequence
  • the method includes administering an effective amount of a cell -penetrating peptide-therapeutic agent conjugate as described herein to a subject in need thereof.
  • a cell-penetrating peptide-therapeutic agent conjugate may be administered to the subject in need thereof to treat a disease or a condition in the subject.
  • a therapeutic agent that may be delivered into a cell by a cellpenetrating peptide of the present disclosure may include an antibody, an antibody fragment, an Fc domain, a single chain Fv, an intrabody, or a nanobody.
  • the therapeutic agent may be a cystine-dense peptide, an affibody, a B-hairpin, an avimer, an adnectin, a stapled peptide, a nannofittin, a kunitz domain, a fynomer, or a bicyclic peptide.
  • the therapeutic agent may be an anti -cancer agent and may be delivered into a cancer cell.
  • the therapeutic agent may be a peptide (e.g., a cystine- dense peptide), a transcription factor, an RNA, a Cas enzyme or other CRISPR component, an immunomodulating agent, or a hormone.
  • an active agent such as a cystine-dense peptide, that binds to a transcription factor (such as a forkhead box class O transcription factor (FOXO), Nuclear factor E2 -related factor 2 (NRF2), runt-related transcription factor 1 (RUNX1), methyl CpG binding protein 2 transcription factor (MECP2), maturity onset diabetes of the young transcription factor (MODY), forkhead box P3 (FOXP3), p53, p65, signal transducer and activator of transcription (STAT), homeodomain transcription factor (HOX), or SOX9 transcription factor) may be conjugated to a cell -penetrating peptide and delivered to a nucleus of a cell to alter the expression level of a target gene.
  • a transcription factor such
  • a cellpenetrating peptide is conjugated to NRF2 to carry NRF2 to the nucleus, facilitate transcription of antioxi dative genes, and mediate recovery from acetaminophen-induced hepatotoxicity.
  • a cell-penetrating peptide is conjugated to RUNX1 to carry RUNX1 to the nucleus, regulate gene expression, and mediate cartilage deposition.
  • a cell-penetrating peptide complex of the present disclosure may target AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, -catenin, IKK, TEAD, NF-KB, PKC, AP-1, Jun, Fos, REL, a transcription factor, Ras, Rho, Ran, Rab, Arf, androgen receptor, ikaros, aiolos, nuclear receptors, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-3, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CS
  • ADP- Ribosylation Factor (Arf) GTPases are part of the Ras superfamily used in regulation of vesicular cellular trafficking, lipid modification, cytokinesis and cell adhesion.
  • Arf GTPases are tightly regulated by specific guanine nucleotide exchange factors (GEFs) with a conserved Sec7 domain, and GTPase-activating proteins (GAPs) with a conserved zinc finger domain. Like all Ras superfamily members, the GEFs switch on/activate Arfs while the GAPS switch off/inactivate them.
  • GEFs guanine nucleotide exchange factors
  • GAPs GTPase-activating proteins
  • ADP-Ribosylation Factor (Arf) GTPases, guanine nucleotide exchange factors (GEFs) with a conserved Sec7 domain, and GTPase-activating proteins (GAPs), using the cellpenetrating peptide of the invention can be used in cancer therapy, including metastatic phases of cancer.
  • one or more CRISPR components e g., guide RNA, a tracrRNA, a crRNA, or a Cas nuclease
  • an immunomodulating agent may be conjugated to a cell-penetrating peptide and delivered into a cell to modulate or inhibit an immune response.
  • a therapeutic agent for treatment of neurodegeneration may be conjugated to a cell-penetrating peptide and delivered across the blood brain barrier to treat a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS)).
  • a neurodegenerative disorder e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS)
  • the therapeutic agent may facilitate degradation of excess proteins in a target cell upon delivery to the target cell by the cell-penetrating peptide.
  • a method of treating a condition may comprise targeted degradation of a target molecule, such as a target protein.
  • the condition is associated with increased activity, increased expression, or novel activity brought on by gene amplification, missense mutation, nonsense mutation, aberrant splicing, or fusion protein brought on by chromosomal translocation of the target molecule.
  • a method of treatment may comprise administering to a subject a cell-penetrating peptide complex comprising a cellpenetrating peptide linked or fused to a cargo molecule comprising a target-binding peptide (e.g., a target-binding CDP) and a ubiquitin ligase-binding molecule.
  • a target-binding peptide e.g., a target-binding CDP
  • a method of treatment can also comprise administering to a subject a cell-penetrating peptide complex comprising a cellpenetrating peptide linked or fused to a cargo molecule comprising a target-binding peptide (e.g., a target-binding CDP) and a molecule that binds any part of the ubiquitin-proteasome system (UPS) or cytosolic protein degradation machinery, such as the 26S proteosome, or any component thereof such as the 19S ubiquitin recognition proteins Rpn-13 and Rpn-10. Binding to the UPS could be through an inhibitor, such as KDT-11, which inhibits Rpn-13.
  • a target-binding peptide e.g., a target-binding CDP
  • UPS ubiquitin-proteasome system
  • cytosolic protein degradation machinery such as the 26S proteosome, or any component thereof such as the 19S ubiquitin recognition proteins Rpn-13 and Rpn-10. Binding
  • the peptide complex may cross a cellular layer of a cell (e.g., a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, or bone marrow cell) to access the target and the ubiquitin ligase in a cellular compartment (e.g., the cytosol or the nucleus) or intercellular compartment (e.g., a nanolumen, intercellular space, or paracellular space).
  • a cell e.g., a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, or bone marrow cell
  • a cellular compartment e.g., the cytosol or the nucleus
  • intercellular compartment e.g., a nanolumen, intercellular space, or paracellular space.
  • the cargo molecule may bind to the target and the ubiquitin ligase, forming a ternary complex.
  • the ubiquitin ligase may ubiquitinate the target, labeling it for proteasomal degradation, thereby treating the condition.
  • a method of treatment may comprise administering to a subject a cell -penetrating peptide complex comprising a cell-penetrating peptide linked or fused to a cargo molecule comprising a PROTAC, a molecular glue, a targetbinding peptide, or other cargos that cause targeted protein degradation.
  • the cargo molecule may comprise an IMiD, a Boc3Arg, an adamantyl group, or a carborane.
  • the peptide complex may cross a cellular layer of a cell, such as by entering the cytosol, and bind, label, target, traffic, or otherwise direct a protein or other target for degradation, such as by the ubiquitin-proteosome system.
  • a method of treating a condition may comprise inhibition of a target molecule.
  • the condition is associated with increased activity, increased expression, or novel activity brought on by gene amplification, missense mutation, nonsense mutation, aberrant splicing, or fusion protein brought on by chromosomal translocation of the target molecule, such as a target protein.
  • Inhibiting the target molecule may comprise inhibiting a conformational change, inhibiting an enzymatic activity, inhibiting ligand binding, inhibiting a protein-protein interaction, or inhibiting a protein-nucleic acid interaction of the target molecule.
  • a method of treating a condition may comprise administering to a subject a cell-penetrating peptide complex comprising a cell -penetrating peptide linked or fused to a cargo molecule that binds to and inhibits a target.
  • the cargo molecule may comprise a target-binding peptide (e.g., a target-binding CDP).
  • the peptide complex may cross a cellular layer of a cell (e g , a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, or bone marrow cell) to access the target in a cellular compartment (e g., the cytosol or the nucleus) or intercellular compartment (e g., a nanolumen, intercellular space, or paracellular space).
  • a cellular compartment e g., the cytosol or the nucleus
  • intercellular compartment e g., a nanolumen, intercellular space, or paracellular space.
  • the term “effective amount,” as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. Compositions containing such agents or compounds can be administered for prophylactic, enhancing, and/or therapeutic treatments. An appropriate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study.
  • the methods, compositions, and kits of this disclosure may comprise a method to prevent, treat, arrest, reverse, or ameliorate the symptoms of a condition.
  • the treatment may comprise treating a subject (e.g., an individual, a domestic animal, a wild animal, or a lab animal afflicted with a disease or condition) with a peptide of the disclosure.
  • the disease may be a cancer or tumor.
  • the peptide may contact the tumor or cancerous cells.
  • the subject may be a human.
  • Subjects can be humans; non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
  • a subject can be of any age.
  • Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants, and fetuses in utero.
  • Treatment may be provided to the subject before clinical onset of disease. Treatment may be provided to the subject after clinical onset of disease. Treatment may be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years or more after clinical onset of the disease. Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years or more after clinical onset of disease. Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may also include treating a human in a clinical trial.
  • a treatment can comprise administering to a subject a pharmaceutical composition, such as one or more of the pharmaceutical compositions described throughout the disclosure.
  • a treatment can comprise a once daily dosing.
  • a treatment can comprise delivering a peptide of the disclosure to a subject, either intravenously, subcutaneously, intramuscularly, by inhalation, dermally, topically, by intra-articular injection, orally, sublingually, intrathecally, intravitreally, transdermally, intranasally, via a peritoneal route, directly into a tumor, e.g., injection directly into a tumor, directly into the brain, e.g., via and intracerebral ventricle route, or directly onto a joint, e.g., via topical, intra-articular injection route.
  • a treatment can comprise administering a peptide-active agent conjugate to a subject, either intravenously, subcutaneously, intramuscularly, by inhalation, by intra-articular injection, dermally, topically, orally, intrathecally, intravitreally, transdermally, intransally, parenterally, orally, via a peritoneal route, nasally, sublingually, or directly onto cancerous tissues.
  • compositions comprising any one of the cell-penetrating peptides described herein conjugated to a therapeutic agent (e.g., an anti-cancer agent), or a pharmaceutical composition thereof, can be used in a method of treating a cancer, tumor progression, and/or dysregulated cell growth.
  • a therapeutic agent e.g., an anti-cancer agent
  • Exemplary diseases, disorder, or condition include: multiple myeloma, plastic anemia, myelodysplasia, and related bone marrow failure syndromes, myeloproliferative diseases, acute and chronic myeloid leukemia, malignancies of lymphoid cells, hematologic malignancies, plasma cell disorders, skeletal muscle disorder, myopathy, muscular dystrophy (e.g., Becker muscular dystrophy, Duchenne muscular dystrophy, Emery -Dreifuss muscular dystrophy, Facioscapulohumoeral muscular dystrophy, Myotonia congentia, and myotonic dystrophy), Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS), and chronic obstructive pulmonary disorder.
  • multiple myeloma plastic anemia, myelodysplasia, and related bone marrow failure syndromes
  • myeloproliferative diseases acute and chronic myeloid leukemia, mal
  • compositions/peptides disclosed herein are used to treat dysregulated cell growth, cancer, tumor, and/or metastasis associated with any of the following cell, tissue, or organ types: brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cell, pancreatic, colon, stomach, cervix, breast, endometrial, prostate, testicle, ovarian, skin, head and neck, esophageal, oral tissue, and bone marrow.
  • compositions/peptides disclosed herein are used to treat any of the following: osteosarcoma, hepatocellular carcinoma, malignant mesothelioma, schwannoma, meningioma, renal carcinoma, cholangiocarcinoma, bile duct hamartoma, soft tissue carcinoma, ovarian carcinoma, colonic adenoma, T cell acute lymphoblastic leukaemia, gastrointestinal hyperplasia, fibrosarcoma, pancreatic ductal metaplasia, squamous cell carcinoma, kaposis sarcoma, and HIV-induced non-Hodgkin’s lymphoma.
  • the peptides of this disclosure (e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) conjugated to a therapeutic agent can be used to access and treat these disorders by delivering the therapeutic agent across a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) to a cellular compartment.
  • a cellular layer e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen
  • Delivery of a therapeutic agent using a cell -penetrating peptide of the present disclosure may increase the efficacy of the therapeutic agent by increasing access of the therapeutic agent to intracellular targets (e g., cytosolic proteins or target nucleic acid sequences) relative to the therapeutic agent alone.
  • intracellular targets e g., cytosolic proteins or target nucleic acid sequences
  • the cell penetrating peptides of this disclosure e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, and SEQ ID NO: 195 - SEQ ID NO: 254
  • complexes described herein can deliver a cargo molecule to the cytosol or the nucleus of a cell.
  • the cargo molecule is delivered to the cytosol to achieve a cytosolic concentration of at least 1 nM, at least 5 nM, at least 10 nM, at least 25 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1000 nM, at least 1100 nM, at least 1200 nM, at least 1300 nM, at least 1400 nM, at least 1500 nM, at least 1600 nM, at least 1700 nM, at least 1800 nM, or at least 2000 nM.
  • the cargo molecule is delivered to the cytosol to achieve a cytosolic concentration of from about 1 nM to about 50 nM, from about 10 nM to about 100 nM, from about 50 nm to about 100 nm, from about 50 nm to about 200 nm, from about 50 nm to about 500 nm, from about 50 nm to about 700 nm, from about 50 nm to about 1000 nm, from about 50 nm to about 1200 nm, from about 50 nm to about 1500 nm, from about 50 nm to about 2000 nm, from about 100 nm to about 200 nm, from about 100 nm to about 500 nm, from about 100 nm to about 700 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 1200 nm, from about 100 nm to about 1500 nm, from about 100 nm to about 2000 nm, from about 100 nm to about 200
  • the cargo molecule is delivered to the nucleus to achieve a nuclear concentration of at least 1 nM, at least 5 nM, at least 10 nM, at least 25 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1000 nM, at least 1100 nM, at least 1200 nM, at least 1300 nM, at least 1400 nM, at least 1500 nM, at least 1600 nM, at least 1700 nM, at least 1800 nM, or at least 2000 nM.
  • the cargo molecule is delivered to the nucleus to achieve a nuclear concentration of from about 1 nM to about 50 nM, from about 10 nM to about 100 nM, from about 50 nm to about 100 nm, from about 50 nm to about 200 nm, from about 50 nm to about 500 nm, from about 50 nm to about 700 nm, from about 50 nm to about 1000 nm, from about 50 nm to about 1200 nm, from about 50 nm to about 1500 nm, from about 50 nm to about 2000 nm, from about 100 nm to about 200 nm, from about 100 nm to about 500 nm, from about 100 nm to about 700 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 1200 nm, from about 100 nm to about 1500 nm, from about 100 nm to about 2000 nm, from about 500 nm to about 1000 nm,
  • a peptide (e.g., a cell-penetrating peptide) may be linked, conjugated, complexed with, or fused to a nucleotide via various chemistries resulting in peptide oligonucleotide complexes that may form either a cleavable or stable linkage to deliver the oligonucleotide to a cell.
  • a cell-penetrating peptide may delivery an oligonucleotide to the cytoplasm or nucleus, or both, of a cell.
  • nucleotides within the peptide oligonucleotide complex can function within the nucleus of a cell, including gapmers, ASO splice blockers, and U1 adapters. Others function within the cytosol, including siRNA and anti- miRs. Aptamers are unique in that they do not function through hybridization or base paring interactions with nucleic acid targets. Instead, aptamers form secondary structures to bind to proteins or other macromolecules. Aptamers may function wherever the target protein or macromolecule is located.
  • the nucleotide portion of the peptide oligonucleotide complexes described herein may target specific RNAs (e.g., mRNAs or pre-mRNAs) from genes expressed in cancer and other diseases.
  • the nucleotide sequence in the complex may be complementary to any target provided in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6.
  • the nucleotide sequence in the complex may be complementary to the target RNA, or in the case of an aptamer, may bind a target protein or other macromolecule.
  • the a nucleotide sequence may be single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or Ul Adapter.
  • ssDNA, ssRNA double stranded
  • dsDNA double stranded
  • dsRNA double stranded
  • a combination of single and double stranded for example with a mismatched sequence, hairpin or other structure
  • an antisense RNA
  • a target of the nucleotide in a peptide oligonucleotide complex may be a gastrointestinal target, such as a gene with pro-inflammatory, extracellular matrixmodifying, or immune cell recruitment functionality.
  • Peptide oligonucleotide complexes described herein e.g., a peptide oligonucleotide complex comprising a cell-penetrating peptide and a nucleotide that binds a gene target mRNA
  • target gastrointestinal gene targets may be used to treat various gastrointestinal disorders, including inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease.
  • a target of the nucleotide in a peptide oligonucleotide complex may be a cancer target, such as a gene involved in oncogenic signaling, anti-apoptotic genes, pro-inflammatory signaling genes, protein homeostasis genes, developmental regulatory genes, or adapter protein genes that initiate downstream cell growth signaling.
  • a cancer target such as a gene involved in oncogenic signaling, anti-apoptotic genes, pro-inflammatory signaling genes, protein homeostasis genes, developmental regulatory genes, or adapter protein genes that initiate downstream cell growth signaling.
  • HER2 and other RTK e.g., EGFR, ERBB3
  • Knockdown of Grb2 can halt signaling in a way that is difficult to mutationally compensate as Grb2 loss is epistatic to HER2 activity.
  • a pro-inflammatory cytokine may be delivered via an mRNA in a peptide oligonucleotide complex, or an antisense construct targeting an anti-inflammatory signal may be delivered.
  • Peptide oligonucleotide complexes described herein e.g., a peptide oligonucleotide complex comprising a cell-penetrating peptide and a nucleotide that binds a gene target mRNA
  • target cancer gene targets may be used to treat various cancers, including solid tumors.
  • Developmental regulators such as transcription factors involved in early cell fate and pluripotency, and chromatin remodeling enzymes, may be targeted to specifically harm dedifferentiated cells which may be present in advanced tumors and associated with a more mobile and/or mitotic cell state.
  • a peptide oligonucleotide construct targeting a cancer target may treat or prevent cancer by reducing oncogenic signaling, reducing target over-expression, reducing oncogenic antisense activity (e g., miRNAs targeting tumor suppressors), and/or eliminating the source of the oncogenic signaling cascade.
  • gene targets e.g., gastrointestinal, or cancer gene targets
  • an oligonucleotide may target a gene for downregulation.
  • An example of an antagomir that may be complexed with a cell-penetrating peptide to target a gene includes cobomarsen.
  • An example of an aptamer that may be complexed with a cell-penetrating peptide to target a gene includes pegaptanib.
  • Examples of gapmers that may be complexed with a cell-penetrating peptide to target a gene include fomivirsen, mipomersen, inotersen, volanesorsen, tofersen, tominersen, pelacarsen, alicaforsen, apatorsen, and trabedersen.
  • splice blockers that may be complexed with a cell-penetrating peptide to target a gene include nusinersen, eteplirsen, golodirsen, viltolarsen, casimersen, and sepofarsen.
  • An example of a translation blocker that may be complexed with a cell-penetrating peptide to target a gene includes prexigebersen.
  • any targets for the nucleic acid portion of the peptide oligonucleotide complex described herein can be used in conjunction with a U1 adapter to degrade targeted mRNAs.
  • the target recognition (or complementary nucleic acid to the target mRNA) portion directs the peptide oligonucleotide complex to the targeted mRNA selected for degradation, while the U1 portion prevents the addition of polyA to the mRNA resulting in degradation of the targeted mRNA.
  • U1 adapters can comprise any nucleotide sequence complementary to the ssRNA component of the U1 small nuclear ribonucleoprotein (U1 snRNP). In some embodiments, the U1 adapter sequences engage the U1 snRNP near its poly A site.
  • the length of the U1 adapter is 15 to 25 nt in length, or about 20 nt in length. In some embodiments, the U1 adapter is above 40% in its G/C content.
  • Exemplary U1 adapters are shown in TABLE 13, in conjunction with a target nucleic acid “target recognition” portion which comprises a nucleotide sequence is single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, or splice blocker ASO.
  • Exemplary U1 adapters include: UCCCCUGCCAGGUAAGUAU (SEQ ID NO: 488); CCCUGCCAGGUAAGUAU (SEQ ID NO: 489); CUGCCAGGUAAGUAU (SEQ ID NO: 490); UGCCAGGUAAGUAU (SEQ ID NO: 491); GCCAGGUAAGUAU (SEQ ID NO: 492); CCAGGUAAGUAU (SEQ ID NO: 493); CAGGUAAGUAU (SEQ ID NO: 494); and CAGGUAAGUA (SEQ ID NO: 495).
  • peptides described herein can be provided as a kit.
  • peptide complexes described herein can be provided as a kit.
  • a kit comprises amino acids encoding a peptide described herein, a vector, a host organism, and an instruction manual.
  • a kit includes written instructions on the use or administration of the peptides.
  • the peptide sequence was reverse-translated into DNA, synthesized, and cloned in-frame with siderocalin using standard molecular biology techniques (M R. Green, Joseph Sambrook. Molecular Cloning. 2012 Cold Spring Harbor Press). The resulting construct was packaged into a lentivirus, transduced into HEK-293 cells, expanded, isolated by immobilized metal affinity chromatography (IMAC), cleaved with tobacco etch virus protease, and purified to homogeneity by reverse-phase chromatography. Following purification, each peptide was lyophilized and stored frozen.
  • IMAC immobilized metal affinity chromatography
  • This example describes generation of pSNAPf and pSNAPf-H2B cell lines.
  • a plasmid vector for SNAP -tag (pSNAPf) was obtained from New England Biolabs and histone H2B- tagged SNAP -tag (pSNAPf-H2B) was obtained from Addgene. Plasmids were sequence validated and purified by Endofree Maxiprep before transfection into NIH3T3, HeLa or HEK293 cells using Lipofectamine 2000. Following a period of recovery, selection for positive transfectants was initiated using geneticin over a period of 2-3 weeks. The pool of stable transfectants was expanded and banked in liquid nitrogen.
  • This example describes conjugation and purification of BG-peptides and generation of BG-substrate positive control reagents.
  • Peptides were produced and purified as described in EXAMPLE 1.
  • SNAP substrate conjugation lyophilized peptides were resuspended in PBS to an approximate 1 mg/mL concentration.
  • a benzylguanine SNAP substrate (BG-GLA-NHS, also referred to as BG-substrate) was attached to reactive amine groups on peptides viaNHS- ester reaction. Reactive amine groups in peptides may include the N-terminus and may include one or more lysine residues.
  • FIG. 1A schematically illustrates the SNAP substrate tagging reaction between a benzylguanine NHS-ester and a reactive amine group of a peptide to generate a SNAP substrate-tagged peptide (also referred to as BG-peptide).
  • BG- GLA-NHS Benzylguanine-NHS
  • DMSO dimethyl sulfoxide
  • One molar equivalent of BG-GLA-NHS per mole of peptide was added to the resuspended peptide and the reaction was allowed to proceed while stirring at room temperature for one hour. Samples of starting material and crude reaction mixture were analyzed by HPLC/MS to assess reaction progress.
  • BG-GLA-NHS was added as needed. Generally, for peptides with only one reactive amine group, a total of three molar equivalents of BG-GLA-NHS was added over the course of three hours. For peptides with multiple reactive amine groups, reactions were halted when the presence of multi-BG-species became apparent, with of goal of producing a reaction product that was primarily a single SNAP substrate tag per peptide. Additionally, if precipitation was noted, addition of BG-GLA-NHS was halted.
  • BG-peptides were separated and eluted over 1.5 column volumes. Fractions with notable 280 nm/214 nm/200 nm absorbance were pooled and concentrated through a 3 kDa molecular weight cut off spin filter. Individual BG-peptides eluted in distinct volumes, though they generally occurred around 1 column volume. BG-peptides were thus purified and separated from any residual BG-GLA-OH. BG-peptides were verified using HPLC/MS. BG-peptide concentration was then assessed using the bicinchoninic acid assay. [0403] Representative images of RP-HPLC and SEC associated with a typical SNAP substrate conjugation reaction and purification process are shown in FIG.
  • Fhe first image (top) shows multiple elution peaks from an HPLC run on the crude reaction mixture representing SNAP substrate BG-GLA-NHS, hydrolyzed BG-GLA-NHS (hydrolyzed to BG-GLA-OH), unconjugated KR CTX (SEQ ID NO: 71) and KR CTX conjugated to SNAP substrate (BG- KR CTX, BG-SEQ ID NO: 71), showing absorbance at 214 nm. Peaks were validated by mass calculated by mass spectrometry (MS). The crude reaction mixture was then run through a size exclusion column (SEC). The SEC absorbance trace is shown in the second image. Details of the individual SEC peaks are shown in the bottom images. Fractions were pooled and concentrated before verifying identity using HPLC/MS.
  • MS mass spectrometry
  • BG-GLA-NHS Hydrolyzed BG-GLA-NHS, which is BG-GLA-OH, was created by incubating BG- GLA-NHS in PBS, pH 7.4, at room temperature for at least 10 minutes. BG-GLA-OH was used in assays as a positive control.
  • BG-GLA-NHS contains a reactive NHS ester, which can react with amine-containing molecules and with hydroxyl-containing molecules.
  • BG-GLA-NHS can react with amines present in peptides to form a BG-peptide conjugate.
  • BG-GLA-NHS can also react with water, becoming hydrolyzed, yielding BG-GLA-OH, which no longer reacts with amine or hydroxyl groups. If BG-GLA-NHS is added to an aqueous buffer, it will typically hydrolyze to form BG- GLA-OH. If BG-GLA-NHS is added to an amine-containing peptide within an aqueous buffer, some of it may react to form BG -peptide and some of it may hydrolyze to form BG-GLA-OH.
  • SNAPPA SNAP Penetration Assay
  • This example describes a SNAP penetration assay (SNAPPA) to identify cell-penetrating peptides.
  • the SNAP penetration assay is illustrated schematically in FIG. IB.
  • Cell-penetrating peptide candidates were labeled with a benzylguanine SNAP substrate as described in EXAMPLE 4.
  • the BG-peptides were incubated with cells expressing a SNAP-tag-protein, which will covalently bind to BG-peptides that enter the cell and reach the cytosol or nucleus where the SNAP-tag-protein is expressed.
  • Cells expressing the SNAP-tag-protein were generated as described in EXAMPLE 3.
  • BG-peptides and controls were diluted in standard cell growth media (DMEM +10% FBS +pen/strep).
  • FBS means fetal bovine serum.
  • 2 pM hydrolyzed BG-GLA-NHS (hydrolyzed to BG-GLA-OH) was used as a positive control for cell penetration, while PBS served as a negative control.
  • BG-GLA-OH is able to diffuse into cells and reach the cytosol, thus representing a positive control of the level of SNAPPA signal obtained when a molecule is cell penetrant.
  • an additional mock conjugation/purification of BG-GLA-NHS also served as a quality control for purification.
  • the mock reaction was performed by adding 1 molar equivalent (250 pM BG-GLA-NHS) to PBS and incubating at room temperature for 1 hour, which allows hydrolysis of BG-GLA-NHS. This was repeated until the concentration reached 750 pM.
  • This mixture of PBS and BG-GLA-NHS (which is hydrolyzed to BG-GLA-OH) was then run on SEC and equivalent fractions where BG-peptides have been found were pooled and concentrated as would be for BG-peptide production. This served as a control to demonstrate adequate purification of free BG-GLA-OH.
  • pSNAPf and pSNAPf-H2B cell lines were plated on collagen-coated 96-well plates, 20,000 cells/well, and allowed to recover in standard cell growth media overnight. Plates were rinsed with PBS and incubated with diluted BG-peptides and controls in standard cell growth media for 2 hours at 37° C. Peptides capable of cell penetration entered the cytosol and reached the SNAP- tag and then covalently bound to the SNAP-tag-protein expressed in the cells (FIG. IB, left path), while peptides that did not penetrate the cell did not bind to the SNAP-tag-protein (FIG. IB, right path).
  • BG-fluorophore SNAP-Cell TMR-Star dye; or SNAP-dye
  • BG-fluorophore 600nM SNAP-Cell TMR-Star
  • Hoescht 33342 1 pg/mL Hoescht 33342, which labels nuclei and is excited at 377 nm excitation
  • SNAP-tag-protein that was already bound to benzylguanine-peptides (BG-peptide) did not react with the BG-fluorophore.
  • the labelling media was removed, and cells were incubated with fresh growth media for 30 minutes at 37° C as a washout phase for excess SNAP-Cell TMR-Star dye. Excess BG-fluorophore was washed away, and the remaining fluorescence, detected by exciting at 562 nm, was inversely related to the amount of peptide that penetrated the cell membrane. Molecules in cells with unbound SNAP-tag-protein exhibited fluorescence from the reaction between the SNAP-tag-protein and BG-fluorophore.
  • Molecules in cells with SNAP-tag bound to BG-peptide did not exhibit fluorescence as the BG-fluorophore did not bind SNAP-tag-protein and was washed away. The media was replaced one last time with phenol red-free OptiMEM for imaging. Imaging occurred on the Molecular Devices ImageXpress Nano, utilizing DAPI and Texas Red filters. For scoring, cells were segmented using ImageXpress software to generate counts for nuclei and average cytoplasmic or nuclear intensity of SNAP-Cell TMR-Star dye. The average intensity was then normalized using the positive control of 2 pM hydrolyzed BG-GLA-NHS (BG-GLA-OH) as 100% cell penetration and PBS as 0% penetration to determine normalized SNAP-tag-protein occupancy.
  • BG-GLA-OH 2 pM hydrolyzed BG-GLA-NHS
  • BG-peptides comprising cystine-dense peptides after SNAPPA in HeLa cells stably transfected with pSNAPf captured on ImageXpress Nano are shown in FIG. 3.
  • Eight BG-peptides comprising cystine-dense peptides that were tested include: KR_KTxl5.8 (BG-SEQ ID NO: 74), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG-SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59).
  • BG-KR_IpTxa (BG- SEQ ID NO: 59) showed a higher level of cell penetration and BG-KR_KTxl5.8 (BG-SEQ ID NO: 74) showed a lower level of penetration, as shown in FIG. 3.
  • SNAPPA SNAP penetration assay
  • SNAP -tag expressing cells expressing either pSNAPf to quantify cytosolic access or pSNAPf-H2B to quantify nuclear access, were generated as described in EXAMPLE 3.
  • Hydrolyzed SNAP reagent BG-GLA-OH was used as a positive control and set to 100% (1.0 fraction) penetration or protein occupancy.
  • the cell penetrance of BG-peptides comprising cystine-dense peptides was normalized to the BG-GLA-OH positive control.
  • the SNAPPA measured the relative percentage penetrance of various BG-peptides comprising cystine-dense peptides tested in the various cell lines
  • BG-peptides comprising cystine-dense peptides were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously.
  • BG-peptides comprising cystine-dense peptides were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf-H2B, which encodes for nuclear expression of the SNAP -tag.
  • BG-peptides comprising cystine- dense peptides were assayed for nuclear penetration including: KR_KTxl5.8 (BG-SEQ ID NO: 74), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG-SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59).
  • BG-MCa_varl SEQ ID NO: 67
  • BG-KR_HwTx-IV SEQ ID NO: 66
  • BG-KR_IpTxa SEQ ID NO: 59
  • Uptake of BG-KR_Txl5.8 BG- SEQ ID NO: 74
  • BG-KR_KTx2.2 BG-SEQ ID NO: 73
  • BG-Tx677 BG-SEQ ID NO: 93
  • BG-KR CTI BG-SEQ ID NO: 72
  • BG-KR CTX BG-SEQ ID NO: 71
  • This example describes assaying cytosolic and nuclear access of cell-penetrating peptide complexes using SNAPPA.
  • Peptides identified using SNAPPA as described in EXAMPLE 5, were conjugated by recombinant fusion to an additional cystine-dense peptide with lower cell penetration to determine if the cell-penetrating peptide could carry an additional peptide into the cytosol or nucleus.
  • Cytosolic and nuclear access of the cell-penetrating peptide complexes was assayed by SNAPPA in three different SNAP -tag expressing cell lines.
  • SNAP-tag expressing cells expressing either pSNAPf to quantify cytosolic penetration or pSNAPf-H2B to quantify nuclear penetration, were generated as described in EXAMPLE 3.
  • BG-peptides comprising cystine-dense peptide complexes were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP-tag ubiquitously.
  • BG-peptides comprising cystine-dense peptide complexes were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf-H2B and expressing the SNAP-tag in the nucleus.
  • Cystine-dense peptide complexes were constructed by linking a cell-penetrating cystine-dense peptide to an additional cystine-dense peptide with lower cell penetration using a (GGGS) 3 linker (GGGSGGGSGGGS, SEQ ID NO: 255).
  • Cell-penetrating peptides MCa (SEQ ID NO: 197) and KR IpTxa (SEQ ID NO: 1, corresponding to SEQ ID NO: 59 without an N- terminal GS) were tested, and low-cell-penetrating cystine-dense peptides KTx3.10 (AQEPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ; SEQ ID NO: 294, corresponding to SEQ ID NO: 296 without an N-terminal GS) and elafin (GVPINVKCRGSRDCLDPCKKAGMRFGKCINSKCHCTP; SEQ ID NO: 293, corresponding to SEQ ID NO: 297 without an N-terminal GS) were conjugated by recombinant fusion to the cell-penetrating peptides, and tagged with SNAP substrate as described in EXAMPLE 4.
  • the peptide complexes that were tested in this assay are show in FIG. 5.
  • FIG. 6A An assay to measure cytosolic access of cell-penetrating peptide complexes is shown in FIG. 6A.
  • uptake of BG-cargo peptides was measured in the absence of conjugation to a cell-penetrating peptide.
  • BG-KR IpTxa was tested as a comparator to assess the level of penetration of the cell-penetrating peptide in the absence of conjugation to a cargo peptide.
  • BG-KR_IpTxa (BG-SEQ ID NO: 59) showed comparable high cytosolic access as the positive control BG-GLA-OH, to which all data was normalized
  • conjugating MCa or KR_IpTxa to KTx3.10 or to elafin greatly increased the level of KTx3.10 or elafin that reached the cytosol of each of the three different cell lines
  • KR_IpTxa reached the cytosol at levels comparable to the cell penetrant small molecule BG-GLA-OH.
  • BG-peptide complexes comprising cell-penetrating peptide were tested at 10 pM, 3 pM, and 1 pM using SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously.
  • Titration of the BG-peptide complexes cell-penetrating peptide demonstrated that peptide complexes incorporating the IpTxa cellpenetrating peptide (FIG. 7D and FIG. 7B) and the peptide complexes incorporating the MCa cell-penetrating peptide (FIG. 7C and FIG.
  • the peptide complexes incorporating the IpTxa cell-penetrating peptide demonstrated higher cytosolic delivery than the peptide complexes incorporating the MCa cell-penetrating peptide (SEQ ID NO: 197).
  • the data demonstrate a dose-dependent and cell-type dependent effect of the various peptide complexes on SNAP tag occupancy.
  • FIG. 6B An assay to measure nuclear access of cell-penetrating peptide complexes is shown in FIG. 6B.
  • BG-KTx3.10 BG-SEQ ID NO: 296
  • BG-elafin BG-SEQ ID NO: 297
  • Peptide complexes incorporating the KR IpTxa cell-penetrating peptide (SEQ ID NO: 1), BG-KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) and BG-KR_IpTxa- elafin (BG-SEQ ID NO: 317) also showed higher levels of nuclear access as compared to the lower penetrating peptides alone.
  • BG-KR IpTxa (BG-SEQ ID NO: 59) was tested as a comparator to assess the level of penetration of the cell-penetrating peptide in the absence of conjugation to a cargo peptide.
  • BG-KR_IpTxa (BG-SEQ ID NO: 59) showed comparable high cytosolic access as the positive control BG-GLA-OH, to which all data was normalized.
  • the data showed that conjugating MCa (SEQ ID NO: 212) or KR IpTxa (SEQ ID NO: 59) to KTx3.10 (SEQ ID NO: 293) or to elafin (SEQ ID NO: 294) greatly increased the level of KTx3.10 or elafin that reached the nucleus of each of the three different cell lines.
  • KR IpTxa (SEQ ID NO: 59) reached the nucleus at levels comparable to the cell penetrant small molecule BG-GLA-OH.
  • the peptide complexes incorporating the IpTxa cell-penetrating peptide (SEQ ID NO: 1) exhibited higher cytosolic delivery than the peptide complexes incorporating the MCa cell-penetrating peptide (SEQ ID NO: 197), but in other cell lines or concentrations, the peptides conjugates incorporating the MCa cell-penetrating peptide (SEQ ID NO: 197) showed comparable or higher levels of deliver than those incorporating the IpTxa cell-penetrating peptide (SEQ ID NO: 1).
  • One alternative strategy included protein engineering an existing loop from a MCa peptide into KTx3.10 (SEQ ID NO: 293) via recombinant fusion to generate cell-penetrating peptide fusions such as the following SNAP substrate-peptides: BG-MCa(loop)-KTx3.10 (BG-SEQ ID NO: 314), and to elafin (SEQ ID NO: 294) to generate: BG-MCa(loop)-elafin (BG-SEQ ID NO: 318).
  • FIG. 10A shows results of a SNAP penetration assay to measure cytosolic access of the cell-penetrating peptide complexes illustrated in FIG.
  • BG-cargo peptides without additional cellpenetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration. Data was normalized to positive control BG-GLA-OH. The level of cargo peptide delivered to the cytosol varied with different cell lines, species of cargo peptide, and species of cell-penetrating peptide.
  • FIG. 10B shows results of a SNAP penetration assay to measure nuclear access of the cell-penetrating peptide complexes illustrated in FIG. 9 in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus.
  • BG-cargo peptides without additional cell-penetrating peptides BG- KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration.
  • the level of cargo peptide delivered to the cytosol varied with different cell lines, species of cargo peptide, and species of cell-penetrating peptide.
  • TEAD-binding cystine-dense peptide complexes This example describes cell penetration of TEAD-binding cystine-dense peptide complexes.
  • a TEAD-binding cystine-dense peptide (PDEYIERAKECCKKQDIQCCLRIFDESKDPNVMLICLFCW; SEQ ID NO: 295, corresponding to SEQ ID NO: 298 without an N-terminal GS) was conjugated to various cellpenetrating peptides to determine whether the cell-penetrating peptides were capable of facilitating cytosolic or nuclear localization of the TEAD-binding peptides.
  • the tested peptide complexes are illustrated in FIG. 11.
  • BG-peptides comprising cell-penetrating cystine-dense peptide fusions containing the TEAD-binding peptide were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells. Cells were stably transfected with pSNAPf expressing the SNAP-tag ubiquitously to test cytosolic access.
  • Cell-penetrating cystine-dense peptide fusions were constructed by linking a short N-terminal sequence derived from either MCa (C3A_MCa(l-9), SEQ ID NO: 210) or Had (C5A_Had(l-l 1), SEQ ID NO: 82) and fused to the N-terminus of the TEAD-binding peptide (GSPDEYIERAKECCKKQDIQCCLRIFDESKDPNVMLICLFCW; SEQ ID NO: 298) to generate a C3A_MCa(l-9)-TEAD-binder fusion (SEQ ID NO: 320) or a C5A_Had(l-l 1)- TEAD-binder fusion (SEQ ID NO: 321).
  • Peptide fusions were labeled with SNAP substrate as described in EXAMPLE 4.
  • the TEAD-binding cystine-dense peptide without an additional cell-penetrating peptide tag was tested as a comparator to assess the basal level of penetration.
  • BG-KR IPTxa (BG-SEQ ID NO: 59) was also tested and BG-GLA-OH was included as a positive control for normalization.
  • the data showed that conjugating C3A_MCa(l- 9) (SEQ ID NO: 195) or C5A_Had(l-l 1) (SEQ ID NO: 41) to the TEAD-binding peptide greatly increased the level of the TEAD-binding peptide that reaches the cytosol of each of three different cell lines.
  • BG-peptides complexes comprising cell-penetrating cystine-dense peptide fusions containing the TEAD-binding peptide were tested as with cytosolic access, but in cells stably transfected with pSNAPf-H2B expressing the SNAP -tag in the nucleus to test nuclear access.
  • BG-TEAD-binder (BG-SEQ ID NO: 298) was low, but nuclear access of BG-C3A_MCa(l-9)-TEAD-binder (BG-SEQ ID NO: 320) and BG-C5A_Had(l-l 1)-TEAD- binder (BG-SEQ ID NO: 321) was significantly increased relative to the TEAD-binding cystine- dense peptide without a cell-penetrating peptide, as shown in FIG. 12B.
  • Penetration of the BG- peptides comprising BG-KR_IPTxa (BG-SEQ ID NO: 59) was also tested and BG-GLA-OH was included as a positive control for normalization.
  • the data showed that conjugating C3A MCa(l-9) (SEQ ID NO: 195) or C5A Had(l-11) (SEQ ID NO: 41) to the TEAD-binding peptide greatly increased the level of the TEAD-binding peptide that reached the nucleus of each of three different cell lines.
  • the data also showed that KR IpTxa (SEQ ID NO: 59) reached the nucleus at levels comparable to the cell penetrant small molecule BG-GLA-OH.
  • This example describes cell penetration of cystine-dense peptides and peptide fragments.
  • the ability of various cell-penetrating cystine-dense peptides and cell-penetrating peptide fragments to enter cells, alone or with cargo cystine-dense peptides was measured as described in EXAMPLE 5 - EXAMPLE 8.
  • Peptides were qualitatively ranked based on their ability to access a cell cytoplasm, nucleus, or both, as shown in TABLE 14. “+++” indicates strong cell uptake, and denote decreasing cell uptake, as measured by SNAPP A, though results varied with different cell lines, cargo peptides, and other conditions.
  • the cell-penetrating cystine-dense peptides KR_HwTx-IV (SEQ ID NO: 8, corresponding to SEQ ID NO: 66 with no N-terminal GS), MCa_varl (SEQ ID NO: 18, corresponding to SEQ ID NO: 67 with no N-terminal GS), and MCa (SEQ ID NO: 212, corresponding to SEQ ID NO: 197 with no N-terminal GS) were also able to access the cytosolic and nuclear compartment of cells at significant levels. Some cystine-dense peptides exhibit lower levels of cellular uptake from the media, and some appeared have little to no access in certain cell lines.
  • This example describes endocytotic profiling via SNAP penetration assay.
  • pSNAPf cells were preincubated with a variety of endocytosis inhibitors to assess mechanisms responsible for uptake and cytosolic access of various BG-peptides.
  • Macropinocytosis was inhibited using 50 pM ethylisopropyl amiloride (EIP A), which may block Na+/H+ exchangers and prevents formation of macropinosomes.
  • EIP A ethylisopropyl amiloride
  • Clathrin-mediated endocytosis was inhibited using either 20 pM nocodazole, which may inhibit microtubule polymerization, 3 pM cytochalasin D, which can interfere with actin polymerization, or 80 pM dynasore, which can inhibit dynamin GTPase.
  • Endosomal acidification or lysosomal maturation can be inhibited using 50 nM bafilomycin A, which can inhibit the vacuolar ATPase complex, or 50 pM chloroquine, which can diffuse into endosomes as a weak base.
  • pSNAPf cells were preincubated with the specified concentration of inhibitor in growth media for 1 hour prior to SNAPP A, which was conducted as described in EXAMPLE 5
  • BG-peptides comprising KR IpTxa (SEQ ID NO: 59) in measured in pSNAPf cells preincubated with various chemicals inhibiting different mechanisms of endocytosis.
  • BG-peptides comprising MCa_varl SEQ ID NO: 67
  • cellular uptake of the BG-peptide comprising cell-penetrating peptide BG-MCa_varl was significantly decreased upon inhibition of macropinocytosis with 50 pM EIPA in HeLa cells, though only moderately with 3T3 cells or 293 cells.
  • This example describes the potential for using additional endosomal escape peptides to further promote cell-penetrating peptides for promoting endosomal escape of cell-penetrating peptide fusions.
  • Peptides S19 PFVIGAGVLGALGTGIGGI; SEQ ID NO: 359), CM18 (KWKLFKKIGAVLKVLTTG; SEQ ID NO: 360), PAS (FFLIPKG; SEQ ID NO: 361), Aureinl.2 (GLFDIIKKIAESF; SEQ ID NO: 362), and B18 (LGLLLRHLRHHSNLLANI; SEQ ID NO: 363) purported to promote endosomal escape were conjugated by fusion to KR IpTxa (SEQ ID NO: 1, corresponding to SEQ ID NO: 59 without an N-terminal GS) in order to potentially further promote delivery of peptide to the cytosol, and then labeled with SNAP substrate as described in EXAMPLE 4.
  • BG-KR_IpTxa-PAS (BG-SEQ ID NO: 322) and BG- KR_IpTxa-S19 (BG-SEQ ID NO: 323) were tested using SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously.
  • the addition of the PAS and S19 sequences did not negatively affect cellular uptake and delivery to the cytosol, as shown in FIG.
  • BG-peptide constructs showed high delivery including BG-peptides comprising KR IpTxa (SEQ ID NO: 59), KR_IpTxA-PAS (SEQ ID NO: 322), and KR_IpTxa-S19 (SEQ ID NO: 323) and as compared to the normalizing positive control BG- GLA-OH.
  • the data showed that KR IpTxa without or with the addition of PAS or S19 had high levels of cell penetration in all cell lines tested.
  • KR_IpTxa SEQ ID NO: 59
  • KR_IpTxA-PAS SEQ ID NO: 322
  • KR_IpTxa-S19 SEQ ID NO: 323
  • KR IpTxa SEQ ID NO: 1
  • FIG. 16A and FIG. 16B illustrates peptide cargos that can be conjugated, such as by chemical conjugation or recombinant fusion, to a cellpenetrating cystine-dense peptide or peptide fragment of the present disclosure.
  • a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 1 4, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is conjugated to, linked to, or fused to a peptide cargo and expressed recombinantly or chemically synthesized as a cell-penetrating peptide complex.
  • the peptide cargo is conjugated to, linked to, or fused to the N-terminus or the C-terminus of the cell-penetrating cystine-dense peptide or peptide fragment.
  • the peptide cargo and the cell-penetrating cystine-dense peptide or peptide fragment are connected by a linker of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485, or any other linker, or no linker.
  • the peptide cargo is a cystine-dense peptide, an affibody, a 13-hairpin, an avimer, an adnectin, a stapled peptide, a nannofittin, a kunitz domain, a fynomer, or a bicyclic peptide, as shown in FIG. 16A.
  • the peptide is a nanobody, an antibody scFc fragment, or an antibody FAb fragment, as shown in FIG. 16B.
  • One or more cell-penetrating peptides of this disclosure may be conjugated to any of these cargos.
  • One or more of these cargos may be conjugates to a cell-penetrating peptide of this disclosure.

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Abstract

Described herein are peptides and variants thereof capable of penetrating a cellular layer. These cell-penetrating peptides may be conjugated, linked, or fused to active agents or detectable agents to deliver the agents into a cellular compartment from which they may otherwise be excluded. The cell-penetrating peptides may be used to facilitate delivery of a therapeutic agent to a patient to treat a disease or a condition in the patient.

Description

CELL-PENETRATING PEPTIDES AND PEPTIDE COMPLEXES AND METHODS OF
USE
CROSS-REFERENCE
[0001] The present application claims the benefit of U.S. Provisional Application No. 63/123,406, entitled “CELL PENETRATING PEPTIDES AND METHODS OF USE,” filed on December 9, 2020, which application is herein incorporated by reference in its entirety for all purposes.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 7, 2021, is named 108406-702530_SL.txt and is 521,277 bytes in size.
BACKGROUND
[0003] A major objective of drug development is ensuring that a therapeutic agent can reach its intended target. Frequently, this means designing therapeutics that can reach the cytosol, nucleus, or other subcellular compartments of a cell. Drugs with poor cell permeation may not reach an intended cytosolic, nuclear, or other intracellular drug target, resulting in decreased efficacy. Moreover, drugs with poor cell permeation may also require higher dosages than those with more targeted or more successful cell permeation, resulting in a higher incidence of toxicity or side effects. Biologic drugs, such as those made of peptides, proteins, knotted peptides, or miniproteins, RNA, and DNA frequently are unable to enter the cytosol and therefore these classes of drugs are not able to drug intracellular targets. There is a need for methods to deliver drugs with low permeation or absorption across cellular membranes to reach cytosolic and nuclear drug targets.
SUMMARY
[0004] In various aspects, the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising a sequence that has: at least 80% sequence identity with any one of SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20 - SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 44 - SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 75, SEQ ID NO: 77, or SEQ ID NO: 80, or a fragment thereof; at least 85% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40 - SEQ ID NO: 42, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 81 - SEQ ID NO: 83, or a fragment thereof; at least 90% sequence identity with any one of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 43, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 72, or SEQ ID NO: 84, or a fragment thereof; or at least 95% sequence identity with any one of SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 61, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 73, or SEQ ID NO: 74, or a fragment thereof.
[0005] In some aspects, the peptide complex comprises a sequence that has at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, or a fragment thereof. In some aspects, the peptide complex comprises a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84. In some aspects, the peptide complex comprises a sequence of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 8; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 40; SEQ ID NO: 41; or SEQ ID NO: 42. In some aspects, the cell-penetrating peptide is fused or linked to a cargo molecule.
[0006] In various aspects, the present disclosure provides a peptide complex comprising a cellpenetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof.
[0007] In various aspects, the present disclosure provides a peptide complex comprising a cellpenetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, SEQ ID NO: 195 - SEQ ID NO: 254, or a fragment thereof, and wherein the cargo molecule is a cystine-dense peptide, a DNA- binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or combinations thereof.
[0008] In some aspects, the cell-penetrating peptide comprises a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254. In some aspects, the peptide complex comprises a sequence of: SEQ ID NO: 195; SEQ ID NO: 197; or SEQ ID NO: 198. In some aspects, the cellpenetrating peptide comprises a sequence of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 8; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 40; SEQ ID NO: 41; or SEQ ID NO: 42.
[0009] In various aspects, the present disclosure provides a peptide complex comprising a cellpenetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence of any one of SEQ ID NO: 325 - SEQ ID NO: 342 or SEQ ID NO: 343 - SEQ ID NO: 351, and wherein the cargo molecule wherein the cargo molecule is a cystine-dense peptide, a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or combinations thereof.
[0010] In some aspects, the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, at least 3.5, at least 3.7, or at least 4.0 arginine amino acid residues per ten amino acid residues. In some aspects, the cell-penetrating peptide comprises no more than 0.5, no more than 0.8, no more than 0.9, no more than 1.0, no more than 1.1, no more than 1.2, no more than 1.3, no more than 1.4, no more than 1.5, no more than 1.6, no more than 1.7, no more than 1.8, no more than 1.9, no more than 2.0, no more than 2.1, no more than 2.2, no more than 2.3, no more than 2.4, no more than 2.5, no more than 3.0, no more than 3.3, no more than 3.5, no more than 3.7, or no more than 4.0 arginine amino acid residues per ten amino acid residues. In some aspects, the cell-penetrating peptide comprises no arginine amino acid residues.
[0011] In some aspects, the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, at least 3.5, at least 3.7, or at least 4.0 lysine amino acid residues per ten amino acid residues. In some aspects, the cell-penetrating peptide comprises no more than 0.5, no more than 0.8, no more than 0.9, no more than 1.0, no more than 1.1, no more than 1.2, no more than 1.3, no more than 1.4, no more than 1.5, no more than 1.6, no more than 1.7, no more than 1.8, no more than 1.9, no more than 2.0, no more than 2.1, no more than 2.2, no more than 2.3, no more than 2.4, no more than 2.5, no more than 3.0, no more than 3.3, no more than 3.5, no more than 3.7, or no more than 4.0 lysine amino acid residues per ten amino acid residues. In some aspects, the cell-penetrating peptide comprises no lysine amino acid residues. [0012] In some aspects, the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least
0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least
1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least
2.5, at least 3.0, at least 3.3, at least 3.5, at least 3.7, or at least 4.0 positively charged amino acid residue per 10 amino acid residues at a pH of about 7.4. In some aspects, the cell-penetrating peptide comprises no more than 4.0, no more than 3.9, no more than 3.8, no more than 3.7, no more than 3.6, no more than 3.5, no more than 3.4, no more than 3.3, no more than 3.2, no more than 3.1, no more than 3.0, no more than 2.9, no more than 2.8, no more than 2.7, no more than
2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, no more than 2.0, no more than 1.5, no more than 1 .2, or no more than 1 .0 positively charged amino acid residue per 10 amino acid residues at a pH of about 7.4. In some aspects, the cellpenetrating peptide comprises at least 0.03, at least 0.05, at least 0. 1, at least 0.2, at least 0.5, at least 0.8, at least 0.9, at least 1 .0, at least 1 . 1, at least 1 .2, at least 1 .3, at least 1 .4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, negatively charged amino acid residues per ten amino acid residues at a pH of about 7.4. In some aspects, the cell-penetrating peptide comprises a ratio of positively charged amino acid residues to negatively charged amino acid residues of at least 1.0, at least 1.5, at least 2, at least 2.5, at least 2.75, at least 3, at least 3.5, at least 4, at least 5, at least 6, or at least 7. In some aspects, the cell-penetrating peptide comprises a ratio of negatively charged amino acid residues to positively charged amino acid residues of no more than 1.0, no more than 1.5, no more than 2, no more than 2.5, no more than 2.75, no more than 3, no more than 3.5, or no more than 4. In some aspects, the cell-penetrating peptide comprises at least 1, at least 2, at least 3, or at least 4 negatively charged amino acids.
[0013] In some aspects, the cell-penetrating comprises at least 1, at least 2, at least 3, or at least 4 histidine amino acid residues. In some aspects, the cell-penetrating comprises at least 1, at least 2, at least 3, or at least 4 proline amino acid residues. In some aspects, the cell-penetrating peptide comprises an amphipathic a-helix. In some aspects, the cell-penetrating peptide comprises at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues. In some aspects, the cell-penetrating peptide comprises no cysteine amino acid residues.
[0014] In various aspects, the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising: at least 1.0, at least 1.8, at least 1.9, at least 2.0, at least 2.2, at least 2.5, at least 3.0, at least 3.3, or at least 3.5 arginine amino acid residues per 10 amino acid residues; and at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues; wherein the cell -penetrating peptide is fused or linked to a cargo molecule.
[0015] In some aspects, the cell-penetrating peptide comprises at least four cysteine amino acid residues. In some aspects, the cell-penetrating peptide comprises no more than 3.3, no more than
3.2, no more than 3.1, no more than 3.0, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, or no more than 2.0 positively charged amino acid residue per 10 amino acid residues at a pH of 7.4.
[0016] In various aspects, the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising: at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, or at least 2.7 arginine amino acid residues per 10 amino acid residues; and no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than
2.2, no more than 2.1, or no more than 2.0 positively charged amino acid residue per 10 amino acid residues at a pH of 7.4; wherein the cell-penetrating peptide is fused or linked to a cargo molecule.
[0017] In various aspects, the present disclosure provides a peptide complex comprising a cellpenetrating peptide comprising: at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, or at least 3.5 arginine amino acid residues per 10 amino acid residues; and no more than 3.3, no more than 3.2, no more than 3.1, no more than 3.0, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, or no more than 2.0 positively charged amino acid residue per 10 amino acid residues at a pH of 7.4; wherein the cell-penetrating peptide is fused or linked to a cargo molecule.
[0018] In some aspects, the positively charged residues are arginine, lysine, or any combination thereof. In some aspects, the cell-penetrating peptide comprises no cysteine amino acid residues. In some aspects, the cell-penetrating peptide comprises at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues. In some aspects, the cell-penetrating peptide comprises a disulfide through disulfide knot. In some aspects, the cell-penetrating peptide comprises a plurality of disulfide bridges formed between cysteine residues. In some aspects, the cell-penetrating peptide comprises three or more disulfide bridges formed between cysteine residues, wherein one of the disulfide bridges passes through a loop formed by two other disulfide bridges. In some aspects, the cell-penetrating peptide comprises at least 1.1 arginine amino acid residues per 10 amino acid residues. In some aspects, the cell-penetrating peptide comprises no more than 2.7 positively charged amino acid residue per 10 amino acid residues.
[0019] In some aspects, the cell-penetrating peptide is derived from maurocalcin, imperatoxin, hadrucalcin, hemicalcin, opicalcin-1, opicalcin-2, midkine, MCoTI-II, chlorotoxin, huwentoxin, vejocalcin, intrepicalcin, or urocalcin. In some aspects, the cell-penetrating peptide or the fragment thereof comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least, 8, at least
9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 40, at least 50, at least 60, at least 70, or at least 80 residues. In some aspects, the cell-penetrating peptide or the fragment thereof comprises no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than
10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, no more than 30, no more than 31, no more than 32, no more than 33, no more than 34, no more than 35, no more than 40, no more than 50, no more than 60, no more than 70, or no more than 80 residues. In some aspects, the cellpenetrating peptide comprises an isoelectric point within a range from about 6.0 to about 12.0, from about 6.0 to about 10.0, from about 6.5 to about 7.5, from about 7.0 to about 10.0, or from about 8.0 to about 10.0. In some aspects, the cell-penetrating peptide is stable at pH of from 6.5 to 7.5. In some aspects, the cell-penetrating peptide is stable at pH values within a range from pH 5.0 to pH 7.0.
[0020] In some aspects, the cargo molecule comprises a cargo peptide comprising four or more cysteine amino acid residues and at least two disulfide bonds. In some aspects, the cargo molecule is fused or linked to the cell-penetrating peptide at an N-terminus or a C-terminus of the cell-penetrating peptide. In some aspects, the cargo molecule comprises at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors. In some aspects, the cargo molecule comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors. In some aspects, the cargo molecule comprises a molecular weight of at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, or at least 1000 Da. In some aspects, the cargo molecule comprises a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0.
[0021] In some aspects, the cargo molecule comprises an antibody, an antibody fragment, an Fc domain, a single chain Fv, an intrabody, or a nanobody. In some aspects, the cargo molecule comprises a cystine-dense peptide, an affibody, a B-hairpin, an avimer, an adnectin, a stapled peptide, a nanofittin, a kunitz domain, a fynomer, or a bicyclic peptide. In some aspects, the cargo molecule comprises an immunomodulatory imide drug, a Boc3Arg tag, an adamantyl group, or a carborane. In some aspects, the cargo molecule comprises a target-binding molecule. In some aspects, the target-binding molecule comprises a target-binding peptide. In some aspects, the target-binding peptide comprises a cystine-dense peptide. In some aspects, the target-binding peptide is capable of binding a transcription factor or a tyrosine kinase. In some aspects, the target-binding peptide is capable of binding TEAD, cold-inducible RNA-binding protein, androgen receptor, ikaros, aiolos, a nuclear receptor, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, tau, AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, P-catenin, IKK, TEAD, NF-KB, PKC, AP-1, Jun, Fos, or REL. In some aspects, the target-binding peptide comprises a sequence with at least 90% or at least 95% sequence identity to any one of SEQ ID NO: 295, SEQ ID NO: 298, or SEQ ID NO: 364 - SEQ ID NO: 407. In some aspects, the targetbinding peptide comprises a sequence of any one of SEQ ID NO: 295, SEQ ID NO: 298, or SEQ ID NO: 364 - SEQ ID NO: 407. In some aspects, the target-binding peptide comprises a sequence of SEQ ID NO: 295.
[0022] In some aspects, the peptide complex comprises a sequence of SEQ ID NO: 307 or SEQ ID NO: 308. In some aspects, the target-binding molecule binds a ubiquitin ligase. In some aspects, the ubiquitin ligase comprises cereblon, cellular inhibitor of apoptosis protein 1, MDM2, DCAF15, DCAF16, cullin-4A, a Cul2-Rbxl-EloN/C-VHL E3 ubiquitin ligase, APC/C activator protein CDH1, or von Hippel -Lindau protein. In some aspects, the target-binding molecule comprises an immunomodulatory imide drug. In some aspects, the target-binding molecule comprises a thalidomide, a pomalidomide, a lenalidomide, a methyl bestatin, a bestatin, a nutlin-3, or a VHL ligand 1. In some aspects, the cargo molecule comprises an anticancer agent, a transcription factor binding agent, an inhibitor of protein-protein interactions, a promoter of protein-protein interactions, a transcription factor, an RNA, a CRISPR component, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or an immunomodulating agent.
[0023] In some aspects, the oligonucleotide comprises a DNA, an RNA, an antisense oligonucleotide, an aptamer, an miRNA, an alternative splicing modulator, an mRNA-binding sequence, an miRNA-binding sequence, an siRNA-binding sequence, an RNaseHl -binding oligonucleotide, a RISC-binding oligonucleotide, a polyadenylation modulator, a gapmer, a RIG-I ligand, an mRNA, an antisense RNA, a small interfering RNA, a guide RNA, a U1 adaptor, a micro RNA, or a combination thereof. In some aspects, the oligonucleotide comprises: a sequence of any one of SEQ ID NO: 488 - SEQ ID NO: 573; a sequence that binds to any one of SEQ ID NO: 574 - SEQ ID NO: 611; a sequence of any one of SEQ ID NO: 574 - SEQ ID NO: 611, or a fragment thereof; or a sequence targeting or encoding a gene target provided in TABLE 12. In some aspects, the CRISPR component is a guide RNA, a tracrRNA, a crRNA, or a Cas nuclease. In some aspects, the cargo molecule comprises a detectable agent or a therapeutic agent. In some aspects, the detectable agent is a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a radioisotope, or a radionuclide chelator.
[0024] In some aspects, the peptide complex further comprises one or more chemical modifications. In some aspects, the chemical modification extends the half-life or modifies a pharmacokinetics of the peptide complex. In some aspects, the chemical modification blocks an N-terminus of the peptide complex. In some aspects, the chemical modification comprises methylation, acetylation, or acylation. In some aspects, the chemical modification comprises: methylation of one or more lysine residues or analogue thereof; methylation of the N-terminus; or methylation of one or more lysine residue or analogue thereof and methylation of the N- terminus. In some aspects, the peptide complex further comprises a half-life modifying agent, a nuclear localization signal, or an endosomal escape motif. In some aspects, the half-life modifying agent comprises a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin.
[0025] In some aspects, the peptide complex further comprises an additional active agent. In some aspects, the additional active agent is fused to the cell-penetrating peptide or the cargo molecule at an N-terminus, at the epsilon amine of an internal lysine residue, at the carboxylic acid of an aspartic acid or glutamic acid residue, or a C-terminus of the cell-penetrating peptide or the cargo molecule by a linker. In some aspects, the linker comprises an amide bond, an ester bond, a carbamate bond, a carbonate bond, a hydrazone bond, an oxime bond, a disulfide bond, a thioester bond, a thioether bond, or a carbon-nitrogen bond. In some aspects, the linker comprises a peptide linker. In some aspects, the peptide linker comprises a sequence of any one of SEQ ID NO: 255 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485. In some aspects, the linker is a cleavable linker or a pH sensitive linker. In some aspects, the cleavable linker comprises a cleavage site for matrix metalloproteinases, thrombin, cathepsins, or betaglucuronidase. In some aspects, the linker is a hydrolytically labile linker. In some aspects, the linker is a stable linker.
[0026] In some aspects, the additional active agent comprises at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors. In some aspects, the additional active agent comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors. In some aspects, the additional active agent comprises a molecular weight of at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, or at least 1000 Da. In some aspects, the additional active agent comprises a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0. In some aspects, the additional active agent is a detectable agent or a therapeutic agent. In some aspects, the detectable agent is a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a radioisotope, or a radionuclide chelator.
[0027] In some aspects, the cell-penetrating peptide is a membrane-penetrating peptide. In some aspects, the cell-penetrating peptide is a nuclear envelope-penetrating peptide. In some aspects, the cell-penetrating peptide is a blood brain barrier-penetrating peptide. In some aspects, the cell-penetrating peptide is arranged in a multimeric structure with at least one other peptide. In some aspects, the cell-penetrating peptide lacks an immunogenic sequence. In some aspects, the cell-penetrating peptide is modified to increase homology to a human protein sequence. In some aspects, the cell-penetrating peptide is modified to increase resistance to degradation. In some aspects, the cell-penetrating peptide is modified to reduce an affinity of the peptide for a human leukocyte antigen complex, a major histocompatibility complex, or both. In some aspects, the cell-penetrating peptide is an active agent.
[0028] In various aspects, the present disclosure provides a pharmaceutical composition comprising a peptide complex of the present disclosure, or a salt thereof, and a pharmaceutically acceptable carrier. [0029] In some aspects, the pharmaceutical composition is formulated for administration to a subject. In some aspects, the pharmaceutical composition is formulated for oral administration, intravenous administration, subcutaneous administration, intramuscular administration, or a combination thereof.
[0030] In various aspects, the present disclosure provides a method of delivering a cargo molecule across a cellular layer of a cell, the method comprising: contacting the cell with a peptide complex comprising a cell-penetrating peptide fused or linked to cargo molecule, wherein the cell-penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof; penetrating the cellular layer with the cell -penetrating peptide; and delivering the cargo molecule across the cellular layer.
[0031] In various aspects, the present disclosure provides a method of delivering a cargo molecule across a cellular layer of a cell, the method comprising: contacting the cell with a peptide complex of the present disclosure comprising a cell-penetrating peptide fused or linked to cargo molecule; penetrating the cellular layer with the cell-penetrating peptide; and delivering the cargo molecule across the cellular layer, thereby treating the condition.
[0032] In various aspects, the present disclosure provides a method of treating a condition in a subject in need thereof, the method comprising: administering to the subject a composition comprising a peptide complex comprising a cell-penetrating peptide fused or linked to cargo molecule, wherein the cell -penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof; penetrating a cellular layer of a cell of the subject with the cell-penetrating peptide; and delivering the cargo molecule across the cellular layer, thereby treating the condition.
[0033] In various aspects, the present disclosure provides a method of treating a condition in a subject in need thereof, the method comprising: administering to the subject a composition comprising a peptide complex of the present disclosure comprising a cell-penetrating peptide fused or linked to cargo molecule; penetrating a cellular layer of a cell of the subject with the cell-penetrating peptide; and delivering the cargo molecule across the cellular layer.
[0034] In some aspects, the disease or condition is cancer, a neurological disorder, an inflammatory disorder, an immune disorder, a neurodegenerative disorder, or a genetic disorder. In some aspects, the cancer is liver cancer, breast cancer, colon cancer, lung cancer, prostate cancer, brain cancer, skin cancer, pancreatic cancer, leukemia, or lymphoma. In some aspects, the composition is administered by inhalation, intranasally, orally, topically, intravenously, subcutaneously, intramuscularly administration, intraperitoneally, intratumorally, intrathecally, intravitreally, or a combination thereof. In some aspects, the composition is administered intravenously as a bolus, injection, infusion, or prolonged infusion. In some aspects, the cellular layer is a cell membrane, a nuclear envelope, an endosomal membrane, a lysosomal membrane, or a blood brain barrier.
[0035] In some aspects, the method comprises penetrating a cell membrane of the cell. In some aspects, the method comprises penetrating a nuclear envelope of the cell. In some aspects, the method comprises delivering the cargo molecule to a cytosol of the cell. In some aspects, the method comprises producing a cargo molecule concentration of at least 10 nM, at least 50 nM, at least 100 nM, at least 300 nM, at least 500 nM, at least 700 nM, at least 1000 nM, at least 1200 nM, at least 1400 nM, or at least 1600 nM in the cytosol of the cell. In some aspects, the method comprises delivering the cargo molecule to a nucleus of the cell. In some aspects, the method comprises producing a cargo molecule concentration of at least 10 nM, at least 50 nM, at least 100 nM, at least 300 nM, at least 500 nM, at least 700 nM, at least 1000 nM, at least 1200 nM, at least 1400 nM, or at least 1600 nM in the nucleus of the cell.
[0036] In some aspects, the cargo molecule comprises an oligonucleotide that binds a target sequence, and wherein the method further comprises modulating alternative splicing of the target sequence, dictating the location of a polyadenylation site of the target sequence, inhibiting translation of the target sequence, inhibiting binding of the target sequence to a secondary target sequence, recruiting RISC to the target sequence, recruiting RNaseHl to the target sequence, inducing cleavage of the target sequence, or regulating the target sequence upon binding of the oligonucleotide to the target sequence.
[0037] In some aspects, the method comprises delivering the cargo molecule into an intracellular space or a paracellular space. In some aspects, the intracellular space is a nanolumen. In some aspects, the cell has uncontrolled or dysregulated cell growth. In some aspects, the cell is a cancerous cell or a tumor cell. In some aspects, the cell is a pancreatic cell, liver cell, colon cell, smooth muscle cell, ovarian cell, breast cell, lung cell, brain cell, skin cell, ocular cell, blood cell, lymph cell, immune system cell, reproductive cell, reproductive organ cell, prostate cell, fibroblast, kidney cell, adenocarcinoma cell, glioma stem cell, tumor cell, or any combination thereof.
[0038] In some aspects, the method further comprises binding the cargo molecule to a target molecule. In some aspects, the target molecule comprises a transcription factor or a tyrosine kinase. In some aspects, the target molecule comprises TEAD, cold-inducible RNA-binding
-l i protein, androgen receptor, ikaros, aiolos, a nuclear receptor, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-5, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, tau, AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, P-catenin, IKK, TEAD, NF-KB, PKC, AP-1, Jun, Fos, or REL.
[0039] In some aspects, the method further comprises inhibiting the target molecule. In some aspects, the method further comprises binding the cargo molecule to a ubiquitin ligase. In some aspects, the ubiquitin ligase comprises cereblon, cellular inhibitor of apoptosis protein 1, MDM2, DCAF15, DCAF16, cullin-4A, a Cul2-Rbxl-EloN/C-VHL E3 ubiquitin ligase, APC/C activator protein CDH1, or von Hippel -Lindau protein. In some aspects, the cargo molecule comprises an immunomodulatory imide drug, a thalidomide, a pomalidomide, a lenalidomide, a methyl bestatin, a bestatin, a nutlin-3, or a VHL ligand 1. In some aspects, the method further comprises ubiquitinating the target molecule upon binding of the cargo molecule to the target molecule and the ubiquitin ligase.
[0040] In some aspects, the cargo molecule comprises a detectable agent. In some aspects, the method further comprises imaging the cell. In some aspects, the method further comprises detecting a presence, absence, location, or a combination thereof of the detectable agent in the cell. In some aspects, the method comprises delivering at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% of the peptide complex across the cellular layer.
INCORPORATION BY REFERENCE
[0041] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0043] FIG. 1A schematically illustrates a process of covalently tagging peptides with a benzylguanine SNAP substrate by using BG-GLA-NHS. The NHS-ester of a benzylguanine SNAP substrate (BG-GLA-NHS) reacts with a reactive amine group, such as at the N-terminus or at a lysine residue, of a peptide to generate a SNAP substrate-tagged peptide (referred to as a BG-peptide, a BG-peptide complex, a BG-cystine-dense peptide, or a BG-cell-penetrating peptide).
[0044] FIG. IB schematically illustrates a SNAP penetration assay (SNAPP A) to quantify cell penetration of a BG-peptide. BG-peptides generated as illustrated in FIG. 1A were incubated with cells expressing a SNAP -tag protein in the cytosol. BG-peptides that enter the cell and reach the cytosol will covalently bind to the cytosolic SNAP -tag protein which reacts specifically and rapidly with benzylguanine (BG) derivatives, including conjugated BG- peptides, leading to irreversible covalent labeling of the SNAP -tag with the benzylguanine- derivatized cell penetrant BG-peptide. The cells were then incubated with a SNAP substrate- tagged fluorophore that can diffuse across the cell membrane (SNAP-Cell TMR-Star, New England Biolabs, referred to as BG-fluorophore). Free SNAP -tag protein in the cell will bind to the BG-fluorophore, while SNAP -tag protein that is bound to BG-peptide cannot bind to the fluorescent SNAP substrate because the BG-peptide already occupies the SNAP -tag. Excess BG-fluorophore was washed away, and the remaining fluorescence was inversely related to the amount of peptide that penetrated the cell membrane. The cells on the left, contacted with a cell penetrant SNAP substrate-tagged peptide (BG-peptide), have a lower fluorescence and the cells on the right, contacted with a non-cell penetrant SNAP substrate-tagged peptide have a higher fluorescence. Thus, a cell penetrant peptide blocks the SNAP -tag from binding fluorescent SNAP substrate and therefore reduces the fluorescent signal in the assay. This assay may also be used to assess penetration into other cellular compartments, such as the nucleus, by expressing a SNAP -tag protein fusion that localizes to the cellular compartment of interest.
[0045] FIG. 2 shows representative images of reversed-phase high performance liquid chromatography (RP-HPLC, top) and size exclusion chromatography (SEC, second, third, and bottom) associated with a typical SNAP substrate conjugation reaction with peptide and subsequent purification process. The bottom two images show RP-HPLC traces of concentrated fractions following SEC, demonstrating separation and purification of BG-peptides and unreacted peptides. Peaks in the top panel correspond to, from left to right, BG-GLA-NHS hydrolyzed to BG-GLA-OH, BG-GLA-NHS ligand, KR CTX (SEQ ID NO: 71), and KR CTX BG-peptide (BG-SEQ ID NO: 71). The primary peak in the third panel corresponds to KR CTX BG-peptide (BG-SEQ ID NO: 71), and the primary peak in the bottom panel corresponds to KR CTX (SEQ ID NO: 71). [0046] FIG. 3 shows representative fluorescence images of the SNAPPA assay after contacting various BG-peptides comprising cystine-dense peptides to HeLa cells stably transfected with pSNAPf and expressing SNAP -tag, followed by addition of BG-fluorophore and washing away unbound BG-fluorophore. Eight BG-peptides comprising cystine-dense peptides, KR_KTxl5.8 (BG-SEQ ID NO: 74), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG-SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59), were assayed for cell penetration. Phosphate buffered saline (PBS) was used as a negative control. Hydrolyzed SNAP substrate, BG-GLA-OH, formed when BG-GLA-NHS is hydrolyzed, was used as a positive control.
[0047] FIG. 4A shows the results of a SNAP penetration assay measuring cellular penetration into the cytosol by various BG-peptides comprising cystine-dense peptides exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag protein in the cytosol. The BG-peptides comprising cystine-dense peptides, KR_KTxl5.8 (BG-SEQ ID NO: 74), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG-SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59), were assayed for cell penetration to reach the cytosol. The cell penetrant moiety BG-GLA-OH, formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
[0048] FIG. 4B shows the results of a SNAP penetration assay measuring cellular penetration into the nucleus by various BG-peptides comprising cystine-dense peptides exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag protein in the nucleus. The BG-peptides comprising cystine-dense peptides, KR_KTxl5.8 (BG-SEQ ID NO: 74), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG-SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59), were assayed for cell penetration to reach the nucleus. The cell penetrant moiety BG-GLA-OH, formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
[0049] FIG. 5 schematically illustrates BG-peptides complexes comprising cell-penetrating peptides and cargo peptides assayed by SNAPPA in FIG. 6A - FIG. 8D.
[0050] FIG. 6A shows results of a SNAP penetration assay to measure cellular penetration into the cytosol of the cell-penetrating peptide complexes illustrated in FIG. 5 exposed to NIH3T3, HeLa and HEK293 cells. Cellular penetration into the cytosol of penetrating peptide complexes BG-MCa-KTx3.10 (BG-SEQ ID NO: 312), BG-MCa-elafin (BG-SEQ ID NO: 316), BG- KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) and BG-KR_IpTxa-elafin (BG-SEQ ID NO: 317) was measured, along with BG-cargo peptides without cell-penetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297). BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration of these cargo peptides with the addition of further cell-penetrating peptides. The cell penetrant moiety BG-GLA-OH, formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization. BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
[0051] FIG. 6B shows results of a SNAP penetration assay to measure cellular penetration into the nucleus of the cell-penetrating peptide complexes illustrated in FIG. 5 exposed to NIH3T3, HeLa and HEK293 cells. Nuclear access of cell-penetrating peptide complexes BG-MCa- KTx3.10 (BG-SEQ ID NO: 312), BG-MCa-elafin (BG-SEQ ID NO: 316), BG-KR_IpTxa- KTx3.10 (BG-SEQ ID NO: 313) and BG-KR_IpTxa-elafm (BG-SEQ ID NO: 317) was measured, along with cargo peptides without additional cell-penetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297). BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration. The cell penetrant moiety BG-GLA-OH, formed when BG-GLA-NHS (BG- substrate) is hydrolyzed, was used as a positive control and for normalization. BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
[0052] FIG. 7A shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-KTx3.10, with increasing concentrations of a BG- MCa-KTx3.10 (BG-SEQ ID NO: 312) cell-penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
[0053] FIG. 7B shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR_IpTxa-KTx3.10, with increasing concentrations of a BG-KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) cell-penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
[0054] FIG. 7C shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-elafin, with increasing concentrations of a BG- MCa-elafin (BG-SEQ ID NO: 316) cell-penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
[0055] FIG. 7D shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR IpTxa-elafin, with increasing concentrations of a BG-KR_IpTxa-elafin (BG-SEQ ID NO: 317) cell -penetrating peptide complex exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
[0056] FIG. 8A shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-KTx3.10, with increasing concentrations of a BG- MCa-KTx3.10 (BG-SEQ ID NO: 312) cell-penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
[0057] FIG. 8B shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR_IpTxa-KTx3.10, with increasing concentrations of a BG-KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) cell-penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
[0058] FIG. 8C shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide MCa-elafin, with increasing concentrations of a BG- MCa-elafin (BG-SEQ ID NO: 316) cell-penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
[0059] FIG. 8D shows results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptide KR IpTxa-elafin, with increasing concentrations of a BG-KR IpTxa-elafm (BG-SEQ ID NO: 317) cell -penetrating peptide complex in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus. The data was normalized to cells exposed to 10 pM BG-GLA-OH.
[0060] FIG. 9 schematically illustrates BG-cargo peptides and BG-cell penetrating peptide- cargo peptide complexes assayed by SNAPPA in FIG. 10A and FIG. 10B.
[0061] FIG. 10A shows results of a SNAP penetration assay to measure cellular penetration to reach the cytosol of the cell-penetrating peptide complexes illustrated in FIG. 9 in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptides complexes BG-MCa(loop)-KTx3.10 (BG-SEQ ID NO: 314), BG-peptide of MCa(loop)-elafin (BG-SEQ ID NO: 318), BG-C3A_MCa(l-9)-KTx3.10 (BG-SEQ ID NO: 315), and BG-C3A_MCa(l-9)-elafin (BG-SEQ ID NO: 319) was measured, along with BG-cargo peptides without cell-penetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297). BG-KTx3 10 (BG-SEQ ID NO: 296) and BG-elafm (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration of these cargo peptides with the addition of further cell-penetrating peptides. The cell penetrant moiety BG-GLA-OH, formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
[0062] FIG. 10B shows results of a SNAP penetration assay to measure cellular penetration to reach the nucleus of the cell-penetrating peptide complexes illustrated in FIG. 9 exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus. SNAP substrate-tagged peptide (BG-peptide) comprising CDP peptides complexes BG-MCa(loop)-KTx3.10 (BG-SEQ ID NO: 314), BG-MCa(loop)-elafm (BG-SEQ ID NO: 318), BG-C3A_MCa(l-9)-KTx3.10 (BG- SEQ ID NO: 315), and BG-C3A_MCa(l-9)-elafin (BG-SEQ ID NO: 319) was measured, along with BG-cargo peptides without cell-penetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafm (BG-SEQ ID NO: 297). BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafm (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration of these cargo peptides with the addition of further cell-penetrating peptides. The cell penetrant moiety BG-GLA-OH, formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
[0063] FIG. 11 schematically illustrates BG-peptides complexes comprising a TEAD-binding peptide (“TEAD-binder”), with or without a cell-penetrating peptide, assayed by SNAPPA in FIG. 12A, FIG. 12B, and FIG. 13
[0064] FIG. 12A shows results of a SNAP penetration assay to measure cellular penetration to reach the cytosol of the cell-penetrating peptides conjugated to a TEAD-binding peptide illustrated in FIG. 11 contacted to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm. SNAP substrate-tagged peptide (BG-peptide) comprising CDP TEAD-binding peptides complexes BG-C3A_MCa(l-9)-TEAD-binder (BG-SEQ ID NO: 320) and BG- C5A_Had(l-l l)-TEAD-binder (BG-SEQ ID NO: 321) was measured, along with BG-TEAD- binding peptide without cell-penetrating peptides BG- TEAD-binder (BG-SEQ ID NO: 298). BG- TEAD-binder (BG-SEQ ID NO: 298) was used as a comparator to assess the basal level of penetration of this cargo peptides with the addition of further cell-penetrating peptides. The cell penetrant moiety BG-GLA-OH, formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization. The SNAP substrate-tagged peptide (BG- peptide) comprising BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
[0065] FIG. 12B shows results of a SNAP penetration assay to measure cellular penetration to meet the nucleus of the cell-penetrating peptides conjugated to a TEAD-binding peptide illustrated in FIG. 11 exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus. A SNAP substrate-tagged peptide (BG-peptide) comprising CDP TEAD-binding peptides complexes BG-C3A_MCa(l-9)-TEAD-binder (BG-SEQ ID NO: 320) and BG- C5A_Had(l-l l)-TEAD-binder (BG-SEQ ID NO: 321) was measured, along with BG-TEAD- binding peptide without cell-penetrating peptides (BG-SEQ ID NO: 298). BG-TEAD-binder (BG-SEQ ID NO: 298) was used as a comparator to assess the basal level of penetration of this cargo peptides with the addition of further cell-penetrating peptides. The cell penetrant moiety BG-GLA-OH, formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization. BG-KR_IpTxa (BG-SEQ ID NO: 59) was used as a comparator to assess the level of penetration in the absence of a cargo peptide.
[0066] FIG. 13 shows results of a SNAP penetration assay to measure cellular penetration into the nucleus of the cell-penetrating peptides conjugated to a TEAD-binding peptide (“TEAD- binder”) illustrated in FIG. 11 exposed to primary GSC cells transiently transfected with pSNAPf-H2B and expressing SNAP -tag in the nucleus. SNAP substrate-tagged peptide (BG- peptide) comprising CDP TEAD-binding peptides complexes BG-C3A_MCa(l-9)-TEAD- binder (BG-SEQ ID NO: 320) and BG-C5A_Had(l-l l)-TEAD-binder (BG-SEQ ID NO: 321) was measured, along with BG-TEAD-binding peptide without cell-penetrating peptides BG- TEAD-binder (BG-SEQ ID NO: 298). BG-TEAD-binder (BG-SEQ ID NO: 298) was used as a comparator to assess the basal level of penetration of this cargo peptides with the addition of further cell-penetrating peptides. The cell penetrant moiety BG-GLA-OH, formed when BG- GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization. [0067] FIG. 14A shows the results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR_IpTxa in the presence of a variety of endocytosis inhibitors to assess mechanisms responsible for uptake of a BG-KR_IpTxa cellpenetrating peptide (BG-SEQ ID NO: 59). Cytoplasmic penetration of BG-KR IpTxa was measured when exposed to NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm while various inhibitors were added. BG-KR_IpTxa (BG-SEQ ID NO: 59) with no inhibitor was used as a comparator to assess the level of penetration in the absence of inhibitor. BG-GLA-OH, formed when BG-GLA-NHS is hydrolyzed, was used as a positive control for normalization. [0068] FIG. 14B shows the results of a SNAP penetration assay of a SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide MCa_varl in the presence of a variety of endocytosis inhibitors to assess mechanisms responsible for uptake of a BG-MCa_varl cellpenetrating peptide (BG-SEQ ID NO: 67). Cytoplasmic access of BG-MCa_varl was measured in NIH3T3, HeLa and HEK293 cells expressing SNAP-tag in the cytoplasm. BG-MCa_varl with no inhibitor was used as a comparator to assess the level of penetration in the absence of inhibitor. BG-GLA-OH, formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control for normalization.
[0069] FIG. 15A shows the results of a SNAP penetration assay of various SNAP substrate- tagged peptide (BG-peptide) comprising cell-penetrating peptides to measure cellular penetration into the cytosol of the cell-penetrating peptides fused to additional peptides that may further promote endosomal escape. Cytosolic penetration of BG-KR_IpTxa-PAS (BG-SEQ ID NO: 322) and BG-KR_IpTxa-S19 (BG-SEQ ID NO: 323) was measured in NIH3T3, HeLa and HEK293 cells expressing the SNAP-tag in the cytoplasm, along with BG-KR_IpTxa (BG-SEQ ID NO: 59). BG-KR_IpTxa (BG-SEQ ID NO: 59) without an endosomal escape peptide was used as a comparator to assess the baseline level of penetration in the absence of an endosomal escape peptide. The cell penetrant moiety BG-GLA-OH, formed when BG-GLA-NHS (BG- substrate) is hydrolyzed, was used as a positive control and for normalization.
[0070] FIG. 15B shows the results of a SNAP penetration assay of various SNAP substrate- tagged peptides (BG-peptides) comprising cell-penetrating peptides to measure cellular penetration to the nucleus of the cell-penetrating peptides fused to additional peptides that may further promote endosomal escape. Nuclear penetration of BG-KR_IpTxa-PAS (BG-SEQ ID NO: 322) and BG-KR_IpTxa-S19 (BG-SEQ ID NO: 323) was measured in NIH3T3, HeLa and HEK293 cells expressing the SNAP-tag in the nucleus, along with BG-KR_IpTxa (BG-SEQ ID NO: 59). BG-KR_IpTxa (BG-SEQ ID NO: 59) without an endosomal escape peptide was used as a comparator to assess the baseline level of penetration in the absence of an endosomal escape peptide. The cell penetrant moiety BG-GLA-OH, formed when BG-GLA-NHS (BG-substrate) is hydrolyzed, was used as a positive control and for normalization.
[0071] FIG. 16A schematically illustrates various types of cargo peptides that may be conjugated to cell -penetrating cystine-dense peptides or peptide fragments.
[0072] FIG. 16B schematically illustrates various types of antibody fragments that may be conjugated to cell -penetrating cystine-dense peptides or peptide fragments.
[0073] FIG. 17 illustrates a multiple sequence alignment of calcin variants MCa_varl (SEQ ID NO: 67), KR Urocalcin (SEQ ID NO: 39), KR IpTxa (SEQ ID NO: 59), KR Hemicalcin (SEQ ID NO: 34), KR_Opicalcin-l (SEQ ID NO: 37), KR_Opicalcin-2 (SEQ ID NO: 38), KR Vejocalcin (SEQ ID NO: 35), and KR Intrepicalcin (SEQ ID NO: 36).
[0074] FIG. 18 shows the results of mass spectrometry analysis identifying tryptic fragments of the SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR_IpTxa (SEQ ID NO: 59; BG-KR_IpTxa) conjugated to SNAP -tag following a cell penetration assay in HeLa cells. HeLa cells expressing GFP-SNAP-tag protein were incubated with 10 pM BG- KR IpTxa before subsequent trypsinization, lysis in mammalian protein extract reagent, and immunoprecipitation with magnetic beads. The recovered GFP-SNAP-tag conjugates were then submitted for mass spectrometry analysis. The data confirms that BG-KR_IpTxa penetrated the intact cells and reached SNAP -tag protein. FIG. 18 discloses SEQ ID NO: 616, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
[0075] FIG. 19 shows the results of mass spectrometry analysis identifying tryptic fragments of BG-KR_IpTxa conjugated to SNAP -tag following a cell penetration assay in HeLa cells to determine limits of sensitivity. HeLa cells expressing GFP-SNAP-tag were exposed to either BG-KR_IpTxa or BG-GLA-OH. One seventh of the cells were incubated with BG-KR_IpTxa and six sevenths were incubated with BG-GLA-OH. The cells were pooled and prepared as above for mass spectrometry analysis, demonstrating the ability to detect uptake of BG-KR- IpTxa at this ratio. FIG. 19 discloses SEQ ID NO: 617, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
[0076] FIG. 20 shows the results of mass spectrometry analysis identifying tryptic fragments of the SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR_IpTxa (SEQ ID NO: 59) (BG-KR_IpTxa) conjugated to GFP-SNAP-tag protein. HeLa GFP-SNAP cells were lysed with M-PER supplemented with protease and phosphatase inhibitors before incubating with 10 pM BG-KR_IpTxa for 2 hours at 4C. Detection of the modified GFP-SNAP- tag tryptic fragment TALSGNPVPILIPCHR (SEQ ID NO: 613) was detected and identified by MS2, serving as a positive control. FIG. 20 discloses SEQ ID NO: 617, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
[0077] FIG. 21 shows the results of mass spectrometry analysis identifying additional tryptic fragments of the SNAP substrate-tagged peptide (BG-peptide) comprising cell-penetrating peptide KR IpTxa (SEQ ID NO: 59; BG-KR_IpTxa) conjugated to GFP-SNAP-tag protein in a cell free environment. Prototypic peptides from BG-KR_IpTxa were also detected under the previously described conditions with the tryptic fragment highlighted detected and identified by MS2, indicating the presence of intact BG-KR_IpTxa. FIG. 21 discloses SEQ ID NO: 618 (ADNDCCGR) [0078] FIG. 22 shows the results of mass spectrometry analysis identifying tryptic fragments of BG-KR_IpTxa (SEQ ID NO: 59) in a cell free environment. HeLa GFP-SNAP cells were lysed with M-PER supplemented with protease and phosphatase inhibitors before incubating with IpM BG-KR_IpTxa for 2 hours at 4 °C. The modified GFP-SNAP-tag was detected and identified by MS2. FIG. 22 discloses SEQ ID NO: 619, SEQ ID NO: 613, and SEQ ID NO: 613, respectively, in order of appearance.
[0079] FIG. 23 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with PBS in the SNAPPA. Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP -Cell TMR-Star dye). [0080] FIG. 24 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with 2 pM hydrolyzed BG-GLA-NHS (BG-GLA-OH) in the SNAPPA. Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP-Cell TMR-Star dye).
[0081] FIG. 25 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with 10 pM BG-KR_IpTxa in the SNAPPA. Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP- Cell TMR-Star dye).
[0082] FIG. 26A shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the cytosol of 3T3 cells. SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP -substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the cytosol, which was quantified as 936 nM based on these results. The maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
[0083] FIG. 26B shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the cytosol of HeLa cells. SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP -substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the cytosol, which was quantified as 700 nM based on these results. The maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH. [0084] FIG. 26C shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the cytosol of HEK-293 cells. SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the cytosol, which was quantified as 1378 nM based on these results. The maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
[0085] FIG. 27A shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the nucleus of 3T3 cells. SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the nucleus, which was quantified as 1632 nM based on these results. The maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
[0086] FIG. 27B shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the nucleus of HeLa cells. SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the nucleus, which was quantified as 471 nM based on these results. The maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
[0087] FIG. 27C shows quantification of fluorescence measurements to measure concentrations of GFP-SNAP-tag protein expressed in the nucleus of HEK-293 cells. SNAP penetration assays were performed with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH) to determine the maximum concentration of GFP-SNAP-tag protein expressed in the nucleus, which was quantified as 1523 nM based on these results. The maximum concentration calculation assumes that that normalized fluorescence would be zero when GFP-SNAP-tag protein is fully occupied by BG-GLA-OH.
[0088] FIG. 28A shows the results of SNAP penetration assays measuring the cytosolic uptake of BG-SEQ ID NO: 320 in the presence of various chemical inhibitors of different mechanisms of endocytosis. Uptake was measured in 3T3 (“3T3 GFP-SNAP”), HeLa (“HeLa GFP-SNAP”), and HEK-293 (“293 GFP-SNAP”) cell lines expressing GFP-SNAP-tag protein in the cytosol. Uptake is shown as the fraction of GFP-SNAP-tag protein that was occupied, where a fraction of 1.0 indicates 100% of the protein was occupied. The cell penetrant moiety BG-GLA-OH was used for normalization to 100% and PBS was used for normalization to 0%. The results show that inhibitors of various mechanisms of endocytosis inhibited uptake to various extents, though none caused complete inhibition, indicating that various endocytic mechanisms are involved in cellular penetration of BG-SEQ ID NO: 320 with some variation in mechanism between cell types.
[0089] FIG. 28B shows the results of SNAP penetration assays measuring the cytosolic uptake of BG-SEQ ID NO: 321 in the presence of various chemical inhibitors of different mechanisms of endocytosis. Uptake was measured in 3T3 (“3T3 GFP-SNAP”), HeLa (“HeLa GFP-SNAP”), and HEK-293 (“293 GFP-SNAP”) cell lines expressing GFP-SNAP-tag protein in the cytosol. Uptake is shown as the fraction of GFP-SNAP-tag protein that was occupied, where a fraction of 1.0 indicates 100% of the protein was occupied. The cell penetrant moiety BG-GLA-OH was used for normalization to 100% and PBS was used for normalization to 0%. The results show that inhibitors of various mechanisms of endocytosis inhibited uptake to various extents, though none caused complete inhibition, indicating that various endocytic mechanisms are involved in cell penetration of BG-SEQ ID NO: 321, with variation in mechanism between cell types. The inhibition was, in general, less than the inhibition seen for BG-SEQ ID NO: 320, indicating that other mechanisms than those inhibited here may play a larger role in cell penetration of BG-SEQ ID NO: 321 than for BG-SEQ ID NO: 320.
[0090] FIG. 29A illustrates examples of components for use in building a targeted degradation complex for targeted degradation of TEAD. Components include a cell-penetrating peptide, a TEAD-binding cystine-dense peptide (CDP), a linker, and an immunomodulatory imide drug (IMiD). The TEAD-binding CDP is capable of binding TEAD and the IMiD is capable of binding cereblon (CRBN), an E3 ubiquitin ligase.
[0091] FIG. 29B illustrates construction of a targeted degradation complex for targeted degradation of TEAD. The IMiD is linked to the cell-penetrating peptide/TEAD-binding CDP complex via a linker, thereby forming a targeted degradation complex.
[0092] FIG. 29C illustrates binding of the targeted degradation complex described in FIG. 29B to TEAD.
[0093] FIG. 29D illustrates ubiquitination and subsequent degradation of TEAD facilitated by the targeted degradation complex described in FIG. 29B. The targeted degradation complex forms a ternary complex with TEAD and CRBN. Recruitment of CRBN to TEAD facilitates ubiquitination of TEAD, which targets TEAD for degradation.
[0094] FIG. 30 illustrates examples of molecules capable of binding an E3 ubiquitin ligase that may be incorporated into a targeted degradation complex. Molecules capable of binding an E3 ubiquitin ligase include thalidomide or pomalidomide which bind cereblon (CRBN), methyl bestatin or bestatin, which bind cellular inhibitor of apoptosis protein 1 (cIAPl), nutlin-3 which binds MDM2, and VHL ligand 1 which binds von Hippel-Lindau protein (VHL).
[0095] FIG. 31 shows a western blot of cell lysates generated from HeLa GFP-SNAP cells that were treated with BG-peptides and then washed and lysed, where the blot was stained with an anti-GFP antibody. Intact cells were incubated with 10 pM BG-SEQ ID NO: 320 or 10 pM BG- SEQ ID NO: 321 for 15, 30 or 60 minutes prior to washing, lysis, and sample preparation. The band slightly below the 55 kDa marker may represent GFP-SNAP protein. The band between the 55 kDa and the 72 kDa markers may represent the BG-peptide of SEQ ID NO: 320 or SEQ ID NO: 321 occupying the GFP-SNAP protein, thus causing a shift upward in molecular size versus the GFP-SNAP protein alone. The fainter band between 95 and 130 kDa may represent a dimer of two GFP-SNAP proteins linked and occupied by a BG-peptide that contains at least two BG moieties (Lys residues and the N-terminus may react with BG-GLA-NHS). Occupancy of GFP-SNAP protein indicates the peptides of SEQ ID NO: 320 and SEQ ID NO: 321 penetrated the cells and entered the cytosol. The loss of the shifted GFP-SNAP band (between 55 and 72 kDa) over time suggests some intracellular degradation of exposed BG-peptide (where the cleavage could be on the N-terminal GS, the cell penetrating MCa or Had sequences, the GGS linker, or the TEAD binding peptide) or residues of the SNAP protein proximal to the reactive site may be occurring, resulting in the continued occupation of GFP-SNAP -tag protein and preventing subsequent BG-fluorophore binding.
[0096] FIG. 32 illustrates examples of structures of various peptide oligonucleotide complexes (e.g., a CPP-oligonucleotide complexes in which the peptide portion comprises a CDP or a fragment thereof) containing alternative and nonconventional bases, as represented in singlestranded, double-stranded, and hairpin structures. Examples of oligonucleotides include an aptamer, a gapmer, an anti-miR, an siRNA, a splice blocker ASO, and a U1 adapter. The CPP portion of the CPP-oligonucleotide complex can be used to guide the oligonucleotide sequence to a specific tissue, target, or cell, or to cause endocytosis, cytosolic or nuclear penetration, or endosomal escape of the oligonucleotide sequence by a cell. The legend is as follows: grey circle with black border = 2’-H (DNA); white circle with black border = 2 ’-OH (RNA); circle with horizontal stripes and black border = 2’-0-ME; circle with vertical stripes = 2’-0-M0E; black circle with grey border = 2’-F; spotted circle with grey border = LNA; hatched circle with grey border = morpholino (unique phosphorodiamidate linkages not shown); grey angle = PO linkage; black angle = PS linkage.
[0097] FIG. 33A - FIG. 33E illustrate incorporation of the shown groups on RNA or DNA. [0098] FIG. 33A illustrates structures of oligonucleotides containing a 5 ’-thiol (thiohexyl; C6) modification (left), and a 3 ’-thiol (C3) modification (right).
[0099] FIG. 33B illustrates an MMT-hexylaminolinker phosphoramidite. [0100] FIG. 33C illustrates a TFA-pentylaminolinker phosphoramidite. [0101] FIG. 33D illustrates RNA residues incorporating amine or thiol residues. [0102] FIG. 33E illustrates oligonucleotides with aminohexyl modifications at the 5’ (left) and 3’ ends (right).
[0103] FIG. 34 illustrates generation of a cleavable disulfide linkage between a peptide (e.g., a cell-penetrating peptide of SEQ ID NO: 1) and a cyclic dinucleotide.
DETAILED DESCRIPTION
[0104] Drug design is often limited by the ability of a drug to access intracellular targets. Small molecule therapeutics can often diffuse through the cell membrane to reach intracellular targets, but small molecules may be unable to specifically and potently inhibit protein-protein interactions, or those interactions lacking well-defined binding pockets. Protein- or peptide- based therapeutics, such as antibodies, antibody fragments, and peptides, are more capable of specifically and potently disrupting protein-protein interactions, targeting poorly-defined binding pockets, or drive the formation of protein-protein interactions or otherwise have a therapeutic effect, but protein- or peptide-based drugs may have a difficult time reaching the intracellular milieu. Proteins and peptides are often too large or too hydrophilic to diffuse across the cell membrane. Some proteins or peptides are taken up into endosomes but are then trafficked to lysosomes for degradation, or the proteins or peptides are released back to the extracellular space without ever reaching the cytosol, the nucleus, or other subcellular compartments at sufficient concentrations for therapeutic effect. Likewise, RNA and DNA are large, hydrophilic, and charged and are often unable to diffuse across the cell membrane or unable to exit the endosome in order to access the cytosol, nucleus, or other subcellular compartments at levels sufficient for therapeutic effect
[0105] Described herein are cell -penetrating peptides capable of penetrating cellular layers and accessing intracellular spaces, such as the cytoplasm, nucleus, or other subcellular compartments and in some aspects, intercellular compartments, such as nanolumen. These cell-penetrating peptides can carry cargo molecules, such as peptide-based therapeutics (e.g., cystine-dense peptide-based therapeutics), protein-based therapeutics, nucleotide-based therapeutics, or small molecule therapeutics with low permeation or absorption, across cell membranes, thereby delivering the cargo into intracellular or intercellular spaces. Also described herein are methods delivering peptides or other cargoes across cell membranes using cell-penetrating peptides. In some embodiments, the cell-penetrating peptides of the present disclosure, and complexes or conjugates thereof, penetrate cells and reach an intracellular concentration, or reach an intracellular space, at a sufficient amount to exert a prophylactic or therapeutic effect.
[0106] Delivery of peptides (e.g., proteins, peptides, or cystine-dense peptides) and other cargos (e.g., target-binding molecules, small molecules, RNAs, mRNAs, DNAs, active agents, macromolecular agents, detectable agents, therapeutic agents, or drugs) across cell membranes may facilitate interactions between the cargo and various intracellular targets. For example, delivery of therapeutic agents across the cell membrane may increase access of the therapeutic agent to intracellular drug targets. Many therapeutically relevant targets have been deemed “undruggable targets” in part due to their location in the nucleus as well as the nature of their functional interactions. For example, transcription factors responsible for driving many types of cancer, such as KRAS, MYC, MYB, FOS, JUN, ABL, NF-KB, RAS, RHO, RAN, RAB, and TEAD reside in the nucleus, and their functional protein-protein and protein-nucleic acid interactions may be difficult to target using small molecule therapeutics, which may penetrate cells but may not specifically or potently interact with the transcription factor or block proteinprotein interactions (PPIs). Transcription factors residing in the nucleus may be inaccessible to protein and peptide drugs that are capable of specifically and potentially interacting with the transcription factor or blocking protein-protein interactions. Exemplary transcription factors used for targeting the cell -penetrating peptides of the disclosure include transcription factors disclosed in “Targeting Transcription Factors for Cancer Treatment” Molecules. 2018 Jun; 23(6): 1479, which is hereby incorporated by reference. Dysregulation of these transcription factors is responsible for driving many cancers and have been difficult to target with small molecules as they interact and coordinate large protein complexes. Peptide therapeutics that modulate transcription factor interactions may be delivered to the nucleus using the cellpenetrating peptides of disclosed herein, enabling modulation of targets previously considered “undruggable”. Scaffold proteins in the cytoplasm coordinate multiple proteins into complexes through similar mechanisms and are also difficult to target using small molecule therapeutics. The cell-penetrating peptides of the present disclosure can be used to deliver agents that modulate these protein complex interactions to drug targets in the cytoplasm. For example, a cell-penetrating peptide of the present disclosure may deliver a therapeutic agent into the cytoplasm to target the inflammasome, which may be responsible for activating inflammatory responses and coordinated by the Nod-like receptor (NLR) family of proteins. Low-grade inflammation driven by NLR family pyrin domain-containing protein 3 (NLRP3) contributes to diabetes and aging. The ability to modulate inflammasome activation is an appealing cytoplasmic target.
[0107] The cell-penetrating peptides of the present disclosure may facilitate delivery of small peptides or other therapeutic molecules into intracellular cellular compartments (e.g., the cytoplasm, the nucleus, lysosomes, or other subcellular compartments) or intercellular compartments (e.g., nanolumen, intercellular space, or paracellular space). Drug makers often adhere to the “rule of five” to design small molecule drugs with acceptable pharmacological properties, including permeation or absorption, restricting drug design to molecules with five or fewer hydrogen bond donors, ten or fewer hydrogen bond acceptors, a molecular weight of less than 500 Da (i.e., 500 g/mol), and a log of the partition coefficient of less than 5. The cellpenetrating peptides of the present disclosure may be used to deliver drugs that do not adhere to the “rule of five” and may not otherwise be able to reach intracellular drug targets. For example, the peptides of the present disclosure may be used to deliver heterobifunctional targeted degradation molecules, such as PROTAC molecules, which often do not follow the rule of five. A targeted degradation molecule may be around 1 kDa in size and comprise an E3 ubitiquitin ligase recruiting ligand linked to a target-binding molecule that binds to a protein of interest. These targeted degradation molecules can recognize and promote the ubiquitination and subsequence degradation of specific proteins. Delivery of small reactive antibody fragments of various size, such as camelid-derived nanobodies (~15kDa), scFv (~27kDa), Fab (~50kDa), VHH, VH, Fv, diabodies, and minobodies, would enable the modulation of a vast array of PPIs or other targets. Delivery of other small miniproteins such as cystine-dense peptides (including knottins and hitchins and cyclotides), adnectins, affibodies, affilins, anticalins, atrimers, avimers, fynomers, kunitz domains, humabodies, Obodies, nanofittins, centyrins, DARPins, stapled peptides, cyclic peptides, bicyclic peptides, pronectins, macroclycics, solomers, VNARs, IgNARs, and lasso peptides would also enable the modulation of a vast array of protein-protein interactions (PPIs) or other targets.
[0108] Delivery of CRISPR Cas9 proteins (~160kDa) and other CRISPR components (e.g., guide RNAs, tracrRNAs, or crRNAs) using the cell-penetrating peptides of the present disclosure could facilitate gene editing. Delivery of antisense RNA (>30kDa) using the cellpenetrating peptides of the present disclosure could be used to inhibit translation of problematic proteins. In some embodiments, the active agent is an anti-cancer agent, a molecule that binds a transcription factor, a molecule that blocks intracellular protein-protein interactions, a molecule that causes the formation or stabilization of intracellular protein-protein interactions, a transcription factor, an RNA, a Cas enzyme or other CRISPR component, an immunomodulating agent, a molecular glue, a targeted degradation molecule, an inhibitor of protein-protein interactions, an inhibitor of enzymatic activity, or a neurotransmitter.
[0109] In some embodiments, a cell-penetrating peptide may be complexed with a RIG-I ligand or a receptor for a RIG-I-like receptor (e.g., MDA5 or TLR3). The ligand may be a double stranded RNA (dsRNA) and may be delivered to a desired cellular compartment (e.g., the cytoplasm or an endosome) to activate the receptor. Delivery of RIG-I ligands across a cellular layer may promote anti -tumor or anti-viral activity in a subject and may be used to treat a cancer or viral infection in a subject.
[0110] The cell-penetrating peptides of the present disclosure may deliver agents (e.g., cargos, cargo peptides, cargo proteins, or cargo molecules) that drive the formation of protein-protein interactions or that block the formation of protein-protein interactions. In some embodiments, the cell-penetrating peptides may deliver agents to the cytoplasm. In some embodiments, the cell-penetrating peptides may deliver agents to the nucleus. In some embodiments, the cellpenetrating peptides may deliver agents to a nanolumen. In some embodiments, a cellpenetrating peptide may deliver an agent to the nucleus that promotes formation of a transcription factor complex or disrupts formation of a transcription factor complex. In some embodiments, the agent may activate or inhibit the transcription factor. For example, a cellpenetrating peptide of the present disclosure may deliver an agent to the nucleus that promotes or disrupts protein-protein interactions within transcription factor complexes or protein-nucleic acid interactions AP-2, ARID/BRIGHT, ARID/B RIGHT (RFX), AT hook, BED ZF, bHLH, Brinker, bZIP, C2H2 ZF, C2H2 ZF (KRAB), C2H2 ZF (non-KRAB), C2H2 ZF (AT hook), C2H2 ZF (BED ZF), C2H2 ZF (Homeodomain), C2H2 ZF (Myb/SANT), CBF/NF-Y, CCCH ZF, CENPB, CG-1, CSD, CSL, CUT (Homeodomain), CxxC, CxxC (AT hook), DM, E2F, EBF1, Ets, Ets (AT hook), FLYWCH, Forkhead, FYVE-type ZF, GAT A, GCM, Grainyhead, GTF2I-like, HMG/Sox, HMG/Sox (bHLH), Homeodomain, Homeodomain (Paired box), Homeodomain (POU), HSF, IRF, LYAR-type C2H2 ZF, MADF, MADS box, MBD, MBD (AT hook), MBD (CxxC ZF), mTERF, Myb/SANT, Myb/SANT (GAT A), MYM-type ZF, MYND- type ZF, Ndt80/PhoG, NFX, NOA36-type ZF, Nuclear receptor, p53, Paired box, Pipsqueak, Prospero, Rel, RFX, Runt, SAND, SART-1, SBP, SMAD, STAT, T-box, TBP, TCR/CxC, TEA, THAP finger, or ZZ-type ZF transcription factor. In some embodiments, a cell-penetrating peptide of the present disclosure may deliver an agent to the nucleus that promotes or disrupts protein-protein interactions with MLL, GABP, RUNX1, KLF8, SIX1, RUNX2, PML, RARu, FOXO, TALI, or Myc [oni] The cell-penetrating peptides of the present disclosure may deliver agents (e g., cargos, cargo peptides, cargo proteins, or cargo molecules) that can cause targeted protein degradation, for example by functioning as molecular glues, proteolysis-targeting chimeras (PROTACs), inhibitors of protein-protein interactions, or targeted degradation complexes. For example, a cell-penetrating peptide may deliver a thalidomide-like molecule to the cytosol. Thalidomidelike molecules that may function as immunomodulatory imide drugs (IMiDs), such as thalidomide, pthalidomide (a-(N-phthalimide)glutarimide), phthalimide derivatives, thalidomide analogues, thalidomide hybrids (e.g., N-phenyl-phthalimide sulfonamides (3a-e), isosters biphenyl -phthalimide amides (4a-e)), pomalidomide, and lenalidomide, can bind to cereblon, cause a conformational change in cereblon, and drive the formation of a complex between cereblon and proteins such as Ikaros and Aiolos, whereby they are ubiquitinated by the CRL4 complex and undergo degradation by the ubiquitin-proteosome system (UPS). In this context, IMiDs may be called molecular glues, which cause the formation of PPIs and cause the targeted degradation of proteins. Similarly, the cell-penetrating peptides of the present disclosure may deliver agents to the cytosol that bind to cereblon, VHL, DCAF15, DCAF16, other molecules in the CRL4 E3 ligase family, other molecules in the E3 ligase family, or other molecules in the UPS system, drive the formation of a complex with another substrate or neosubstrate, and thereby cause ubiquitination and degradation of the substrate or neosubstrate by the UPS, acting as molecular glues. Non-limiting examples of agents that can cause targeted protein degradation and can be delivered by a cell -penetrating peptide of the present disclosure may be found in Collins, et al (Biochem J. 2017 Apr 1; 474(7): 1127-1147), Chopra, et al (Drug Discov Today Technol, 2019 Apr; 31 :5-13), and Chamberlain, et al (Nat Chem Biol, 2019 Oct; 15(10):937- 944) each of which is incorporated by reference in its entirety. Molecular glues can be small molecules, but small molecules may not be able to induce formation of ternary complexes between proteins desired for degradation and the E3 ligase complex. In some embodiments, a cell-penetrating peptide can deliver an agent that functions as a molecular glue to the cytosol. In some embodiments, a cell-penetrating peptide can deliver an agent that functions as a targeted degradation complex to the cytosol. Targeted degradation complexes may comprise a molecule that binds to a ubiquitin ligase (e.g., cereblon, VHL, DCAF15, DCAF16, other molecules in the CRL4 E3 ligase family, other molecules of the Cul2-Rbxl-EloN/C-VHL E3 ligase, the MDM2 E3 ligase, cIAPl, cIAP, APCZC(CDHl), or other molecules in the E3 ligase family), a linker, and target-binding molecule that binds to a protein targeted for degradation. By bringing the E3 ligase and the target together, the targeted protein is ubiquitinated and then degraded by the UPS. In some embodiments, a targeted degradation complex may comprise a small molecule that does not fit the “rule of five” often used to constrain molecule size and solubility parameters for successful small molecule therapeutics. Protein degradation can also be caused by molecules that bind the protein targeted for degradation and that also contain an IMiD, a Boc3-Arg tag, an adamantyl group, or a carborane as hydrophobic tag that causes binding to the 20S proteosome or to HSP70. The cell-penetrating peptides of this disclosure may act as molecular glues or as PROTACS or otherwise cause targeted protein degradation. Alternatively or in addition, the cell-penetrating peptides of this disclosure may serve to deliver active agents that act as molecular glues or targeted degradation complexes or that otherwise drive targeted degradation to the cytosol, thereby allowing those active agents to reach their targets and cause targeted protein degradation. Proteins that can targeted for degradation by a cell-penetrating peptide or a cell-penetrating peptide complex may include TEAD, cold-inducible RNA-binding protein (also referred to as CIRP or CIRBP), androgen receptor, ikaros, aiolos, nuclear receptors, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, or tau. In some embodiments, a cellpenetrating peptide complex of the present disclosure may target AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, P-catenin, IKK, TEAD, NF-KB, PKC, AP-1, Jun, Fos, REL or a transcription factor for degradation. In some embodiments, a cell -penetrating peptide or an active agent for targeted protein degradation can have one or more lysine residues removed or replaced with other amino acids to prevent labeling of the lysine residues with ubiquitin. By targeting proteins for degradation, they may be reduced or removed from cells, thereby removing functions that contribute to disease. Proteins that can be degraded using the cell -penetrating peptides or cellpenetrating peptide complexes of the present disclosure may be transcription factors, kinases, zinc finger domain containing, may perform a scaffolding function, or may be adaptor proteins. [0112] The cell-penetrating peptides of the presence disclosure may themselves be agents that cause a therapeutic effect by binding to or interacting with various molecules within the cytosol, nucleus, or other subcellular, intracellular, or paracellular compartments. A cell-penetrating peptide may promote or disrupt a protein-protein interaction in the subcellular, intracellular, or paracellular compartment. Alternatively or in addition, a cell -penetrating peptide may deliver an agent that cause a therapeutic effect to the cytosol, nucleus, or other subcellular compartment. [0113] Described herein are cell -penetrating peptides, including, but not limited to, designed or engineered peptides, recombinant peptides, synthetic peptides, and small cystine-dense peptides (or disulfide-knotted peptides, knottins, knotted peptides, or hitchins), that are capable of penetrating cellular layers (e.g., cell membranes, nuclear envelopes, endosomes, lysosomes, or other subcellular compartments) to access intracellular or intercellular spaces. Cell-penetrating peptides can include calcines (e.g., imperatoxin A (IpTxa), maurocalcin (MCa), hadrucalcin (Had), hemicalcin, vejocalcin, intrepicalcin, opicalcin-1, opicalcin-2, or urocalcin), toxins (e.g., chlorotoxin (CTX), huwentoxin IV (HwTx-IV), potassium channel toxin-like Tx677, potassium channel toxin KTx2.2, potassium channel toxin KTxl5.8), CTI, and variants and fragments thereof. A cell-penetrating peptide of the present disclosure may be conjugated to, linked to, or fused with a cargo molecule (e g., a peptide, a cystine-dense peptide, a small molecule, a protein, an RNA, an mRNA, a DNA, an active agent, a detectable agent, a therapeutic agent, or a drug) to generate a cell-penetrating peptide complex. In some embodiments, the cellpenetrating peptide may itself be an active agent. These cell-penetrating peptide complexes may be used to deliver cargo molecules across cellular layers (e.g., cell membranes, nuclear envelopes, intercellular spaces, paracellular spaces, endosomal membranes, lysosomal membranes, blood brain barriers, or nanolumen) and into cellular, intercellular, or paracellular compartments or spaces (e.g., cytosols, nuclei, or nanolumen). The cell-penetrating peptides and cell-penetrating peptide complexes of the present disclosure may be used in various methods to deliver cargo molecules to intracellular and intercellular targets (e.g., drug targets). For example, a cell-penetrating peptide of the present disclosure may be conjugated to a therapeutic agent (e.g., a small molecule drug, a peptide drug, a protein drug, a biologic drug, a transcription factor, a gene editing agent, or a disruptor of PPIs or a driver of PPIs) that would otherwise be excluded from intracellular spaces, and the resulting cell-penetrating peptide complex may be administered to a subject (e.g., a human subject) to deliver the therapeutic agent to a target of the therapeutic agent (e g., a protein or a nucleic acid) located in a cellular, intercellular, or paracellular compartment or space (e.g., cytoplasm, nucleus, or nanolumen).
[0114] Additional aspects and advantages of the present disclosure will become apparent to those skilled in this art from the following detailed description, wherein illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
[0115] As used herein, the abbreviations for the natural L-enantiomeric amino acids are conventional and are as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Vai). Typically, Xaa can indicate any amino acid. In some embodiments, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R). In some embodiments, a peptide of the present disclosure can comprise a nonnatural amino acid, wherein the non-natural amino acid can be an insertion, appendage, or substitution for another amino acid. A non-natural amino acid may comprise a D-isomer, a homo-amino acid, a B-homo-amino acid, an N-methyl amino acid, an a-methyl amino acid. In some embodiments, a non-natural amino acid can comprise citrulline (Cit), hydroxyproline (Hyp), norleucine (Nle), 3-nitrotyrosine, nitroarginine, ornithine (Om), naphtylalanine (Nal), aminobutyric acid (a, Abu), di aminobutyric acid (Dab), methionine sulfoxide, or methionine sulfone.
[0116] The present disclosure refers to amino acid residues and positions of amino acid residues within a sequence. As referred to herein, a position can be referred to as “position X,” wherein X is the position number within a sequence not counting, if present, the N-terminal GS. For example, position 5 in SEQ ID NO: 59 (GSDCLPHLRRCRADNDCCGRRCRRRGTNAERRCR) refers to the “H” residue at position 5 within the sequence, wherein position 1 does not count the N-terminal “GS,” and is, thus, the first D residue. As another example, position 9 in SEQ ID NO: 66 (GSECLEIFRACNPSNDQCCRSSRLVCSRRTRWCRYQIG) refers to the “A” residue within the sequence at position 9, wherein position 1 does not count the N-terminal “GS,” and is, thus, the first E residue. In some embodiments, the N-terminal GS is included in the sequence as a byproduct of proteolytic cleavage during recombinant protein production or a spacer. As yet another example, position 11 in SEQ ID NO: 18 (GDCLPHLRLCKENRDCCSRRCKRRGTNIERRCR) refers to the “K” residue at position 11 within the sequence, wherein position 1 is the very first G residue and there is no N-terminal GS. As also referred to herein, a position can be referred to as “XY,” wherein X is the single-letter abbreviation for an amino acid and wherein Y is the position number within a sequence not counting, if present, the N-terminal GS. For example, G15 refers to the “G” residue at position 15 within a sequence, not counting, if present, an N-terminal GS. As another example, Y23 refers to the “Y” residue at position 23 within a sequence, not counting, if present, an N-terminal GS. As yet another example, E25 refers to the “E” residue at position 25 within a sequence, not counting, if present, an N-terminal GS. As also referred to herein, a position can be referred to as “XYZ,” wherein X and Z are the single-letter abbreviation for an amino acid and wherein Y is the position number within a sequence not counting, if present, the N-terminal GS. For example, L23A refers to a “L” residue at position 23 within a sequence (not counting, if present, an N- terminal GS), which has been substituted with a “A” residue. As another example, E25A refers to an “E” residue at position 25 within a sequence (not counting, if present, an N-terminal GS), which has been substituted with an “A” residue. As yet another example, F27A refers to an “F” residue at position 27 within a sequence (not counting, if present, an N-terminal GS), which has been substituted with an “A” residue.
[0117] Some embodiments of the disclosure contemplate D-amino acid residues of any standard or non-standard amino acid or analogue thereof. When an amino acid sequence is represented as a series of three-letter or one-letter amino acid abbreviations, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy terminal direction, in accordance with standard usage and convention.
[0118] As used herein, the term “peptide complex” may refer to a peptide that is fused to, recombinantly fused to, linked to, conjugated to, chemically conjugated to, recombinantly expressed with, complexed with, or is otherwise connected to an additional agent (e g., a protein, a peptide, a cystine-dense peptide, a small molecule, an RNA, an mRNA, a DNA, an active agent, macromolecular agent, a detectable agent, a therapeutic agent, or a drug). In some embodiments, a peptide complex may be a peptide construct. In some embodiments, a peptide complex may be a peptide conjugate. In some embodiments, a peptide complex of the present disclosure may comprise a cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254, or a variant or fragment thereof) that is fused, linked, conjugated, recombinantly expressed with, or is otherwise connected to a cargo molecule (e.g., a peptide, a peptide, a cystine-dense peptide, a small molecule, an RNA, an mRNA, a DNA, an active agent, macromolecular agent, a detectable agent, a therapeutic agent, or a drug), and may be referred to herein as a cell-penetrating peptide complex. In some embodiments, a peptide complex may comprise a linker (e.g., a peptide linker of any one SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485, or a small molecule linker as described herein).
[0119] As used herein, a “cargo,” “cargo peptide,” “cargo protein,” or “cargo molecule” may be any protein, peptide, or molecule that is fused, linked, conjugated, recombinantly expressed with, or is otherwise connected to a cell-penetrating peptide. A cargo, cargo peptide, cargo protein, or cargo molecule may be carried across a cellular layer (e.g., a plasma membrane, a vesicular membrane, an endosomal membrane, a nuclear envelope, or a blood brain barrier) by a cell-penetrating peptide of the present disclosure. A cargo, cargo peptide, cargo protein, or cargo molecule may be carried within the vesicles of a cell's secretory system (e.g., within the endoplasmic reticulum, Golgi apparatus, lysosomes, or other subcellular compartment, whether to or from the plasma membrane) or within a vesicle as a structure within or outside a cell, comprising liquid or cytoplasm enclosed by a lipid bilayer. A cargo peptide, cargo protein, or cargo molecule may be a protein, a peptide, a cystine-dense peptide, a small molecule, an RNA, an mRNA, a DNA, an active agent, macromolecular agent, a detectable agent, a therapeutic agent, or a drug.
[0120] In this disclosure, “penetration” (e.g., membrane penetration, cytosolic penetration, nuclear penetration, or blood brain barrier penetration) includes movement of a molecule from the extracellular space to the cytosol, the nucleus, or other subcellular compartments by any means, and may also be described as cytosolic access or cytosolic delivery. Cell penetration may also include endosomal uptake, such as by macropinocytosis, clathrin-mediated endocytosis, calveolae-dependent endocytosis, or other endocytosis, followed by endosomal escape such that the molecule leaves the endosome and enters the cytosol. It is understood that cell penetration is not limited to a molecule solely entering endosomes not further accessing or entering the cytosol.
Peptides
[0121] Disclosed herein are peptide sequences, such as those listed in TABLE 1, capable of cell penetration. A peptide capable of penetrating a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, a cell membrane, an endosomal membrane, a lysosomal membrane, a nuclear envelope, or other subcellular compartment membrane, a blood brain barrier, or a nanolumen) may also be referred to herein as a cellpenetrating peptide. A cell -penetrating peptide may enter the cytosol. Alternatively or in addition, a cell-penetrating peptide may enter the nucleus. In some embodiments, the cellpenetrating peptides disclosed herein can penetrate or enter target cells, such as cancerous or tumor cells, liver cells, pancreas cells, colon cells, ovarian cells, breast cells, and/or lung cells, or any combination thereof. In this disclosure, cell penetration includes movement of a molecule from the extracellular space to the cytosol, the nucleus, or other subcellular compartments by any means, and may also be described as cytosolic access or cytosolic delivery. Cell penetration may also include endosomal uptake, such as by macropinocytosis, clathrin-mediated endocytosis, calveolae-dependent endocytosis, or other endocytosis, followed by endosomal escape such that the molecule leaves the endosome and enters the cytosol. It is understood that cell penetration is not limited to a molecule solely entering endosomes not further accessing or entering the cytosol.
[0122] In some embodiments, a peptide or a library of peptides is designed in silica without derivation from a naturally occurring knottin scaffold. In other embodiments, a peptide or a library of peptides is designed in silica by derivation, grafting relevant or important proteinbinding residues, or conserved residues, in the protein-binding interface, or structural modeling based on a naturally occurring peptide or protein known to bind to a protein or receptor of interest. In some embodiments, a library of peptides is screened for the ability to access a cellular compartment, such as the cytoplasm or the nucleus, using a fluorescence-based screening method or a mass spectrometry -based screening method, as described herein.
[0123] In some embodiments, a peptide with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is used as a scaffold or base sequence for further modifications, including addition, deletion, or amino acid substitution. For example, a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 may be modified by performing one or more amino acid substitutions to introduce one or more lysine, arginine, proline, or histidine amino acids. In some embodiments, residues GS are added at the N-terminus of a peptide. In some cases, peptides lack GS at the N-terminus. In some instances, peptides undergo one or more post-translational modifications. In some embodiments, peptides are truncated so that they contain fewer amino acids, or are less than 50, less than 40, less than 30, less than 20, less than 15, less than 12, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, or less than 4 amino acids long.
[0124] TABLE 1 lists exemplary peptide sequences according to the present disclosure.
TABLE 1 - Exemplary Cell-Penetrating Peptide Sequences
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
[0125] In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more positively charged amino acid residues (e.g., lysine or arginine). Positively charged amino acid residues may mediate interactions between the cell-penetrating peptide and negatively charged cellular layers (e.g., cell membranes, nuclear envelopes, intercellular spaces, paracellular spaces, endosomal membranes, lysosomal membranes, or other subcellular compartment membrane, blood brain barriers, or nanolumen). A cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positively charged amino acid residues. In some embodiments, a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 positively charged amino acid residues. A cell-penetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 positively charged amino acid residues. Positively charged amino acid residues may include lysine residues and arginine residues.
[0126] In some embodiments, a cell-penetrating peptide may comprise at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1. 1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1 .6, at least 1 .7, at least 1.8, at least 1 .9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3.7, at least 3.8, at least 3.9, or at least 4 positively charged amino acid residues per 10 amino acid residues. In some embodiments, a cell-penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2. 1, no more than 2, no more than 1.9, no more than 1.8, no more than 1.7, no more than 1.6, no more than 1.5, no more than 1.4, no more than 1.3, no more than 1.2, no more than 1.1, or no more than 1 positively charged amino acid residues per 10 amino acid residues. In some embodiments, a cell-penetrating peptide may comprise no more than 0.5, no more than 0.6, no more than 0.7, no more than 0.8, no more than 0.9, no more than 1, no more than 1.1, no more than 1.2, no more than 1.3, no more than 1.4, no more than 1.5, no more than 1.6, no more than 1.7, no more than 1.8, no more than 1.9, no more than 2, no more than 2.1, no more than 2.2, no more than 2.3, no more than 2.4, no more than 2.5, no more than 2.6, no more than 2.7, no more than 2.8, no more than 2.9, no more than 3, no more than 3. 1, no more than 3.2, no more than 3.3, no more than 3.4, no more than 3.5, no more than 3.6, no more than 3.7, no more than 3.8, no more than 3.9, or no more than 4 positively charged amino acid residues per 10 amino acid residues. A cellpenetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 positively charged amino acid residues per 10 amino acid residues. Positively charged amino acid residues may include lysine residues and arginine residues.
[0127] In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more arginine amino acid residues. A cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 arginine amino acid residues. In some embodiments, a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 arginine amino acid residues. A cell-penetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 arginine amino acid residues.
[0128] In some embodiments, a cell-penetrating peptide may comprise at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1. 1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1 .6, at least 1 .7, at least 1.8, at least 1 .9, at least 2, at least 2. 1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.2, at least 3.4, at least 3.6, at least 3.8, or at least 4 arginine amino acid residues per 10 amino acid residues. In some embodiments, a cell -penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2. 1, no more than 2, no more than 1 .9, no more than 1 .8, no more than 1.7, no more than 1.6, no more than 1.5, no more than 1.4, no more than 1.3, no more than 1.2, no more than 1.1, no more than 1, no more than 0.9, no more than 0.8, or no more than 0.5 arginine amino acid residues per 10 amino acid residues. A cell-penetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 arginine amino acid residues per 10 amino acid residues. In some embodiments, the cell-penetrating peptide comprises no arginine amino acids.
[0129] In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more lysine amino acid residues. A cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 lysine amino acid residues. In some embodiments, a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 lysine amino acid residues. A cell-penetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 lysine amino acid residues. In some embodiments, the cellpenetrating peptide comprises no lysine amino acid residues.
[0130] In some embodiments, a cell-penetrating peptide may comprise at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1. 1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1 .6, at least 1 .7, at least 1.8, at least 1 .9, at least 2, at least 2. 1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.2, at least 3.4, at least 3.6, at least 3.8, or at least 4 lysine amino acid residues per 10 amino acid residues. In some embodiments, a cell -penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2. 1, no more than 2, no more than 1 .9, no more than 1 .8, no more than 1.7, no more than 1.6, no more than 1.5, no more than 1.4, no more than 1.3, no more than 1.2, no more than 1.1, or no more than 1 lysine amino acid residues per 10 amino acid residues. A cellpenetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 lysine amino acid residues per 10 amino acid residues.
[0131] In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more negatively charged amino acid residues (e g., aspartic acid or glutamic acid). A cellpenetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 negatively charged amino acid residues. In some embodiments, a cell-penetrating peptide may comprise no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 negatively charged amino acid residues. A cellpenetrating peptide may comprise from 1 to 30, from 2 to 20, from 3 to 30, from 4 to 30, from 5 to 30, from 1 to 20, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 2 to 15, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, or from 10 to 15 negatively charged amino acid residues. Negatively charged amino acid residues may include aspartic acid residues (or aspartate residues) and glutamic acid residues (or glutamate residues).
[0132] In some embodiments, a cell-penetrating peptide may comprise at least 0.03, at least 0.05, at least 0.1, at least 0.2, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least
1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.2, at least 3.4, at least 3.6, at least
3.8, or at least 4 negatively charged amino acid residues per 10 amino acid residues. In some embodiments, a cell-penetrating peptide may comprise no more than 8, no more than 7.5, no more than 7, no more than 6.5, no more than 6, no more than 5.5, no more than 5, no more than 4.5, no more than 4, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, no more than 2, no more than 1.9, no more than 1.8, no more than 1.7, no more than 1.6, no more than 1.5, no more than 1.4, no more than 1.3, no more than 1.2, no more than 1.1, or no more than 1 negatively charged amino acid residues per 10 amino acid residues. A cell-penetrating peptide may comprise from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2, from 1.5 to 5, from 1.5 to 4, from 1.5 to 3, from 1.5 to 2.8, from 1.5 to 2.6, from 1.5 to 2.4, from 1.5 to 2.2, from 1.5 to 2, from 2 to 3, from 2 to 2.8, from 2 to 2.6, from 2 to 2.4, or from 2 to 2.2 negatively charged amino acid residues per 10 amino acid residues. Positively charged amino acid residues may include lysine residues and arginine residues. Negatively charged amino acid residues may include aspartic acid residues (or aspartate residues) and glutamic acid residues (or glutamate residues).
[0133] In some embodiments, a cell-penetrating peptide of the present disclosure may comprise more positively charged amino acid residues than negatively charged amino acid residues. A cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 more positively charged amino acid residues than negatively charged amino acid residues. In some embodiments, a cellpenetrating peptide may comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 more positively charged amino acid residues than negatively charged amino acid residues. A cell-penetrating peptide may comprise from 1 to 30, from 1 to 20, from 1 to 10, from 5 to 30, from 5 to 20, from 5 to 10, from 3 to 30, from 3 to 20, from 3 to 10, from 4 to 30, from 4 to 20, from 4 to 10, from 5 to 30, from 5 to 20, or from 5 to 10 more positively charged amino acid residues than negatively charged amino acid residues.
[0134] In some embodiments, a cell-penetrating peptide of the present disclosure may comprise more negatively charged amino acid residues than positively charged amino acid residues. A cell-penetrating peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 more negatively charged amino acid residues than positively charged amino acid residues. In some embodiments, a cellpenetrating peptide may comprise no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, or no more than 30 more negatively charged amino acid residues than positively charged amino acid residues. A cell-penetrating peptide may comprise from 1 to 30, from 1 to 20, from 1 to 10, from 5 to 30, from 5 to 20, from 5 to 10, from 3 to 30, from 3 to 20, from 3 to 10, from 4 to 30, from 4 to 20, from 4 to 10, from 5 to 30, from 5 to 20, or from 5 to 10 more negatively charged amino acid residues than positively charged amino acid residues.
[0135] In some embodiments, a cell-penetrating peptide may comprise a ratio of positively charged amino acid residues to negatively charged amino acid residues that is at least 1.0, at least 1.5, at least 2, at least 2.5, at least 2.75, at least 3, at least 3.5, at least 4, at least 5, at least 6, or at least 7. In some embodiments, a cell-penetrating peptide may comprise a ratio of negatively charged amino acid residues to positively charged amino acid residues that is no more than 1.0, no more than 1.5, no more than 2, no more than 2.5, no more than 2.75, no more than 3, no more than 3.5, or no more than 4.
[0136] Proline amino acid residues may provide structural stability or specific conformations for the cell-penetrating peptides of the present disclosure. For example, proline residues may provide a conformation or stability to short cell-penetrating peptide sequences (e g., cellpenetrating peptides of less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, or less than 4 amino acid residues). A cell-penetrating peptide of the present disclosure (e.g., a short cell-penetrating peptide) may contain a proline amino acid residue. In some embodiments, a cell-penetrating peptide may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 proline amino acid residues.
[0137] Histidine amino acid residues may facilitate membrane penetration of the cellpenetrating peptides of the present disclosure. The side chain of a histidine amino acid may be, on average, neutral at an extracellular pH such as pH 7.4, pH 7.3, pH 7.2, pH 7.1, or pH 7.0. The side chain of a histidine amino acid may, on average, become increasingly positively charged as the pH drops in an early endosome, in a late endosome, or in a lysosome. Histidine residue side chains may contribute more positive charge to the net charge of a peptide as pH decreases, for example as the pH of an endosome goes below pH 6.0. A histidine residue side chain may have a pKa of 6.0, such that, as the pH drops below pH 7.4, the amount of positive charge increases. A histidine residue may develop a positive charge in an endosome or a lysosome, thereby causing a change in the conformation of the molecule or a change in interaction with the endosomal membrane such that the cell-penetrating peptide containing one or more histidine residues disrupts the endosome or otherwise escapes the endosome or lysosome as the pH in the endosome or lysosomes drops below about pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, or lower. A cellpenetrating peptide of the present disclosure may contain a histidine amino acid residue. In some embodiments, a cell-penetrating peptide may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 histidine amino acid residues. For example, a short cell-penetrating peptide sequences (e.g., any one of SEQ ID NO: 4 - SEQ ID NO: 7, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 21 - SEQ ID NO: 29, SEQ ID NO: 41 - SEQ ID NO: 58, SEQ ID NO: 62 - SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 82 SEQ ID NO: 84, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 214, or SEQ ID NO: 216) may contain one proline amino acid residue, one histidine amino acid residue, or one proline amino acid residue and one histidine amino acid residue. In some embodiments, the penetration properties of a cell-penetrating peptide increase as the pH decreases. For example, a cell-penetrating peptide of the present disclosure may penetrate membranes, including the endosomal membrane or lysosomal membrane, when the pH is below pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, or pH 4.5. In some embodiments, the pH change or the ionic strength change can cause changes in conformation of the cell-penetrating peptide or interaction of the cell-penetrating peptide with the endosomal or lysosomal membrane.
[0138] In some embodiments, CDPs or knotted peptides, including engineered, non-naturally occurring CDPs and those found in nature (e g., a target-binding peptide), can be conjugated to, linked to, or fused to the cell-penetrating peptides of the present disclosure, such as those described in TABLE 1, TABLE 2, or TABLE 3, to deliver a target molecule to cellular, intracellular, or paracellular compartment or space. The cell can be a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, bone marrow cell, or any combination thereof. An engineered peptide can be a peptide that is non-naturally occurring, artificial, synthetic, designed, or recombinantly expressed. In some embodiments, a cellpenetrating peptide of the present disclosure, or a peptide complex comprising a cell- penetrating, enables cell-penetrating, and an additional CDP or knotted peptide that is conjugated to, linked to, or fused to the cell-penetrating peptide can be delivered to the cellular, intracellular, or paracellular compartment or space in a cell associated with a disease or condition. In some cases, the cell is a cancer cell. Cancers can include breast cancer, liver cancer, colon cancer, brain cancer, leukemia, acute myeloid leukemia (AML), lymphoma, nonHodgkin lymphoma, myeloma, blood-cell-derived cancer, spleen cancer, cancers of the salivary gland, kidney cancer, muscle cancers, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, sarcoma, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, diffuse intrinsic pontine glioma, lung cancer, bone marrow cell cancers, skin cancer, genitourinary cancer, osteosarcoma, muscle-derived sarcoma, melanoma, head and neck cancer, a neuroblastoma, glioblastoma, astrocytoma, glioma, medulloblastoma, ependymoma, choroid plexus carcinoma, midline glioma, and diffuse intrinsic pontine glioma (DIPG), or a CMYC-overexpressing cancer. In some cases, the cell is associated with a disease, such as lupus, myelodysplastic syndromes (MDS), Alzheimer’s disease, Huntington’s disease, heart disease, or an auto-immune disease.
[0139] TABLE 2 lists exemplary peptide sequences according to the present disclosure.
TABLE 2 -Exemplary Cell-Penetrating Peptide Sequences
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
[0140] In some embodiments, a peptide can be modified with biotin, for example at a lysine residue (e.g., in SEQ ID NO: 102 - SEQ ID NO: 117). In some embodiments, a peptide can be modified with a fluorophore, for example at the N-terminus or the C-terminus of the peptide (e.g., Cy5.5 in SEQ ID NO: 135 - SEQ ID NO: 154). In some embodiments, a peptide can contain a non-proteinogenic amino acid, for example an a-aminobutyric acid, abbreviated “a” or “Abu” (e.g., in SEQ ID NO: 118 - SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 140 - SEQ ID NO: 154, SEQ ID NO: 163 - SEQ ID NO: 166, or SEQ ID NO: 168). [0141] TABLE 3 lists additional exemplary peptide sequences according to the present disclosure. TABLE 3 - Additional Exemplary Cell-Penetrating Peptide Sequences
Figure imgf000052_0001
Figure imgf000053_0001
[0142] In some embodiments, peptides of the present disclosure may comprise one or more cellpenetrating peptides. For example, peptides can comprise at least one or multiple peptides having cell penetration properties (e.g., one or more of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254). In some embodiments, a cell-penetrating peptide may comprise one or more Arg residues (e g., SEQ ID NO: 1 - SEQ ID NO: 8, SEQ ID NO: 16 - SEQ ID NO: 41, SEQ ID NO: 43 - SEQ ID NO: 82, SEQ ID NO: 84 - SEQ ID NO: 120, SEQ ID NO: 124 - SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 153 - SEQ ID NO: 194, SEQ ID NO: 197 SEQ ID NO: 209, SEQ ID NO: 212 SEQ ID NO: 235, SEQ ID NO: 237 - SEQ ID NO: 243, SEQ ID NO: 250 - SEQ ID NO: 254, SEQ ID NO: 408 - SEQ ID NO: 431, or SEQ ID NO: 433 - SEQ ID NO: 456). In some embodiments, a cell -penetrating peptide of the present disclosure may comprise one or more Lys residues. In some embodiments, a cellpenetrating peptide of the present disclosure may comprise one or more Asp residues. In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more Glu residues. In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more His residues. In some embodiments, a cell-penetrating peptide of the present disclosure may comprise one or more Pro residues. In some embodiments, a cellpenetrating peptide of the present disclosure may comprise one or more Cys residues (e g., SEQ ID NO: 1 - SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 18 - SEQ ID NO: 20, SEQ ID NO: 30 - SEQ ID NO: 40, SEQ ID NO: 59 - SEQ ID NO: 61, SEQ ID NO: 64 - SEQ ID NO: 69, SEQ ID NO: 71 - SEQ ID NO: 81, SEQ ID NO: 85 - SEQ ID NO: 117, SEQ ID NO: 119 - SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 169 - SEQ ID NO: 193, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 202 - SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 214 - SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 244, SEQ ID NO: 247, or SEQ ID NO: 408 - SEQ ID NO: 457). In some embodiments, one or more Lys residues may be substituted with Arg residues. In some embodiments, one or more Arg residues may be substituted with Lys residues. In some embodiments, one or more Cys residues may be substituted with Ala residues. In some embodiments, a cell-penetrating peptide may be a short cell-penetrating peptide (e.g., SEQ ID NO: 4 - SEQ ID NO: 7, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 21 - SEQ ID NO: 29, SEQ ID NO: 41 - SEQ ID NO: 58, SEQ ID NO: 62 - SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 82 - SEQ ID NO: 84, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 214, or SEQ ID NO: 216).
[0143] In some embodiments, a cell -penetrating peptide may be a fragment of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254. In some embodiments, a cell-penetrating peptide may be any consecutive fragment of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 SEQ ID NO: 254 that is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids long.
[0144] In some embodiments, a cell -penetrating peptide may comprise a functional fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254. The functional fragment may be a consecutive fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254. In some embodiments, a functional fragment may be a fragment that has cell -penetrating properties. For example, a functional fragment may be capable of penetrating a cellular layer, cell membrane, a nuclear envelope, an endosomal membrane, a lysosomal membrane, other cellular or subcellular membrane, or a blood brain barrier. In some embodiments, a functional fragment may be a fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 18 - SEQ ID NO: 20, SEQ ID NO: 30 - SEQ ID NO: 40, SEQ ID NO: 59 - SEQ ID NO: 61, SEQ ID NO: 64 - SEQ ID NO: 69, SEQ ID NO: 71 - SEQ ID NO: 81, SEQ ID NO: 85 - SEQ ID NO: 117, SEQ ID NO: 119 - SEQ ID NO: 162, SEQ ID NO 166,
SEQ ID NO: 167, SEQ ID NO: 169 - SEQ ID NO: 193, SEQ ID NO: 196, SEQ ID NO: 197,
SEQ ID NO: 202 - SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 214 -
SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 244, or SEQ ID NO: 247 comprising at least
1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 cysteine residues. In some embodiments, a functional fragment may be a fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 positively charged amino acid residues. In some embodiments, a functional fragment may be a fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 8, SEQ ID NO: 16 - SEQ ID NO: 41, SEQ ID NO: 43 - SEQ ID NO: 82, SEQ ID NO: 84 SEQ ID NO: 120, SEQ ID NO: 124 SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 153 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, SEQ ID NO: 197 - SEQ ID NO: 209, SEQ ID NO: 212 - SEQ ID NO: 235, SEQ ID NO: 237 SEQ ID NO: 243, or SEQ ID NO: 250 SEQ ID NO: 254 comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 arginine amino acid residues. In some embodiments, a functional fragment may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 lysine amino acid residues. In some embodiments, a functional fragment may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 histidine amino acid residues. In some embodiments, a functional fragment may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 proline amino acid residues. In some embodiments, a functional fragment may comprise an amphipathic a-helix.
[0145] In some embodiments, a short cell-penetrating peptide may have a sequence of any one of GSXDALPHLKL (SEQ ID NO: 334) or XDALPHLKL (SEQ ID NO: 325), wherein X is any amino acid. In some embodiments, a short cell -penetrating peptide may have a sequence of any one of GSGXALPHLKL (SEQ ID NO: 335) or GXALPHLKL (SEQ ID NO: 326), wherein X is any amino acid. In some embodiments, a short cell -penetrating peptide may have a sequence of any one of GDXLPHLKL (SEQ ID NO: 327) or GSGDXLPHLKL (SEQ ID NO: 336), wherein X is any amino acid. In some embodiments, a short cell -penetrating peptide may have a sequence of any one of GDAXPHLKL (SEQ ID NO: 328) or GSGDAXPHLKL (SEQ ID NO: 337), wherein X is any amino acid. In some embodiments, a short cell-penetrating peptide may have a sequence of any one of GDALXHLKL (SEQ ID NO: 329) or GSGDALXHLKL (SEQ ID NO: 338), wherein X is any amino acid In some embodiments, a short cell -penetrating peptide may have a sequence of any one of GDALPXLKL (SEQ ID NO: 330) or GSGDALPXLKL (SEQ ID NO: 339), wherein X is any amino acid. In some embodiments, a short cell-penetrating peptide may have a sequence of any one of GDALPHXKL (SEQ ID NO: 331) or GSGDALPHXKL (SEQ ID NO: 340), wherein X is any amino acid. In some embodiments, a short cell-penetrating peptide may have a sequence of any one of GDALPHLXL (SEQ ID NO: 332) or GSGDALPHLXL (SEQ ID NO: 341), wherein X is any amino acid. In some embodiments, a short cell -penetrating peptide may have a sequence of any one of GDALPHLKX (SEQ ID NO: 333) or GSGDALPHLKX (SEQ ID NO: 342), wherein X is any amino acid. An amino acid may be a natural amino acid or a non-natural amino acid. [0146] Cell-penetrating peptides may be derived from calcins or calcin variants. For example, the peptides may be derived from the calcin variants shown in FIG. 17. In some embodiments, a cell-penetrating peptide derived from a calcin may have a sequence of DCLX1X2LRX3CX4X5X6X7DCCX8RX9CX10RR, wherein X'-X1'1 are each independently any amino acid or no amino acid (SEQ ID NO: 343). In some embodiments, one or more of amino acids X^X10 in SEQ ID NO: 343 can be deleted, resulting in no amino acid in the position with reference to SEQ ID NO: 343. In some embodiments, known calcin sequences (e.g., naturally occurring calcins) may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 343. In some embodiments, a cell-penetrating peptide derived from a calcin may have a sequence of DCLX1X2LRX3CX4X5X6X7DCCX8RX9CX10RR, wherein X1 is selected from P or A, X2 is selected from H or R, X3 is selected from L or R, X4 is selected from R or K, X5 is selected from E, R, or A, X6 is selected from N or D, X7 is selected from N or R, X8 is selected from S or G, X9 is selected from R or S, and X10 is selected from R, S, or K (SEQ ID NO: 344). In some embodiments, known calcin sequences (e.g., naturally occurring calcins) may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 344. In some embodiments, a cell-penetrating peptide derived from a calcin may have a sequence of DCLPHLRLCRX1X2X3DCCSRRCRRRGT X4ERRCR, wherein X'-X4 are each independently any amino acid (SEQ ID NO: 345), or wherein X1 is E, D, A, R, K, or G, X2 is N or D or E, X3 is R, K, N, and X4 is I, A, P, or G (SEQ ID NO: 346). In some embodiments, known calcin sequences (e g., naturally occurring calcins) may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 345 or SEQ ID NO: 346. In some embodiments, a cell-penetrating peptide derived from a calcin may have a sequence of X1DCLX2X3LRX4CX5X6X7X8DCCX9RX10CX11RRX12X13X14X15X16X17X18X19X20X21, wherein X4-X21 are each independently any amino acid or no amino acid (SEQ ID NO: 347). In some embodiments, known calcin sequences (e.g., naturally occurring calcins) may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 347. In some embodiments, a cellpenetrating peptide derived from a calcin may have a sequence of X1DX2LX3X4LRX5, wherein X1 any amino acid or no amino acid and X2-X5 are each independently any amino acid (SEQ ID NO: 348), or wherein X1 is G, A, S, R, or K, X2 is C, A, G, S, X3 is P, R, K, A, or G, X4 is H, R, or K, and X5 is L, R, or K (SEQ ID NO: 349). In some embodiments, known calcin sequences (e.g., naturally occurring calcins) may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 348 or SEQ ID NO: 349. In some embodiments, a cell-penetrating peptide derived from a calcin may have a sequence of LXLX2LRX3, wherein X4-X3 are each independently any amino acid (SEQ ID NO: 350), or wherein X1 is P, R, K, A, or G, X2 is H, R, or K, and X3 is L, R, or K (SEQ ID NO: 351). In some embodiments, known calcin sequences (e.g., naturally occurring calcins) may be excluded from the cell-penetrating peptides encompassed by SEQ ID NO: 350 or SEQ ID NO: 351. In some embodiments, a cell-penetrating peptide described herein as a consensus sequence or as variants with respect to percent identity may exclude known cell-penetrating sequences.
[0147] Additional cell-penetrating tag peptide sequences can include Tat (GRKKRRQRRR; SEQ ID NO: 225), CysTat (CYRKKRRQRRR; SEQ ID NO: 226), S19-TAT (PFVIGAGVLGALGTGIGGIGRKKRRQRRR; SEQ ID NO: 227), R8 (RRRRRRRR; SEQ ID NO: 228), pAntp (RQIKIWFQNRRMKWKK; SEQ ID NO: 229), Pas-TAT (FFLIPKGGRKKRRQRRR; SEQ ID NO: 230), Pas-R8 (FFLIPKGRRRRRRRR; SEQ ID NO: 231), PasFHV (FFLIPKGRRRRNRTRRNRRRVR; SEQ ID NO: 232), Pas-pAntP (FFLIPKGRQIKIWFQNRRMKWKK; SEQ ID NO: 233), F2R4 (FFRRRR; SEQ ID NO: 234), B55 (KAVLGATKIDLPVDINDPYDLGLLLRHLRHHSNLLANIGDPAVREQVLSAMQEEE; SEQ ID NO: 235), auzurin (LSTAADMQGVVTDGMASGLDKDYLKPDD; SEQ ID NO: 236), IMT-P8 (RRWRRWNRFNRRRCR; SEQ ID NO: 237), BR2 (RAGLQFPVGRLLRRLLR; SEQ ID NO: 238), OMOTAG1 (KRAHHNALERKRR; SEQ ID NO: 239), OMOTAG2 (RRMKANARERNRM; SEQ ID NO: 240), pVEC (LLIILRRRIRKQAHAHSK; SEQ ID NO: 241), SynB3 (RRLSYSRRRF; SEQ ID NO: 242), DPV1047 (VKRGLKLRHVRPRVTRMDV; SEQ ID NO: 243), CY105Y (CSIPPEVKFNKPFVYLI; SEQ ID NO: 244), Transportan (GWTLNSAGYLLGKINLK ALAALAKKIL: SEQ ID NO: 245), MTS (KGEGAAVLLPVLLAAPG; SEQ ID NO: 246), hLF (KCFQWQRNMRKVRGPPVSCIKR; SEQ ID NO: 247), PFVYLI (PFVYLI; SEQ ID NO: 248), and yBBR (VLDSLEFIASKL, SEQ ID NO: 249). For example, in some embodiments, a cell-penetrating peptide can comprise an Arginine patch (Arg patch), for example, an RRRRRRRR (SEQ ID NO: 228), or a variant or fragment thereof In some embodiments, the Arg patch comprises two or more Arg residues, or Argn wherein n is a whole number and can be 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 250). In other embodiments, the cell -penetrating peptide can comprise a Tat peptide (Tat proteins are reviewed in Gump et al. TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med. 2007 Oct;13(10):443-8 and Harada et al. Antitumor protein therapy; application of the protein transduction domain to the development of a protein drug for cancer treatment. Breast Cancer. 2006; 13(1): 16-26). The Tat peptide can have a sequence of, for example, YGRKKRRQRRR (SEQ ID NO: 251), GRKKRRQRRR (SEQ ID NO: 225), or any modification, variant, or fragment thereof. In some embodiments, the Tat peptide sequence can be GRKKRRQRRRPQ (SEQ ID NO: 252), GRKKRRQRRR (SEQ ID NO: 225), or a fragment or variant thereof. Cell-penetrating peptides may be capable of penetrating and transferring a cargo, either covalently or non-covalently attached to the peptides, into a cell. Such cellpenetrating peptides can be synthesized or derived from known proteins, such as penetratin (RQIKIWFQNRRMKWKKGG; SEQ ID NO: 254), VP22 (MTSRRSVKSGPREVPRDEYEDLYYTPSSGMASPDSPPDTSRRGALQTRSRQRGEVRFV QYDESDYALYGGSSSEDDEHPEVPRTRRPVSGAVLSGPGPARAPPPPAGSGGAGRTPTT APRAPRTQRVATKAPAAPAAETTRGRKSAQPESAALPDAPASTAPTRSKTPAQGLARKL HFSTAPPNPDAPWTPRVAGFNKRVFCAAVGRLAAMHARMAAVQLWDMSRPRTDEDL NELLGITTIRVTVCEGKNLLQRANELVNPDVVQDVDAATATRGRSAASRPTERPRAPAR SASRPRRPVE; SEQ ID NO: 101), Tat peptide, pVEC, or chimeric peptides, such as transportan, MPG, Pep-1, or synthetic peptides, such as polyarginines, MAP, and ReWs (SEQ ID NO: 253).
[0148] In some embodiments, a cell-penetrating peptide can may be derived from maurocaline (GDCLPHLKLCKENKDCCSKKCKRRGTNIEKRCR; SEQ ID NO: 197), imperatoxin (GDCLPHLKRCKADNDCCGKKCKRRGTNAEKRCR; SEQ ID NO: 202), hadrucalcin (SEKDCIKHLQRCRENKDCCSKKCSRRGTNPEKRCR; SEQ ID NO: 200), hemicalcin (GDCLPHLKLCKADKDCCSKKCKRRGTNPEKRCR; SEQ ID NO: 205), opicalcin-1 (GDCLPHLKRCKENNDCCSKKCKRRGTNPEKRCR; SEQ ID NO: 206), opicalcin-2 (GDCLPHLKRCKENNDCCSKKCKRRGANPEKRCR; SEQ ID NO: 207), huwentoxin (ECLEIFKACNPSNDQCCKSSKLVCSRKTRWCKYQIG; SEQ ID NO: 203), CTI (SCEPGKTFKDKCNTCRCGADGKSAACTLKACPNQ; SEQ ID NO: 87), intrepicalcin (MRQNTMTIIFIVFIVTFASLTIYGAEASEANFLERRADCLAHLKLCKKNKDCCSKKCSR RGTNPEQRCR; SEQ ID NO: 90), vejocalcin (ADCLAHLKLCKKNNDCCSKKCSRRGTNPEQRCR; SEQ ID NO: 91), urocalcin (MKASTLVVIFIVIFITISSFSIHDVQASGVEKREQKDCLKKLKLCKENKDCCSKSCKRRG TNIEKRCR; SEQ ID NO: 92), potassium channel toxin-like Tx677 (MKISALVMITLLICSMMILCQGQKILSNRCNNSSECIPHCIRIFGTRAAKCINRKCYCYP; SEQ ID NO: 86), potassium channel toxin KTx2.2 (RPTDIKCSASYQCFPVCKSRFGKTNGRCVNGLCDCF; SEQ ID NO: 88), potassium channel toxin KTxl5.8 (QVQTNVKCQGGSCASVCRREIGVAAGKCINGKCVCYRN; SEQ ID NO: 89), midkine (62-104) (CKYKFENWGACDGGTGTKVRQGTLKKARYNAQCQETIRVTKPC; SEQ ID NO: 208), MCoTI-II (SGSDGGVCPKILKKCRRDSDCPGACICRGNGYCG; SEQ ID NO: 209), or chlorotoxin (MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR; SEQ ID NO: 204). [0149] In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any sequence of SEQ ID NO: 1 - SEQ ID NO: 84. In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any sequence of SEQ ID NO: 85 - SEQ ID NO: 194 or SEQ ID NO: 408 - SEQ ID NO: 457. In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any sequence of SEQ ID NO: 195 - SEQ ID NO: 254. In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a fragment of SEQ ID NO: 1 - SEQ ID NO: 84. In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a fragment of SEQ ID NO: 85 - SEQ ID NO: 194 or SEQ ID NO: 408 - SEQ ID NO: 457. In some embodiments, the cell-penetrating peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a fragment of SEQ ID NO: 195 - SEQ ID NO: 254. In some embodiments, a variant may comprise one or more Lys residues replaced with Arg. In some embodiments, a variant may comprise one or more Arg residues replaced with Lys. In some embodiments, a variant may comprise one or more Cys residues replaced with Ala.
[0150] In some embodiments a fragment may be a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46 at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76 residues long, at least 77, at least 78, at least 79, at least 80, or at least 81 residues long, wherein the peptide fragment is selected from any portion of the peptide. In some embodiments a fragment may be a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, no more than 30, no more than 31, no more than 32, no more than 33, no more than 34, no more than 35, no more than 36, no more than 37, no more than 38, no more than 39, no more than 40, no more than 41, no more than 42, no more than 43, no more than 44, no more than 45, no more than 46, no more than 47, no more than 48, no more than 49, no more than 50, no more than 51, no more than 52, no more than 53, no more than 54, no more than 55, no more than 56, no more than 57, no more than 58, no more than 59, no more than 60, no more than 61, no more than 62, no more than 63, no more than 64, no more than 65, no more than 66 no more than 67, no more than 68, no more than 69, no more than 70, no more than 71, no more than 72, no more than 73, no more than 74, no more than 75, no more than 76, no more than 77, no more than 78, no more than 79, no more than 80, or no more than 81 residues long, wherein the peptide fragment is selected from any portion of the peptide. In some embodiments, the peptide sequence is flanked by additional amino acids. One or more additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
[0151] In some embodiments, nuclear localization signals can be coupled to, conjugated to, linked to, or fused to a cell-penetrating peptide or a cell-penetrating peptide complex described herein to promote nuclear localization. In some embodiments, cell-penetrating peptides are conjugated to, linked to, or fused to a nuclear localization signal, such as a four-residue sequence of K-K/R-X-K/R (SEQ ID NO: 352), wherein X can be any amino acid, or a variant thereof. In some embodiments, cell-penetrating peptides are conjugated to, linked to, or fused to a nuclear localization signal as described in Lange et al, J Biol Chem. 2007 Feb 23 ;282(8): 5101 - 5, such as PKKKRRV (SEQ ID NO: 353) or KRPAATKKAGQAKKKK (SEQ ID NO: 354) In some embodiments, a cell-penetrating peptide described herein is conjugated to, linked to, or fused to a nuclear localization signal comprising KxRy (SEQ ID NO: 355), wherein x and y independently can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, such as KKRR (SEQ ID NO: 356), KKKRR (SEQ ID NO: 357), or KKKK (SEQ ID NO: 358). Other cell-penetrating moieties can also be linked to, conjugated to, linked to, or fused to the peptides described herein, including, but not limited to, polycations, polyorganic acids, endosomal releasing polymers, poly(2- propylacrylic acid), poly(2-ethylacrylic acid), or any combination thereof.
[0152] In other embodiments, cell penetration can be increased by using high dosage of a peptide described here, such as up to 10 pM, or 10 pM or more of the cell-penetrating peptide. Up 10 pM, or 10 pM or more cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) may be co-delivered with a peptide to facilitate cell penetration. Protein transfection agents can also be used to increase cell penetration of a peptide. In some embodiments, direct cytosolic expression of a peptide can be used. In other embodiments, physical disruption methods such as electroporation can be used to improve delivery of a peptide into a cell.
[0153] In some embodiments, cell penetrance of a peptide described herein can be improved. For example, the binding interface of a peptide described herein can modified from a cellpenetrating peptide, such as a calcine. Some non-limiting examples of calcines can be imperatoxin-A, maurocalcine, hemicalcin, opiclacin 1, opicalcin 2, and hadrucalcin. The cellpenetrating peptide can comprise at least 60%, 70%, 80%, 90%, 95%, or 98% with any one of SEQ ID NO: 1 - SEQ ID NO: 100, SEQ ID NO: 102 - SEQ ID NO: 224. In some embodiments, the cell penetrance peptide can be calcines, modified calcines, derivatives of calcines, or fragments thereof, or variants thereof, which can be used to increase cell penetration. Modified calcines, derivatives of calcines, or fragments can be screened for cell penetration activity such as activation of sarcoplasmic reticulum ryanodine receptors, activity on ryanodine-sensitive Ca2+ channels RyRl, Ryr2, or both, or as a selective agonist of the foregoing. Alternatively or in addition, peptides, peptide fragments, or peptide variants can be screened for cell penetration using a mass spectrometry-based or fluorescence-based assay (e.g., a SNAP penetration assay), as described herein. Moreover, modified calcines can include substitution, addition or reduction of Lysine residues, addition or reduction of Arginine residues, or other charged residues, within a native calcine in order to modify activity and optimize such calcine cell -penetration activity or activity on the RyRl or RyR2 receptors. In some cases, the residues of a cell-penetrating peptide of this disclosure are modified so they do not act on or have reduced activity on RyRl, RyR2, or other receptors.
[0154] In some cases, cell-penetrating peptides can penetrate into a target cell or into the nucleus of a target cell. Examples of target cells include cancerous cells, tumors, neurons, inflammatory cells, cells of metastases, cancerous stem cells, and other cell types. The target cell can be a human cell, a mammalian cell, a human or mammalian cell line, a cancer cell line, a cell extracted from a subject, in vivo, or in vitro.
[0155] In some instances, the peptide can contain only one lysine residue, or no lysine residues. In some instances, some or all of the lysine residues in the peptide are replaced with arginine residues. In some instances, some or all of the methionine residues in the peptide are replaced by leucine or isoleucine. Some or all of the tryptophan residues in the peptide can be replaced by phenylalanine or tyrosine. In some instances, some or all of the asparagine residues in the peptide are replaced by glutamine. In some embodiments, some or all of the aspartic acid residues can be replaced by glutamic acid residues. In some embodiments, some or all of the cysteine residues are replaced by alanine residues. In some embodiments, some or all of the cysteine residues are replaced by serine residues. In some embodiments, some or all of the cysteine residues are replaced by glycine residues. In some cases, the C-terminal Arg residues of a peptide is modified to another residue such as Ala, Asn, Asp, Gin, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai. For example, the C-terminal Arg residue of a peptide can be modified to He. Alternatively, the C-terminal Arg residue of a peptide can be modified to any non-natural amino acid. This modification can prevent clipping of the C-terminal residue during expression, synthesis, processing, storage, in vitro, or in vivo including during treatment, while still allowing maintenance of a key hydrogen bond. A key hydrogen bond can be the hydrogen bond formed during the initial folding nucleation and is critical for forming the initial hairpin.
[0156] In some cases, the N-terminus of the peptide is blocked, such as by an acetyl group. Alternatively or in combination, in some instances, the C-terminus of the peptide is blocked, such as by an amide group. In some embodiments, the peptide is modified by methylation on free amines. For example, full methylation can be accomplished through the use of reductive methylation with formaldehyde and sodium cyanoborohydride.
[0157] In some cases, GS can be added as the first two N-terminal amino acids, as shown in SEQ ID NO: 59 - SEQ ID NO: 84, SEQ ID NO: 93 - SEQ ID NO: 101, or SEQ ID NO: 210 - SEQ ID NO: 224, or such N-terminal amino acids (GS) can be absent as shown in SEQ ID NO: 1 - SEQ ID NO: 58, SEQ ID NO: 85 - SEQ ID NO: 92, SEQ ID NO: 102 - SEQ ID NO: 209, or SEQ ID NO: 225 - SEQ ID NO: 254, or can be substituted by any other one or two amino acids. In some embodiments, GS is used as a linker or used to couple to a linker to make a protein conjugate or fusion. In some embodiments, the linker comprises a GxSy (SEQ ID NO: 256) peptide, wherein x and y independently can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises (GS)x (SEQ ID NO: 258), wherein x can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises GGSSG (SEQ ID NO: 259), GGGGG (SEQ ID NO: 260), GSGSGSGS (SEQ ID NO: 261), GGGGS (SEQ ID NO: 263), GGGS (SEQ ID NO: 264), or a variant or fragment thereof or any number of repeats and combinations thereof.
Additionally, KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx, and EPKSSDKTHT (SEQ ID NO: 267) from human IgG3 can be used as a peptide linker or any number of repeats and combinations thereof. In some embodiments, the peptide linker comprises GGGSGGSGGGS (SEQ ID NO: 292) or a variant or fragment thereof or any number of repeats and combinations thereof. It is understood that any of the foregoing linkers or a variant or fragment thereof can be used with any number of repeats or any combinations thereof. It is also understood that other peptide linkers in the art or a variant or fragment thereof can be used with any number of repeats or any combinations thereof. The length of the linker can be tailored to maximize binding of the cell-penetrating peptides in the protein conjugate or fusion at the same time including accounting for steric access. In some embodiments, the linker between the cell -penetrating peptides in the protein conjugate or fusion within the selective depletion complex is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36 at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65 residues incrementally up to 100 residues long.
[0158] In some cases, the C-terminal Arg residues of a peptide is modified to another residue such as Ala, Asn, Asp, Gin, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai. For example, the C-terminal Arg residue of a peptide can be modified to He. Alternatively, the C-terminal Arg residue of a peptide can be modified to any non-natural amino acid. This modification can prevent clipping of the C-terminal residue during expression, synthesis, processing, storage, in vitro, or in vivo including during treatment, while still allowing maintenance of a key hydrogen bond. A key hydrogen bond can be the hydrogen bond formed during the initial folding nucleation and is critical for forming the initial hairpin.
[0159] In some cases, the peptide comprises the sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254. A peptide can be a fragment comprising a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is at least 3, at least 4, at least 5, at least 6, at least 7, at least, 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46 at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76 residues long, at least 77, at least 78, at least 79, at least 80, or at least 81 residues long, wherein the peptide fragment is selected from any portion of the peptide. In some embodiments, the peptide sequence is flanked by additional amino acids. One or more additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
[0160] In some embodiments, a peptide can be a fragment comprising a contiguous fragment of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 that is not greater than 3, not greater than 4, not greater than 5, not greater than 6, not greater than 7, not greater than, 8, not greater than 9, not greater than 10, not greater than 11, not greater than 12, not greater than 13, not greater than 14, not greater than 15, not greater than 16, not greater than 17, not greater than 18, not greater than 19, not greater than 20, not greater than 21, not greater than 22, not greater than 23, not greater than 24, not greater than 25, not greater than 26, not greater than 27, not greater than 28, not greater than 29, not greater than 30, not greater than 31, not greater than 32, not greater than 33, not greater than 34, not greater than 35, not greater than 36, not greater than 37, not greater than 38, not greater than 39, not greater than 40, not greater than 41, not greater than 42, not greater than 43, not greater than 44, not greater than 45, not greater than 46 not greater than 47, not greater than 48, not greater than 49, not greater than 50, not greater than 51, not greater than 52, not greater than 53, not greater than 54, not greater than 55, not greater than 56, not greater than 57, not greater than 58, not greater than 59, not greater than 60, not greater than 61, not greater than 62, not greater than 63, not greater than 64, not greater than 65, not greater than 66, not greater than 67, not greater than 68, not greater than 69, not greater than 70, not greater than 71, not greater than 72, not greater than 73, not greater than 74, not greater than 75, not greater than 76 residues long, not greater than 77, not greater than 78, not greater than 79, not greater than 80, or not greater than 81 residues long, wherein the peptide fragment is selected from any portion of the peptide. In some embodiments, the peptide sequence is flanked by additional amino acids. One or more additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
[0161] In some embodiments, peptide sequences comprise primarily beta-sheets and/or alphahelix structures. In some embodiments, cell-penetrating peptides of the present disclosure are small, compact mini-proteins stabilized by intra-chain disulfide bonds (mediated by cysteines) and a well-packed hydrophobic core. In some embodiments, cell-penetrating peptides (e.g., SEQ ID NO: 1 SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) have structures comprising helical bundles with at least one disulfide bridge between each of the alpha helices, thereby stabilizing the peptides. A cell-penetrating peptide can be amphipathic, comprising a hydrophilic region and a hydrophobic region, which can facilitate interactions with cell membranes and insertion dynamics. In some embodiments, a cell-penetrating peptide can comprise an amphipathic a- helix, which can facilitate interactions with cellular membranes. In some embodiments, the amphipathicity of a cell-penetrating peptide is pH dependent and is present to a higher degree at endosomal or lysosomal pHs than at extracellular pH. In some embodiments, cell-penetrating peptides contain one or more positively charged residues or carry a net positive charge. In some embodiments, a cell-penetrating peptide is cyclized peptide or a stapled peptide.
[0162] In other embodiments, peptides can be conjugated to, linked to, or fused to a cargo peptide or cargo molecule to deliver the cargo peptide or cargo molecule to a target of interest in a cell. In other embodiments, peptides can be conjugated to, linked to, or fused to a molecule that extends half-life or modifies the pharmacodynamic and/or pharmacokinetic properties of the peptides, or any combination thereof.
[0163] In some instances, a peptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 positively charged residues, such as Arg or Lys, or any combination thereof. In some instances, one or more lysine residues in the peptide are replaced with arginine residues. In some instances, one or more arginine residues in the peptide are replaced with lysine residues. In some cases, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more Arg or Lys residues are solvent exposed on a peptide.
[0164] The peptides of the present disclosure can further comprise neutral amino acid residues. In some cases, the peptide has 35 or fewer neutral amino acid residues. In other cases, the peptide has 81 or fewer neutral amino acid residues, 70 or fewer neutral amino acid residues, 60 or fewer neutral amino acid residues, 50 or fewer neutral amino acid residues, 40 or fewer neutral amino acid residues, 36 or fewer neutral amino acid residues, 33 or fewer neutral amino acid residues, 30 or fewer neutral amino acid residues, 25 or fewer neutral amino acid residues, or 10 or fewer neutral amino acid residues.
[0165] The peptides of the present disclosure can further comprise negative amino acid residues. In some cases, the peptide has 6 or fewer negative amino acid residues, 5 or fewer negative amino acid residues, 4 or fewer negative amino acid residues, 3 or fewer negative amino acid residues, 2 or fewer negative amino acid residues, or 1 or fewer negative amino acid residues. While negative amino acid residues can be selected from any negatively charged amino acid residues, in some embodiments, the negative amino acid residues are either E, or D, or a combination of both E and D.
[0166] In some cases, a peptide comprises no Cys or disulfides. In some cases, a peptide comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more Cys or disulfides. In other embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Ser residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Thr residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Ala, Gly, or Met residues.
[0167] Generally, the NMR solution or x-ray crystallography structures of related structural homologs can be used to inform mutational strategies that may improve the folding, stability, manufacturability, while maintaining a particular biological function. They can be used to predict the 3D pharmacophore of a group of structurally homologous scaffolds, as wells as to predict possible graft regions of related proteins to create chimeras with improved properties. For example, this strategy was used to identify critical amino acid positions and loops that may be used to design peptides with improved cell-penetrating properties, high expression, high stability in vivo, or any combination thereof.
[0168] In some embodiments, a cell-penetrating peptide capable of penetrating a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with with a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, as provided in TABLE 1, or a fragment thereof. In some embodiments, a cell-penetrating peptide capable of penetrating a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) comprises a sequence with no more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a peptide of any one of SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, as provided in TABLE 2, or a fragment thereof. In some embodiments, a cell -penetrating peptide capable of penetrating a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) comprises a sequence with no more than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a peptide of any one of SEQ ID NO: 195 — SEQ ID NO: 254, as provided in TABLE 3, or a fragment thereof. Two or more peptides can share a degree of sequence identity or homology and share similar properties in vivo. For instance, a peptide can share a degree of sequence identity or homology with any one of the peptides of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254. In some cases, one or more peptides of the disclosure can have up to about 20% pairwise sequence identity or homology, up to about 25% pairwise sequence identity or homology, up to about 30% pairwise sequence identity or homology, up to about 35% pairwise sequence identity or homology, up to about 40% pairwise sequence identity or homology, up to about 45% pairwise sequence identity or homology, up to about 50% pairwise sequence identity or homology, up to about 55% pairwise sequence identity or homology, up to about 60% pairwise sequence identity or homology, up to about 65% pairwise sequence identity or homology, up to about 70% pairwise sequence identity or homology, up to about 75% pairwise sequence identity or homology, up to about 80% pairwise sequence identity or homology, up to about 85% pairwise sequence identity or homology, up to about 90% pairwise sequence identity or homology, up to about 95% pairwise sequence identity or homology, up to about 96% pairwise sequence identity or homology, up to about 97% pairwise sequence identity or homology, up to about 98% pairwise sequence identity or homology, up to about 99% pairwise sequence identity or homology, up to about 99.5% pairwise sequence identity or homology, up to about 99.6% pairwise sequence identity or homology, up to about 99.7% pairwise sequence identity or homology, up to about 99.8% pairwise sequence identity or homology, up to about 99.9% pairwise sequence identity or homology, or up to about 100% pairwise sequence identity or homology. In some cases, one or more peptides of the disclosure can have at least about 20% pairwise sequence identity or homology, at least about 25% pairwise sequence identity or homology, at least about 30% pairwise sequence identity or homology, at least about 35% pairwise sequence identity or homology, at least about 40% pairwise sequence identity or homology, at least about 45% pairwise sequence identity or homology, at least about 50% pairwise sequence identity or homology, at least about 55% pairwise sequence identity or homology, at least about 60% pairwise sequence identity or homology, at least about 65% pairwise sequence identity or homology, at least about 70% pairwise sequence identity or homology, at least about 75% pairwise sequence identity or homology, at least about 80% pairwise sequence identity or homology, at least about 85% pairwise sequence identity or homology, at least about 90% pairwise sequence identity or homology, at least about 95% pairwise sequence identity or homology, at least about 96% pairwise sequence identity or homology, at least about 97% pairwise sequence identity or homology, at least about 98% pairwise sequence identity or homology, at least about 99% pairwise sequence identity or homology, at least about 99.5% pairwise sequence identity or homology, at least about 99.6% pairwise sequence identity or homology, at least about 99.7% pairwise sequence identity or homology, at least about 99.8 pairwise sequence identity or homology, at least about 99.9% pairwise sequence identity or homology, or at least about 100% pairwise sequence identity or homology, with a second peptide.
[0169] Various methods and software programs can be used to determine the homology between two or more peptides, such as NCBI BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method or algorithm. Pairwise sequence alignment is used to identify regions of similarity that may indicate functional, structural and/or evolutionary relationships between two biological sequences (protein or nucleic acid) By contrast, multiple sequence alignment (MSA) is the alignment of three or more biological sequences. From the output of .MSA applications, homology can be inferred and the evolutionary relationship between the sequences assessed. One of skill in the art would recognize as used herein, “sequence homology” and “sequence identity” and “percent (%) sequence identity” and “percent (%) sequence homology” have been used interchangeably to mean the sequence relatedness or variation, as appropriate, to a reference polynucleotide or amino acid sequence.
[0170] In some instances, the peptide is any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or a functional fragment thereof. In other embodiments, the peptide of the disclosure further comprises a peptide with 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99%, 97%, 95%, 90%, 85%, or 80% sequence identity or homology to any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or fragment thereof.
[0171] In other instances, the peptide can be a peptide that is homologous to any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or a functional fragment thereof. The term “homologous” is used herein to denote peptides having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity or homology to a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 or a functional fragment thereof.
[0172] In still other instances, the variant nucleic acid molecules that encode a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408
- SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 can be identified by either a determination of the sequence identity or homology of the encoded peptide amino acid sequence with the amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254, or by a nucleic acid hybridization assay. Such peptide variants of any one of SEQ ID NO: 1
- SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 can be characterized as nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule having the nucleotide sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1 x-0.2xSSC with 0.1% SDS at 50-65° C., and (2) that encode a peptide having at least 70%, at least 80%, at least 90*Eo, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity or homology to the amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254. [0173] Multiple sequence alignment can be used to design peptide variants that retain cellpenetrating properties, or have increase cell-penetrating properties, are shorter or longer, or have increased stability or decreased immunogenicity. In some embodiments, a peptide is modified to increase homology to a human protein sequence. In some embodiments, a peptide is modified to increase resistance to degradation. In some embodiments, a peptide is modified to reduce an affinity of the peptide for a human leukocyte antigen complex, a major histocompatibility complex, or both.
[0174] Multiple peptides that have cell-penetrating capabilities, or that belong to families of peptides where some peptides have been shown to have cell-penetrating capabilities, can be aligned where amino acids that are the same or similar are aligned. Positional amino acid variants at one position in a given peptide may be placed in the same position in a different aligned peptide to create a new peptide. Likewise, fragments of one peptide that is penetrant may identify fragments in aligned peptides that may also be cell penetrant. Amino acids of like charge can be varied, such as changing Lys to Arg and vice versa, and changing Asp to Glu and vice versa. Cysteine residues may be replaced in peptide fragments or where unpaired with other Cys residues to improve manufacturability, shelf-life stability, purity, in vivo stability, in vivo safety, and in vivo activity. For example, one or more cysteine residues may be mutated to alanine. For example, one or more cysteine residues may be mutated to any other amino acid, or to glycine or aminobutyric acid. Histidine residues may be added to facilitate endosomal escape. Proline residues may be added to facilitate endosomal escape. An alignment may be generated using R language and an “MSA” software package, which codes for R language specific for multiple alignments (Bodenhofer, U et al. Bioinformatics, 31 (24): 3997-3999 (2015)). Identified permissive or preferred amino acids at a given location, provide a guide for discovery of novel peptide variants that could be generated and that could retain essential properties such as structure, function, peptide folding, biodistribution, or stability. Furthermore, based on the ability to substitute K residues with R residues and R residues with K residues and other mutations, peptides of the family of sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO 22 - SEQ ID NO: 29, SEQ ID NO: 44 - SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, or SEQ ID NO: 68 may retain essential properties such as structure, function, peptide folding, biodistribution, or stability. Additionally, peptides comprising a segment corresponding to SEQ ID NO: 350 or SEQ ID NO: 351 may have structure, function, peptide folding, biodistribution, binding, accumulation, retention, or stability favorable for cell penetration. In some embodiments, a positively charged region, for example as seen in residues 30-33 of SEQ ID NO: 197 or SEQ ID NO: 202 or residues 32-35 of SEQ ID NO: 200, may improve cell-penetrating properties of a peptide (e.g., a cystine-dense peptide). Other conserved regions within sequences of the present disclosure can be identified.
Alternatively, an alignment may be generated using R language and an “msa” software package, which codes for R language specific for multiple alignments (Bodenhofer, U et al.
Bioinformatics, 31 (24): 3997-3999 (2015)). Using such in silico methods while analyzing the peptides of this disclosure was used to identify additional cell-penetrating peptides.
Cystine-Dense Peptides
[0175] In some embodiments, cell-penetrating peptides, cargo peptides, or cell -penetrating peptide complexes of the present disclosure comprise one or more Cys, or one or more disulfide bonds. In some embodiments, the cell-penetrating peptides are derived from cystine-dense peptides (CDPs), knotted peptides, or hitchins. In some embodiments, the cargo peptides are derived from cystine-dense peptides (CDPs), knotted peptides, or hitchins. As used herein, the term “peptide” is considered to be interchangeable with the terms “knotted peptide”, “cystine- dense peptide”, “CDP”, and “hitchin”. (See e.g., Correnti et al. Screening, large-scale production, and structure-based classification for cystine-dense peptides. Nat Struct Mol Biol. 2018 Mar; 25(3): 270-278).
[0176] In some cases, the peptides of the present disclosure are cystine-dense peptides (CDPs), related to knotted peptides or hitchin-derived peptides or knottin-derived peptides. The cellpenetrating peptides can be cystine-dense peptides (CDPs). Hitchins can be a subclass of CDPs wherein six cysteine residues form disulfide bonds according to the connectivity [1-4], 2-5, 3-6 indicating that the first cysteine residue forms a disulfide bond with the fourth residue, the second with the fifth, and the third cysteine residue with the sixth. The brackets in this nomenclature indicate cysteine residues form the knotting disulfide bond. (See e.g., Correnti et al. Screening, large-scale production, and structure-based classification for cystine-dense peptides. Nat Struct Mol Biol. 2018 Mar; 25(3): 270-278). Knotting can be a subclass of CDPs wherein six cysteine residues form disulfide bonds according to the connectivity 1-4, 2-5, [3-6], Knotting are a class of peptides, usually ranging from about 20 to about 80 amino acids in length that are often folded into a compact structure. Knottins are typically assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks and can contain beta strands and other secondary structures. The presence of the disulfide bonds gives knottins and hitchins remarkable environmental stability, allowing them to withstand extremes of temperature and pH and to resist the proteolytic enzymes of the blood stream. In some cases, the peptides described herein can be derived from knotted peptides. The amino acid sequences of peptides as disclosed herein can comprise a plurality of cysteine residues. In some cases, at least six cysteine residues of the plurality of cysteine residues present within the amino acid sequence of a peptide participate in the formation of disulfide bonds. In some cases, all cysteine residues of the plurality of cysteine residues present within the amino acid sequence of a peptide participate in the formation of disulfide bonds. As described herein, the term “knotted peptide” can be used interchangeably with the terms “cystine-dense peptide”, “CDP”, or “peptide”.
[0177] This disclosure demonstrates the utility of CDPs as a diverse scaffold family that can be screened for applicability to modem drug discovery strategies. CDPs comprise alternatives to existing biologies, primarily antibodies, which can bypass some of the liabilities of the immunoglobulin scaffold, including poor tissue permeability, immunogenicity, and long serum half-life that can become problematic if toxicities arise. Peptides of the present disclosure in the 20-80 amino acid range represent medically relevant therapeutics that are mid-sized, with many of the favorable binding specificity and affinity characteristics of antibodies but with improved cell penetration, stability, reduced immunogenicity, and simpler manufacturing methods. The intramolecular disulfide architecture of CDPs provides particularly high stability metrics, reducing fragmentation and immunogenicity, while their smaller size could improve tissue penetration or cell penetration and facilitate tunable serum half-life. Disclosed herein are peptides representing candidate peptides that can serve as vehicles for delivering target molecules to cellular, intracellular, and paracellular compartments or spaces.
[0178] In some embodiments, cell-penetrating peptides can be engineered peptides. An engineered peptide can be a peptide that is non-naturally occurring, artificial, isolated, synthetic, designed, or recombinantly expressed. In some embodiments, the cell-penetrating peptides of the present disclosure comprise one or more properties of CDPs, knotted peptides, or hitchins, such as stability, resistance to proteolysis, resistance to heat, resistance to denaturation, resistance to reducing conditions, and/or ability to cross the blood brain barrier.
[0179] In some embodiments, cell-penetrating peptides as described herein contain no disulfides or Cys (e g., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 21 - SEQ ID NO: 29, SEQ ID NO: 41 - SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 70, SEQ ID NO: 82 - SEQ ID NO: 84, SEQ ID NO: 188, SEQ ID NO: 163 - SEQ ID NO: 165, SEQ ID NO: 168, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 198 - SEQ ID NO: 201, SEQ ID NO: 210, SEQ ID NO: 213, SEQ ID NO: 225, SEQ ID NO: 227 - SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 246, or SEQ ID NO: 248 - SEQ ID NO: 254). In other embodiments, cell-penetrating peptides may comprise one or more Cys, or one or more disulfide bond (e g., SEQ ID NO: 1 - SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 18 - SEQ ID NO: 20, SEQ ID NO: 30 - SEQ ID NO: 40, SEQ ID NO: 59 - SEQ ID NO: 61, SEQ ID NO: 64 - SEQ ID NO: 69, SEQ ID NO: 71 - SEQ ID NO: 81, SEQ ID NO: 85 - SEQ ID NO: 117, SEQ ID NO: 119 SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 169 - SEQ ID NO: 193, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 202 - SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 214 - SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 244, SEQ ID NO: 247, or SEQ ID NO: 408 SEQ ID NO: 457). In some embodiments, the cell-penetrating peptides are derived from knotted or knottin peptides. In some embodiments, such knotted peptides can penetrate a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen). In some embodiments, a peptide that penetrates a cellular layer comprises one or more properties of some knottin peptides, such as stability, resistance to heat, resistance to denaturation, resistance to proteolysis, and/or resistance to reducing conditions.
[0180] In other embodiments, knotted peptides can be conjugated to, linked to, or fused to cellpenetrating peptides, such as those described in TABLE 1, TABLE 2, or TABLE 3, to deliver the knotted peptide to a target cell, such as a cancer cell, a pancreatic cell, liver cell, colon cell, a smooth muscle cell, ovarian cell, breast cell, lung cell, brain cell, skin cell, ocular cell, blood cell, lymph cell, immune system cell, reproductive cell, reproductive organ cell, prostate cell, fibroblast, kidney cell, adenocarcinoma cell, glioma stem cell, tumor cell, or any combination thereof. In some embodiments, such knotted peptides may perform a function inside a target cell, such a cancer cell, such as a breast cancer, liver cancer, colon cancer, brain cancer, pancreatic cancer, lung cancer, leukemia, lymphoma, myeloma, skin cancer, fibroblastic cancer, kidney cell cancer, adenocarcinoma cell, glioma stem cell, or tumor cell. For example, a knotted peptide may promote or inhibit a protein-protein interaction within a target cell, or a knotted peptide may promote or inhibit transcription of a target gene. In some cases, knotted peptides are conjugated to, linked to, or fused to cell-penetrating peptides that are capable of delivering the knotted peptide across a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) to target cells in the central nervous system. In some embodiments, cell-penetrating peptides can be used to deliver a cytotoxic agent to the target cell. In some embodiments, a knotted cargo peptide may be conjugated to a knotted cell-penetrating peptide to form a diknottin (e g., SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 316, or SEQ ID NO: 317).
[0181] Knottins are a class of peptides, usually ranging from about 11 to about 81 amino acids in length that are often folded into a compact structure. Knottins are typically assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks and may contain beta strands, alpha helices, and other secondary structures. The presence of the disulfide bonds gives knottins remarkable environmental stability, allowing them to withstand extremes of temperature and pH and to resist the proteolytic enzymes of the blood stream. The presence of a disulfide knot may provide resistance to reduction by reducing agents. The rigidity of knottins also allows them to bind to targets without paying the “entropic penalty” that a floppy peptide accrues upon binding a target. For example, binding is adversely affected by the loss of entropy that occurs when a peptide binds a target to form a complex. Therefore, “entropic penalty” is the adverse effect on binding, and the greater the entropic loss that occurs upon this binding, the greater the “entropic penalty.” Furthermore, unbound molecules that are flexible lose more entropy when forming a complex than molecules that are rigidly structured, because of the loss of flexibility when bound up in a complex. However, rigidity in the unbound molecule also generally increases specificity by limiting the number of complexes that molecule can form. The knotted peptides can bind targets with antibody -like affinity, or with nanomolar or picomolar affinity. A wider examination of the sequence structure and sequence identity or homology of knottins reveals that they have arisen by convergent evolution in all kinds of animals and plants. In animals, they are often found in venoms, for example, the venoms of spiders and scorpions and have been implicated in the modulation of ion channels. The knottin proteins of plants can inhibit the proteolytic enzymes of animals or have antimicrobial activity, suggesting that knottins can function in the native defense of plants. [0182] The present disclosure provides peptides that comprise or are derived from these knotted peptides (or knottins). As used herein, the term “knotted peptide” is considered to be interchangeable with the terms “knottin.”
[0183] Knotted peptides of the present disclosure comprise cysteine amino acid residues. In some cases, the peptide has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 cysteine amino acid residues. In some cases, the peptide has at least 8 cysteine amino acid residues. In other cases, the peptide has at least 10 cysteine amino acid residues, at least 12 cysteine amino acid residues, at least 14 cysteine amino acid residues or at least 16 cysteine amino acid residues.
[0184] A knotted peptide can comprise disulfide bridges. A knotted peptide can be a peptide wherein 5% or more of the residues are cysteines forming intramolecular disulfide bonds. A disulfide-linked peptide can be a drug scaffold. In some embodiments, the disulfide bridges form a knot. A disulfide bridge can be formed between cysteine residues, for example, between cysteines 1 and 4, 2 and 5, or, 3 and 6. In some cases, one disulfide bridge passes through a loop formed by the other two disulfide bridges, for example, to form the knot. In other cases, the disulfide bridges can be formed between any two cysteine residues.
[0185] The present disclosure further includes peptide scaffolds that, e g., can be used as a starting point for generating additional peptides. In some embodiments, these scaffolds can be derived from a variety of knotted peptides (or knottins). In certain embodiments, knotted peptides are assembled into a complex tertiary structure that is characterized by a number of intramolecular disulfide crosslinks, and optionally contain beta strands and other secondary structures such as an alpha helix. For example, knotted peptides include, in some embodiments, small disulfide-rich proteins characterized by a disulfide through disulfide knot. This knot can be, for example, obtained when one disulfide bridge crosses the macrocycle formed by two other disulfides and the interconnecting backbone. In some embodiments, the knotted peptides can include growth factor cysteine knots or inhibitor cysteine knots. Other possible peptide structures include peptide having two parallel helices linked by two disulfide bridges without p- sheets (e g., hefutoxin).
[0186] A knotted peptide can comprise at least one amino acid residue in an L configuration. A knotted peptide can comprise at least one amino acid residue in a D configuration. In some embodiments, a knotted peptide is 15-40 amino acid residues long. In other embodiments, a knotted peptide is 11-57 amino acid residues long. In still other embodiments, a knotted peptide is 11-81 amino acid residues long. In further embodiments, a knotted peptide is at least 20 amino acid residues long. [0187] Knotted peptides or peptides can be derived from a class of proteins known to be present or associated with toxins or venoms. In some cases, the peptide can be derived from toxins or venoms associated with scorpions or spiders. The peptide can be derived from venoms and toxins of spiders and scorpions of various genus and species. For example, the peptide can be derived from a venom or toxin of the Leiurus quinquestriatus hebraeus, Buthus occitanus tunetanus, Hottentotta judaicus, Mesobuthus eupeus, Buthus occitanus Israelis, Hadrurus gertschi, Androctonus australis, Centruroides noxius, Heterometrus laoticus, Opistophthalmus carinatus, Haplopelma schmidti, Isometrus maculatus, Haplopelma huwenum, Haplopelma hainanum, Haplopelma schmidti, Agelenopsis aperta, Haydronyche versuta, Selenocosmia huwena, Heteropoda venatoria, Grammostola rosea, Ornithoctonus huwena, Hadronyche versuta, Atrax robustus, Angelenopsis aperta, Psalmopoeus cambridgei, Hadronyche infensa, Paracoelotes luctosus, and Chilobrachys jingzhaoor another suitable genus or species of scorpion or spider. In some cases, a peptide can be derived from a Buthus martensii Karsh (scorpion) toxin.
[0188] In some embodiments, a peptide of the present disclosure (e.g., a cell-penetrating peptide) can comprise a sequence having cysteine residues at one or more of corresponding positions 11, 12, 13, 14, 19, 20, 21, 22, 36, 38, 39, 41, for example with reference to SEQ ID NO: 295. In some embodiments, a peptide comprises Cys at corresponding positions 11, 12, 19, 20, 36, 39, or any combination thereof. For example, in certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 11. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 12. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 13. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 14. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 19. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 20. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 21. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 22. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 36. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 38. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 39. In certain embodiments, a peptide can comprise a sequence having a cysteine residue at corresponding position 41. In some embodiments, the first cysteine residue in the sequence can be disulfide bonded with the 4th cysteine residue in the sequence, the 2nd cysteine residue in the sequence can be disulfide bonded to the 5th cysteine residue in the sequence, and the 3rd cysteine residue in the sequence can be disulfide bonded to the 6th cysteine residue in the sequence. Optionally, a peptide can comprise one disulfide bridge that passes through a ring formed by two other disulfide bridges, also known as a “two-and-through” structure system. In some embodiments, the peptides disclosed herein can have one or more cysteines mutated to serine.
[0189] In some embodiments, peptides of the present disclosure (e g., cell-penetrating peptides or cell-penetrating peptide complexes) comprise at least one cysteine residue. In some embodiments, peptides of the present disclosure comprise at least two cysteine residues. In some embodiments, peptides of the present disclosure comprise at least three cysteine residues. In some embodiments, peptides of the present disclosure comprise at least four cysteine residues. In some embodiments, peptides of the present disclosure comprise at least five cysteine residues. In some embodiments, peptides of the present disclosure comprise at least six cysteine residues. In some embodiments, peptides of the present disclosure comprise at least ten cysteine residues. In some embodiments, a peptide of the present disclosure comprises six cysteine residues. In some embodiments, a peptide of the present disclosure comprises seven cysteine residues. In some embodiments, a peptide of the present disclosure comprises eight cysteine residues. In some embodiments, a peptide of the present disclosure comprises an even number of cysteine residues.
[0190] In some embodiments, a peptide of the present disclosure (e.g., a cell-penetrating peptide or cell-penetrating peptide complex) comprises an amino acid sequence having cysteine residues at one or more positions, for example with reference to SEQ ID NO: 295. In some embodiments, the one or more cysteine residues are located at any one of the corresponding amino acid positions 6, 10, 20, 34, 44, 48, or any combination thereof. In some aspects of the present disclosure, the one or more cysteine (C) residues participate in disulfide bonds with various pairing patterns (e.g., C10-C20). In some embodiments, the corresponding pairing patterns are C6-C48, C10-C44, and C20-C34. In some embodiments, the peptides as described herein comprise at least one, at least two, or at least three disulfide bonds. In some embodiments, at least one, at least two, or at least three disulfide bonds are arranged according to the corresponding C6-C48, C10-C44, and C20-C34 pairing patterns, or a combination thereof. In some embodiments, peptides as described herein comprise three disulfide bonds with the corresponding pairing patterns C6-C48, C10-C44, and C20-C34. [0191] In certain embodiments, a peptide (e.g., a cell-penetrating peptide or a cell-penetrating peptide complex) comprises a sequence having a cysteine residue at corresponding position 6. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 10. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 20. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 34. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 44. In certain embodiments, a peptide comprises a sequence having a cysteine residue at corresponding position 50. In some embodiments, the first cysteine residue in the sequence is disulfide bonded with the last cysteine residue in the sequence. In some embodiments, the second cysteine residue in the sequence is disulfide bonded with the second to the last cysteine residue in the sequence. In some embodiments, the third cysteine residue in the sequence is disulfide bonded with the third to the last cysteine residue in the sequence and so forth.
[0192] In some embodiments, the first cysteine residue in the sequence is disulfide bonded with the 6th cysteine residue in the sequence, the 2nd cysteine residue in the sequence is disulfide bonded to the 5th cysteine residue in the sequence, and the 3rd cysteine residue in the sequence is disulfide bonded to the 4th cysteine residue in the sequence. Optionally, a peptide can comprise one disulfide bridge that passes through a ring formed by two other disulfide bridges, also known as a “two-and-through” structure system. In some embodiments, the peptides disclosed herein have one or more cysteines mutated to serine. In some embodiments, a peptide of the present disclosure has an even number of cysteine residues. In some embodiments, a peptide of the present disclosure comprises no cysteine residues.
[0193] In some embodiments, a peptide (e.g., a cell-penetrating peptide or a cell-penetrating peptide complex) comprises no cysteine or disulfides. In some embodiments, a peptide comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more cysteine or disulfides. In other embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more cysteine residues have been replaced with serine residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more cysteine residues have been replaced with threonine residues.
[0194] In some embodiments, a peptide (e.g., a cell-penetrating peptide or a cell-penetrating peptide complex) comprises no Cys or disulfides. In some embodiments, a peptide comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more Cys or disulfides. In other embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Ser residues. In some embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more Cys residues have been replaced with Thr residues.
[0195] In some instances, one or more or all of the methionine residues in the peptide are replaced by leucine or isoleucine. In some instances, one or more or all of the tryptophan residues in the peptide are replaced by phenylalanine or tyrosine. In some instances, one or more or all of the asparagine residues in the peptide are replaced by glutamine. In some embodiments, the N-terminus of the peptide is blocked, such as by an acetyl group. Alternatively or in combination, in some instances, the C-terminus of the peptide is blocked, such as by an amide group. In some embodiments, the peptide is modified by methylation on free amines.
Sequence Identity and Homology
[0196] Percent sequence identity or homology is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (Id.). The sequence identity or homology is then calculated as: ([Total number of identical matches]/[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).
[0197] Additionally, there are many established algorithms available to align two amino acid sequences. For example, the “FASTA” similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of sequence identity or homology shared by an amino acid sequence of a peptide disclosed herein and the amino acid sequence of a peptide variant The FASTA algorithm is described by Pearson and Lipman, Proc. Nat'lAcad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO: 1) and a test sequence that has either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are “trimmed” to include only those residues that contribute to the highest score. If there are several regions with scores greater than the “cutoff’ value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch- Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, Siam J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. Illustrative parameters for FASTA analysis are: ktup=l, gap opening penalty-! 0, gap extension penalty—!, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file (“S MATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).
[0198] FASTA can also be used to determine the sequence identity or homology of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as described above.
[0199] Some examples of common amino acids that are a “conservative amino acid substitution” are illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine. The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat'lAcad. Sci. USA 89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above), the language “conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
[0200] Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that can be more or less tolerant of change and maintain the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity or homology and computer analysis using available software (e.g., the Insight II.RTM. viewer and homology modeling tools; MSI, San Diego, Calif), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, G.J., Current Opin. Struct. Biol. 5'312-6 (1995) and Cordes, M.H. et al., Current Opin. Struct. Biol. 6:3-10 (1996)). In general, when designing modifications to molecules or identifying specific fragments determination of structure can typically be accompanied by evaluating activity of modified molecules.
[0201] At physiological pH, peptides can have a net charge, for example, of -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, or +5. When the net charge is zero, the peptide can be uncharged or zwitterionic. In some embodiments, the peptide contains one or more disulfide bonds and has a positive net charge at physiological pH where the net charge can be +0.5 or less than +0.5, +1 or less than +1, +1.5 or less than +1.5, +2 or less than +2, +2.5 or less than +2.5, +3 or less than +3, +3.5 or less than +3.5, +4 or less than +4, +4.5 or less than +4.5, +5 or less than +5, +5.5 or less than +5.5, +6 or less than +6, +6.5 or less than +6.5, +7 or less than +7, +7.5 or less than +7.5, +8 or less than +8, +8.5 or less than +8.5, +9 or less than +9.5, +10 or less than +10. In some embodiments, the peptide has a negative net charge at physiological pH where the net charge can be -0.5 or less than -0.5, -1 or less than -1, -1.5 or less than -1.5, -2 or less than -2, -2.5 or less than -2.5, -3 or less than -3, -3.5 or less than -3.5, -4 or less than -4, -4.5 or less than -4.5, -5 or less than -5, -5.5 or less than -5.5, -6 or less than -6, -6.5 or less than -6.5, -7 or less than -7, - 7.5 or less than -7.5, -8 or less than -8, -8.5 or less than -8.5, -9 or less than -9.5, -10 or less than -10.
[0202] In some embodiments, peptides of the present disclosure can have an isoelectric point (pl) value from 3 and 10. In other embodiments, peptides of the present disclosure can have a pl value from 4.3 and 8.9. In some embodiments, peptides of the present disclosure can have a pl value from 3-4. In some embodiments, peptides of the present disclosure can have a pl value from 3-5. In some embodiments, peptides of the present disclosure can have a pl value from 3-6. In some embodiments, peptides of the present disclosure can have a pl value from 3-7. In some embodiments, peptides of the present disclosure can have a pl value from 3-8. In some embodiments, peptides of the present disclosure can have a pl value from 3-9. In some embodiments, peptides of the present disclosure can have a pl value from 4-5. In some embodiments, peptides of the present disclosure can have a pl value from 4-6. In some embodiments, peptides of the present disclosure can have a pl value from 4-7. In some embodiments, peptides of the present disclosure can have a pl value from 4-8. In some embodiments, peptides of the present disclosure can have a pl value from 4-9. In some embodiments, peptides of the present disclosure can have a pl value from 4-10 In some embodiments, peptides of the present disclosure can have a pl value from 5-6. In some embodiments, peptides of the present disclosure can have a pl value from 5-7. In some embodiments, peptides of the present disclosure can have a pl value from 5-8. In some embodiments, peptides of the present disclosure can have a pl value from 5-9. In some embodiments, peptides of the present disclosure can have a pl value from 5-10. In some embodiments, peptides of the present disclosure can have a pl value from 6-7. In some embodiments, peptides of the present disclosure can have a pl value from 6-8. In some embodiments, peptides of the present disclosure can have a pl value from 6-9. In some embodiments, peptides of the present disclosure can have a pl value from 6-10. In some embodiments, peptides of the present disclosure can have a pl value from 7-8. In some embodiments, peptides of the present disclosure can have a pl value from 7-9. In some embodiments, peptides of the present disclosure can have a pl value from 7-10. In some embodiments, peptides of the present disclosure can have a pl value from 8-9. In some embodiments, peptides of the present disclosure can have a pl value from 8-10. In some embodiments, peptides of the present disclosure can have a pl value from 9-10.
[0203] In some cases, the engineering of one or more mutations within a peptide yields a peptide with an altered isoelectric point, charge, surface charge, or rheology at physiological pH. Such engineering of a mutation to a peptide derived from a scorpion or spider can change the net charge of the peptide, for example, by decreasing the net charge by 1, 2, 3, 4, or 5, or by increasing the net charge by 1, 2, 3, 4, or 5. In such cases, the engineered mutation can facilitate the ability of the peptide to penetrate a cell, an endosome, or the nucleus. Suitable amino acid modifications for improving the rheology and potency of a peptide can include conservative or non-conservative mutations.
[0204] A peptide can comprise at most 1 amino acid mutation, at most 2 amino acid mutations, at most 3 amino acid mutations, at most 4 amino acid mutations, at most 5 amino acid mutations, at most 6 amino acid mutations, at most 7 amino acid mutations, at most 8 amino acid mutations, at most 9 amino acid mutations, at most 10 amino acid mutations, or another suitable number as compared to the sequence of the venom or toxin component that the peptide is derived from. In other cases, a peptide, or a functional fragment thereof, comprises at least 1 amino acid mutation, at least 2 amino acid mutations, at least 3 amino acid mutations, at least 4 amino acid mutations, at least 5 amino acid mutations, at least 6 amino acid mutations, at least 7 amino acid mutations, at least 8 amino acid mutations, at least 9 amino acid mutations, at least 10 amino acid mutations, or another suitable number as compared to the sequence of the venom or toxin component that the peptide is derived from. In some embodiments, mutations can be engineered within a peptide to provide a peptide that has a desired charge or stability at physiological pH.
[0205] Generally, the NMR solution structures, the x-ray crystal structures, as well as the primary structure sequence alignment of related structural homologs or in silico design can be used to inform mutational strategies that can improve the folding, stability, and/or manufacturability, while maintaining a particular biological function. The general strategy for producing homologs or in silico designed peptides or proteins can include identification of a charged surface patch or conserved residues of a protein, mutation of critical amino acid positions and loops, and testing of sequences. This strategy can be used to design peptides with improved properties or to correct deleterious mutations that complicate folding and manufacturability. These key amino acid positions and loops can be retained while other residues in the peptide sequences can be mutated to improve, change, remove, or otherwise modify function, such as binding or ability to penetrate a cell, endosome, or nucleus in a cell, homing, or another activity of the peptide.
[0206] The present disclosure also encompasses multimers of the various peptides described herein. Examples of multimers include dimers, trimers, tetramers, pentamers, hexamers, heptamers, and so on. A multimer may be a homomer formed from a plurality of identical subunits or a heteromer formed from a plurality of different subunits. In some embodiments, a peptide of the present disclosure is arranged in a multimeric structure with at least one other peptide, or two, three, four, five, six, seven, eight, nine, ten, or more other peptides. In certain embodiments, the peptides of a multimeric structure each have the same sequence. In alternative embodiments, some or all of the peptides of a multimeric structure have different sequences. [0207] The present disclosure further includes peptide scaffolds that, e.g., can be used as a starting point for generating additional peptides. In some embodiments, these scaffolds can be derived from a variety of knotted peptides or knottins. Some suitable peptides for scaffolds can include, but are not limited to, CDPS, chlorotoxin, brazzein, circulin, stecrisp, hanatoxin, midkine, hefutoxin, potato carboxypeptidase inhibitor, bubble protein, attractin, a-GI, a-GID, p- PIIIA, co-MVIIA, ®-CVID, %-MrIA, p-TIA, conantokin G, contulakin G, GsMTx4, margatoxin, shK, toxin K, chymotrypsin inhibitor (CTI), and EGF epiregulin core. In some embodiments, the peptide sequence is flanked by additional amino acids. One or more additional amino acids can, for example, confer a desired in vivo charge, isoelectric point, chemical conjugation site, stability, or physiologic property to a peptide.
Cargo Molecules and Cell-Penetrating Peptide Complexes
[0208] A cell-penetrating peptide sequence (e.g., a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) can be fused or linked to a cargo molecule, such a small molecule or peptide. For example, a cell-penetrating peptide may be appended to either the N- terminus or the C-terminus of a peptide (e.g., a cargo peptide, an active agent peptide, a therapeutic peptide, or a detectable peptide). In some embodiments, the cell -penetrating peptide can be appended to the N-terminus of a peptide (e.g., a cargo peptide, an active agent peptide, a therapeutic peptide, or a detectable peptide) following an N-terminal GS dipeptide and preceding, for example, a GGGS (SEQ ID NO: 257) linker. In some embodiments, cellpenetrating peptide sequence (e.g., a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254), can be appended to either the N-terminus or C-terminus of a peptide (e.g., a cargo peptide, an active agent peptide, a therapeutic peptide, or a detectable peptide) using a peptide linker such as GxSy (SEQ ID NO: 256) peptide linker, wherein x and y can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises (GS)x (SEQ ID NO: 258), wherein x can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises GGSSG (SEQ ID NO: 259), GGGGG (SEQ ID NO: 260), GSGSGSGS (SEQ ID NO: 261), GSGG (SEQ ID NO: 262), GGGGS (SEQ ID NO: 263), GGGS (SEQ ID NO: 264), GGS (SEQ ID NO: 265), GGGSGGGSGGGS (SEQ ID NO: 255), or a variant or fragment thereof. Additionally, KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx, and EPKSSDKTHT (SEQ ID NO: 267) from human IgG3 can be used as a peptide linker, or any multiple of SEQ ID NO: 266 or SEQ ID NO: 267. In other embodiments, the cell-penetrating peptide can be appended to the peptide at any amino acid residue. In further embodiments, the cell-penetrating peptide can be appended to the peptide at any amino acid residue without interfering with an activity of the peptide (e.g., a therapeutic activity, an enzymatic activity, a binding activity, or a transcription factor activity). In some embodiments, the tag peptide is appended via conjugation, linking, or fusion techniques. In some embodiments, the tag peptide is placed between two other peptides with active functions. In other embodiments, the cell-penetrating peptide can be appended to the peptide at any amino acid residue. In further embodiments, the cell-penetrating peptide can be appended to the peptide at any amino acid residue without interfering with peptide activity (e.g., therapeutic activity, enzymatic activity, binding activity, or transcription factor activity). In some embodiments, the cell-penetrating peptide is appended via conjugation, linking, or fusion techniques. Examples of cell -penetrating peptide complexes that may improve cell penetration of the cargo peptide are provided in TABLE 4.
TABLE 4 - Exemplary Cell-Penetrating Peptide Complexes
Figure imgf000085_0001
Figure imgf000086_0001
[0209] In some embodiments, a cargo molecule is conjugated to, linked to, or fused to one or more cell-penetrating peptides (e.g., a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) for delivery of a cargo molecule across a cellular layer or into the cytoplasm or nucleus of a cell. Conjugation, linking, or fusion can be direct or with a spacer in between (chemical or peptide-based). A spacer can be any peptide linker. For example, a spacer can be GGGSGGGSGGGS (SEQ ID NO: 255), KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx, EPKSSDKTHT (SEQ ID NO: 267) from human IgG3 or any variant or fragment thereof. In some embodiments, a cell-penetrating peptide may be linked to a cargo peptide via a peptide linker (e.g., any of SEQ ID NO: 255 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485). In some embodiments, a cell-penetrating peptide may be linked to a cargo molecule via a small molecule linker (e.g., a linker provided in TABLE 11). [0210] A cargo molecule can be delivered carried across a cellular layer (e g., a plasma membrane, a vesicular membrane, an endosomal membrane, a nuclear envelope, or a blood brain barrier) to a cellular, intracellular, or paracellular space (e.g., a cytosol, a nucleus, or a nanolumen) when linked, fused, conjugated, or otherwise connected to a cell -penetrating peptide of the present disclosure. In some embodiments, a cargo molecule can be an active agent (e.g., a therapeutic agent or a detectable agent). In some embodiments, a cargo molecule can be a cystine-dense peptide, an anti-cancer agent, a transcription factor binding agent, an inhibitor of protein-protein interactions, a promoter of protein-protein interactions, a transcription factor, an RNA, a Cas enzyme or other CRISPR component, a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, a protein (e.g., a protein that inhibits proteinprotein interactions), an oligonucleotide, or an immunomodulating agent. For example, a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254, or a fragment thereof, can be conjugated, fused, linked, or otherwise connected to an active agent comprising a cystine- dense peptide, an anti-cancer agent, a transcription factor binding agent, an inhibitor of proteinprotein interactions, a promoter of protein-protein interactions, a transcription factor, an RNA, a Cas enzyme or other CRISPR component, a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, a protein (e g., a protein that inhibits proteinprotein interactions), an oligonucleotide, or an immunomodulating agent for delivery of the active agent across a cellular layer.
[0211] In some embodiments, one or more cell-penetrating peptides can be fused, linked, or conjugated to a cargo peptide or cargo molecule for improved cell penetration. A cargo molecule may be a target-binding molecule that binds a target of interest, such as a target peptide. In some embodiments, the cargo molecule inhibits the target upon binding. For example, biding of the cargo molecule may inhibit formation of protein-protein or protein-nucleic acid interactions with the target. In another example, binding of the cargo molecule may inhibit a conformation change or enzymatic activity of the target molecule. Examples of target molecules include TEAD, coldinducible RNA-binding protein, androgen receptor, ikaros, aiolos, a nuclear receptor, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, tau, AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, P-catenin, IKK, TEAD, NF-KB, PKC, AP-1, Jun, Fos, or REL. In some embodiments, a cargo molecule that binds a target may comprise a sequence of any one of SEQ ID NO: 293 - SEQ ID NO: 298 or SEQ ID NO: 364 - SEQ ID NO: 407. In some embodiments, a cargo molecule that binds a target may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NO: 293 - SEQ ID NO: 298 or SEQ ID NO: 364 - SEQ ID NO: 407. Examples of cargo-binding molecules are provided in TABLE 5.
TABLE 5 - Exemplary Cargo Peptides and Associated Targets
Figure imgf000088_0001
Figure imgf000089_0001
[0212] In some embodiments, a cargo molecule may comprise a small molecule (e.g., a small molecule ligand) that binds a target. For example, a cargo molecule may comprise a small molecule that binds a ubiquitin ligase. Examples of cargo molecules that binds ubiquitin ligase include thalidomide, pomalidomide, lenalidomide, methyl bestatin, bestatin, nutlin-3, and VHL ligand 1, as well as other immunomodulatory drugs (IMiDs). A cargo molecule, such as a cargo molecule of a targeted degradation complex, may comprise a target-binding peptide (e.g., a peptide that binds TEAD, cold-inducible RNA-binding protein, androgen receptor, ikaros, aiolos, nuclear receptors, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, or tau) and a small molecule that binds an E3 ubiquitin ligase (e.g., thalidomide, pomalidomide, lenalidomide, methyl bestatin, bestatin, nutlin-3, or VHL ligand 1). The cargo molecule may be fused or linked to a cell -penetrating peptide to form a cell-penetrating targeted degradation complex.
[0213] A peptide complex of the present disclosure may be delivered across a cellular layer of a cell by way of the cell-penetrating peptide. The peptide complex may be delivered to a cellular compartment (e g., the cytosol or nucleus) of the cell. The peptide complex may be delivered to an intercellular compartment (e.g., a nanolumen, intercellular space, or paracellular space) across the cellular layer. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 22%, at least about 25%, at least about 27%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of the peptide complex is delivered across the cellular layer. In some embodiments, from about 5% to about 80%, from about 10% to about 80%, from about 15% to about 80%, from about 20% to about 80%, from about 25% to about 80%, from about 30% to about 80%, from about 35% to about 80%, from about 40% to about 80%, from about 45% to about 80%, from about 50% to about 80%, from about 55% to about 80%, from about 60% to about 80%, from about 65% to about 80%, from about 70% to about 80%, from about 5% to about 60%, from about 10% to about 60%, from about 15% to about 60%, from about 20% to about 60%, from about 25% to about 60%, from about 30% to about 60%, from about 35% to about 60%, from about 40% to about 60%, from about 45% to about 60%, from about 50% to about 60%, from about 5% to about 40%, from about 10% to about 40%, from about 15% to about 40%, from about 20% to about 40%, from about 25% to about 40%, from about 30% to about 40%, from about 5% to about 25%, from about 10% to about 25%, from about 15% to about 25%, or from about 20% to about 25% of the peptide complex is delivered across the cellular layer.
Cystine-Dense Peptide-Based Cell-Penetrating Peptide Complexes
[0214] In certain embodiments, the cell-penetrating peptides described herein are attached to another molecule (e.g., a cargo molecule), such as a cystine-dense peptide (CDP) that provides a functional capability. For example, a cell-penetrating peptide attached to a CDP may have a sequence of any one of SEQ ID NO: 299 - SEQ ID NO: 308 or SEQ ID NO: 312 - SEQ ID NO: 321. In some embodiments, cell-penetrating peptides can direct the CDP into the cell. In further embodiments, cell-penetrating peptides can direct the CDP into the nucleus. In further embodiments, cell-penetrating peptides can direct the CDP cross the blood brain barrier. In further embodiments, cell-penetrating peptides can direct the CDP into a nanolumen between cells (e.g., cancer cells). In some embodiments, the CDP has a therapeutic effect inside the cell. In further embodiments, cell-penetrating peptides can direct the CDP into a nanolumen between cells (e.g., cancer cells). In further embodiments, cell-penetrating peptides can direct the CDP into or across a cellular space or compartment (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, or other subcellular compartment membrane, a blood brain barrier, or a nanolumen).
[0215] In some embodiments, the CDP may bind to a cytosolic, nuclear, intracellular, or paracellular protein and modulate an activity or a protein-protein interaction of the protein. For example, the CDP may bind to and modulate a transcription factor, cereblon, VHL, DCAF15, DCAF16, other molecules in the CRL4 E3 ligase family, other molecules in the E3 ligase family, or other molecules in the UPS system. In some embodiments, the CDP may bind to target molecule, such as a target peptide. Binding of the CDP to the target molecule may inhibit the target molecule (e g., inhibit a conformational change, inhibit an enzymatic activity, inhibit ligand binding, inhibit a protein-protein interaction, or inhibit a protein-nucleic acid interaction of the target). In some embodiments, binding of the CDP may recruit additional agents to the target, such as a ubiquitin ligase. In some embodiments, the CDP is a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a protein (e.g., a protein that inhibits protein-protein interactions), or a proteolysis targeting chimera.
Active Agent Peptide Conjugates
[0216] In some embodiments, the cell-penetrating peptides or peptide complexes of the present disclosure (e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) can be conjugated to, linked to, or fused with an active agent (e.g., a cargo molecule, a therapeutic agent, a drug, a biologic, or a peptide). The cell-penetrating peptide may be linked to more than one active agent. For example, the cell-penetrating peptide may be linked to a cargo peptide (e.g., a targetbinding peptide) and an additional active agent (e.g., a target-binding ligand, a therapeutic agent, a drug, a biologic, or a peptide). The cell-penetrating peptide may deliver the active agent into a cellular compartment (e.g., across a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, a blood brain barrier, or a nanolumen). Peptides according to the present disclosure can be conjugated to, linked to, or fused to an agent for use in the treatment of a disease or a condition (e.g., cancer, neurodegeneration, over-expression or under-expression of a gene, inflammation, or protein over-expression or accumulation). For example, in certain embodiments, the peptides described herein are fused to another molecule, such as an active agent that provides an additional functional capability. An active agent may be a small molecule active agent, a peptide active agent, or a nucleic acid active agent. For example, a cell-penetrating peptide may be conjugated to a small molecule therapeutic agent.
[0217] The cell-penetrating peptides of the present disclosure may be fused to an active agent that may otherwise be excluded from a cell or a cellular compartment to facilitate absorption or permeation of the active agent. For example, a cell-penetrating peptide of the present disclosure may facilitate cytosolic delivery of an active agent with multiple hydrogen bond donors or hydrogen bond acceptors, a large molecular weight, or a high partition coefficient, which may lead to poor cytosolic delivery of the active agent in the absence of the cell -penetrating peptide. In some embodiments, a cell-penetrating peptide may be conjugated to an active agent comprising at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors. In some embodiments, the cell-penetrating peptide may be conjugated to an active agent comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors. In some embodiments, the cell-penetrating peptide may be conjugated to an active agent comprising a molecular weight of at least 200 Da, at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, at least 1000 Da, at least 1500 Da, at least 2000 Da, at least 3000 Da, at least 4000 Da, at least 5000 Da, at least 6000 Da, at least 7000 Da, at least 8000 Da, at least 9000 Da, or at least 10,000 Da. In some embodiments, the cell-penetrating peptide may be conjugated to an active agent comprising a molecular weight of up to 1000 Da, up to 2000, up to 3000 Da, up to 4000 Da, up to 5000 Da, up to 6000 Da, up to 7000 Da, up to 8000 Da, up to 9000 Da, up to 10,000 Da, up to 12,000 Da, up to 15,000 Da, up to 20,000 Da, or up to 25,000 Da. In some embodiments, the cell-penetrating peptide may be conjugated to an active agent comprising a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0. The partition coefficient may be a measure of a ratio of the solubility of the agent in a hydrophobic solvent (e.g., octanol) relative to water.
[0218] A peptide can be fused with an active agent through expression of a vector containing the sequence of the peptide with the sequence of the active agent. In various embodiments, the sequence of the peptide and the sequence of the active agent can be expressed from the same Open Reading Frame (ORF). In various embodiments, the sequence of the peptide and the sequence of the active agent can comprise a contiguous sequence. The peptide and the active agent can each retain similar functional capabilities in the fusion peptide compared with their functional capabilities when expressed separately. In certain embodiments, examples of active agents can include other peptides.
[0219] As another example, in certain embodiments, the peptides described herein are attached to another molecule, such as an active agent that provides a functional capability. In some embodiments, cell-penetrating peptides can direct the active agent into the cell. In further embodiments, cell-penetrating peptides can direct the active agent into the nucleus. In further embodiments, cell-penetrating peptides can direct the active agent across the blood brain barrier. In further embodiments, cell-penetrating peptides can direct the active agent into a nanolumen between cells (e.g., cancer cells). In further embodiments, cell-penetrating peptides can direct the active agent into or across a cellular space or compartment (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen). In some embodiments, the active agent has a therapeutic effect inside the cell. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 active agents can be linked to a peptide. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell-penetrating peptides of this disclosure can be linked to an active agent. Multiple active agents can be attached by methods such as conjugating to multiple lysine residues and/or the N-terminus, or by linking the multiple active agents to a scaffold, such as a polymer or dendrimer and then attaching that agent-scaffold to the peptide (such as described in Yurkovetskiy, A. V., Cancer Res 75(16): 3365-72 (2015)) or by recombinant fusion. Examples of active agents include but are not limited to: a peptide, an oligopeptide, a polypeptide, a peptidomimetic, a polynucleotide, a polyribonucleotide, a cysteine-dense peptide, an affibody, an avimer, an adnectin, a B-hairpin, a stapled peptide, a kunitz domain, a nanofttin, a fynomer, a bicycle peptide, a Cas protein, a transcription factor, a DNA, a cDNA, a ssDNA, a RNA, a dsRNA, a micro RNA, a guide RNA, a U1 adaptor, a crRNA, a tracrRNA, an oligonucleotide, an antibody, a single chain variable fragment (scFv), a nanobody, an antibody fragment, an aptamer, a cytokine, an interferon, a hormone, an enzyme, a growth factor, a checkpoint inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, a CD antigen, a chemokine, a neurotransmitter, an ion channel inhibitor, an ion channel activator, a G-protein coupled receptor inhibitor, a G-protein coupled receptor activator, a chemical agent, a radiosensitizer, a radioprotectant, a radionuclide, a therapeutic small molecule, a steroid, a corticosteroid, an anti-inflammatory agent, an immune modulator, a complement fixing peptide or protein, a tumor necrosis factor inhibitor, a tumor necrosis factor activator, a tumor necrosis factor receptor family agonist, a tumor necrosis receptor antagonist, a Tim-3 inhibitor, a protease inhibitor, an amino sugar, a chemotherapeutic, a cytotoxic molecule, a toxin, a tyrosine kinase inhibitor, an anti-infective agent, an antibiotic, an anti-viral agent, an anti-fungal agent, an aminoglycoside, a nonsteroidal anti-inflammatory drug (NS AID), a statin, a nanoparticle, a liposome, a polymer, a biopolymer, a polysaccharide, a proteoglycan, a glycosaminoglycan, polyethylene glycol, a lipid, a dendrimer, a fatty acid, or an Fc region, or an active fragment or a modification thereof. In some embodiments, the peptide is covalently or non-covalently linked to an active agent, e.g., directly or via a linker. For example, cytotoxic molecules that can be used include auristatins, MMAE, MMAF, dolostatin, auristatin F, monomethylaurstatin D, DM1, DM4, maytansinoids, maytansine, calicheamicins, N-acetyl-y-calicheamicin, pyrrolobenzodiazepines, PBD dimers, doxorubicin, vinca alkaloids (4-deacetylvinblastine), duocarmycins, cyclic octapeptide analogs of mushroom amatoxins, epothilones, and anthracy lines, CC-1065, taxanes, paclitaxel, cabazitaxel, docetaxel, SN-38, irinotecan, vincristine, vinblastine, platinum compounds, cisplatin, methotrexate, and BACE inhibitors. Additional examples of active agents are described in McCombs, J. R , AAPS J, 17(2): 339-51 (2015), Ducry, L., Antibody Drug Conjugates (2013), and Singh, S. K., Pharm Res. 32(11): 3541-3571 (2015). Exemplary linkers suitable for use with the embodiments herein are discussed in further detail herein.
[0220] The peptides or fusion peptides of the present disclosure can also be conjugated to, linked to, or fused to other moieties that can serve other roles, such as providing an affinity handle (e.g., biotin) for retrieval of the peptides from tissues or fluids. For example, peptides or fusion peptides of the present disclosure can also be conjugated to, linked to, or fused to biotin. In addition to extension of half-life, biotin can also act as an affinity handle for retrieval of peptides or fusion peptides from tissues or other locations. In some embodiments, fluorescent biotin conjugates that can act both as a detectable label and an affinity handle can be used. Non limiting examples of commercially available fluorescent biotin conjugates include Atto 425- Biotin, Atto 488-Biotin, Atto 520-Biotin, Atto-550 Biotin, Atto 565-Biotin, Atto 590-Biotin, Atto 610-Biotin, Atto 620-Biotin, Atto 655-Biotin, Atto 680-Biotin, Atto 700-Biotin, Atto 725- Biotin, Atto 740-Biotin, fluorescein biotin, biotin-4-fluorescein, biotin-(5-fluorescein) conjugate, and biotin-B-phycoerythrin, ALEXA FLUOR 488 biocytin, ALEXA FLUOR 546, ALEXA FLUOR 549, lucifer yellow cadaverine biotin-X, Lucifer yellow biocytin, Oregon green 488 biocytin, biotin-rhodamine and tetramethylrhodamine biocytin. In some other examples, the conjugates could include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels. In some embodiments, the peptide-active agent fusions described herein can be attached to another molecule. For example, the peptide sequence also can be attached to another active agent (e.g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar, etc.). In some embodiments, the peptide can be fused with, or covalently or non-covalently linked to an active agent. In some other examples, the conjugates can include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels. In some embodiments, the peptide described herein can also be attached to another molecule. For example, the peptide sequence also can be attached to another active agent (e.g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding agents, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar). In some embodiments, the peptide can be conjugated to, linked to, or fused with, or covalently or non- covalently linked to an active agent.
[0221] Additionally, more than one peptide sequence derived from a toxin or venom knottin protein can be present on, conjugated to, linked to, or fused with a particular peptide. A peptide can be incorporated into a biomolecule by various techniques. A peptide can be incorporated by a chemical transformation, such as the formation of a covalent bond, such as an amide bond. A peptide can be incorporated, for example, by solid phase or solution phase peptide synthesis. A peptide can be incorporated by preparing a nucleic acid sequence encoding the biomolecule, wherein the nucleic acid sequence includes a subsequence that encodes the peptide. The subsequence can be in addition to the sequence that encodes the biomolecule or can substitute for a subsequence of the sequence that encodes the biomolecule. [0222] Active agents that may be delivered using the cell -penetrating peptides of the present disclosure include a RIG-I ligand, or ligands targeting a related receptor, such as melanoma differentiation-associated protein 5 (MDA5) or toll-like receptor 3 (TLR3). Ligands for RIG-I, MDA5, and TLR3 that may be complexed with a cell -penetrating peptide include abnormal double stranded RNA (dsRNA) comprising a 5’ diphosphate or, in the case of RIG-I, a 5’ triphosphate, such as a dsRNA associated with viral infection. Activation of RIG-I and MDA5 using RIG-I or MDA5 ligand can be effective at promoting antitumor immunity and apoptosis. Examples of RIG-I, MDA5, or TLR3 ligands that may be complexed with a cell-penetrating peptide of the present disclosure include dsRNA, 5’ diphosphate dsRNA, 5’ triphosphate dsRNA, double stranded hairpin RNA, a benzobisthiazole compound, and polyinosinic:polycytidylic acid. Delivery of a RIG-I, MDA5, or TLR3 ligand across a cellular layer may promote anti -tumor or anti-viral activity in a subject, thereby treating a cancer or viral infection in a subject.
[0223] Because RIG-I is active intracellularly, the present disclosure provides a peptide-RIG-I ligand complex in which the peptide can be capable of cell penetration such that the RIG-I ligand can access the cytoplasm of the target cell. RIG-I ligands by themselves may be able to activate the RIG-I helicase when in contact with it, but when applied to cells in vivo or in vitro, may access the cytoplasm at only low levels and thus not be able to access and activate RIG-I. Without a peptide of this disclosure, RIG-I ligands may require formulation with transfection reagents or other components to access the cytoplasm, which may not be feasible for human cancer therapy, due to toxicity, stability, safety issues, or inability to apply the formulation systemically (such as by intravenous or subcutaneous administration) and deliver sufficient amounts of active agent to the tumor. By combining a peptide of this disclosure with a RIG-I ligand to create a peptide-RIG-I ligand complex, RIG-I can be activated in vivo for anti-cancer therapy. For example, the RIG-I ligand in the peptide- RIG-I complex can access the cytoplasm of the target cell via cleavage of the peptide- RIG-I complex in the endosome or after exit from the endosome into the cytosol and dissociated RIG-I ligand therefrom can access the cytoplasm, or via any other mechanism as described herein. The RIG-I ligand can also optionally access the cytoplasm and RIG-I without cleavage. Cleavage can also occur in the cytosol. The peptide- RIG-I ligand complex can be designed such that the peptide is distal from the end of the RIG-I ligand that activates the helicase, and as such the peptide-RIG-I ligand complex may be active without cleavage. The interaction between RIG-I ligands and the RIG-I helicase, such as shown in the crystal structure in Devarker et al., Proc Natl Acad Sci, 113(3): 596-601, 2016, can be analyzed to design peptide-RIG-I ligand complexes. In the peptide- RIG-I complex, the peptides of this disclosure can be located a number of base pairs away from the 5’ triphosphate end of the RIG-I ligand, such as 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs away from the 5 ’ triphosphate end. In some embodiments, the peptide of this disclosure is conjugated 7-20 base pairs away from the 5’ triphosphate end end. In some embodiments, the peptide of this disclosure is conjugated more than 20 base pairs away from the 5’ triphosphate end. In some embodiments, the RIG-I ligand can be chemically conjugated to the peptide. For example, the peptide and RIG-I ligand can be linked with a cleavable linker, such that the linker can be cleaved selectively once intracellular, such as in the endosome or cytosol, thereby releasing the RIG-I ligand adequately in high concentrations within a cell in order to target intracellular RIG-I. The peptide and RIG-I ligand can be linked such that the RIG-I is inactive or blocked from binding to its receptor by the peptide until the peptide is removed, thereby reducing exposure of noncancerous tissues to the RIG-I ligand. For example, the linker can be a disulfide bond. In other embodiments, the linker can be acid labile. In other embodiments, the linker can be enzymatically cleavable, such that it is cleaved by enzymes in the endosomal- lysosomal pathway, or within the cytosol. In other embodiments, the peptide-RIG-I ligand complex can be co-formulated. In some embodiments, the peptide-RIG-I ligand complex can be formulated in a delivery vehicle, such as a liposome. In other embodiments, the peptide-RIG-I ligand complex comprises a RIG-I ligand that can be encapsulated in a liposome, which can be further coated with a peptide of the present disclosure. In other embodiments, the peptide-RIG-I ligand complex can be linked by a stable linker and is active as a complex. The linker can comprise additional functions as peptides or chemical structures that enhance endosomal escape, endosomal uptake, tissue biodistribution to the tumor, or cell penetration. Cell penetrating or endosomal escape peptide sequences can be added to the linker or to the other end of the peptide. The linker can comprised hydrophobic domains (such as (CFbjx where x = 1-30), cholesterol, LCA, DHA, or DLA, hydrophilic domains (such as hydroxyl groups or oligoethylene glycol), or flexible domains that allow the peptide and RIG-I ligand freedom of movement for interaction with cellular components.
[0224] Receptor-specificity of an RNA ligand-cell-penetrating peptide complex may be achieved by selective delivery of the complex to a compartment where the receptor is found (e.g., the cytoplasm or an endosome), by using a receptor-specific ligand, or a combination thereof. RIG-I and MDA5 are both RNA helicases present in the cytoplasm, while various tolllike receptors (TLRs) may be found in the endosome. In some embodiments, ligand recognized by two or more receptors found in different cellular compartments may be complexed with a cell-penetrating peptide of the present disclosure to specifically deliver the ligand to a desired cellular compartment (e.g., the cytosol or an endosome) to specifically activate one of the receptors. For example, delivery of polyinosinic: poly cytidylic acid to the cytoplasm may specifically activate MDA5, which is present in the cytoplasm, rather than a toll-like receptor found in endosomes (e g., TLR3, TLR7, TLR8, or TLR10). In some embodiments, an RNA ligand can be conjugated to a peptide of this disclosure to form a peptide complex that enters the cytoplasm and activates a cytoplasmic receptor (e g., RIG-I or MDA5). In some embodiments, complexing the ligand to a cell-penetrating peptide capable of endosomal delivery may enable activation of an endosomal RNA-sensing receptor (e.g., an RNA-sensing TLR).
[0225] RIG-I and MDA5 each have a C-terminal domain involved in ligand specificity and two N-terminal CARD domains, which enable mitochondrial antiviral -signaling protein (MAVS)- mediated signal transduction, and both recognize the internal RNA duplex structure.
Additionally, RIG-I can also recognize the 5’ end of dsRNA. Ligand binding of RIG-I or MDA5 may lead to activation of the MAVS -dependent signaling pathway, stimulating production of proinflammatory substances, including Type I interferons that contribute to antiviral and antitumor immunity, and are distinct from the gene expression induced by TLR3 activation. Activation of RIG-I or MDA5 can additionally lead to the activation of the inflammasome resulting in changes to the tumor microenvironment that promotes antitumor immunity, such as secretion of IL-1, IL- 18, and damage-associated molecular patterns (DAMPs). RIG-I and MDA5 both signal through mitochondrial antiviral-signaling (MAVS) proteins, which initiates signaling via IRF3/7 and NFKB factors. MAVS is also important for initiation of tumor cell apoptosis, via RIG-I-like receptor (RLR) activation, which leads to immunogenic cell death (ICD). Additionally, RIG-I can utilize multiple interferon regulatory factors (IRF). Thus, engagement of RIG-I or MDA5 with a RIG-I-specific ligand can activate anti-viral immune mechanisms, which can have therapeutic effects against tumors. In some embodiments, engagement of RIG-I or MDA5 with a RIG-I ligand can induce direct immunogenic cell death (ICD) of tumor cells, but not normal cells. This can result in dendritic cell (DC) presentation of tumor antigens to the immune system. Engagement of RIG-I or MDA5 by the 5’ triphosphate dsRNA can result in the secretion of pro-inflammatory cytokines Type I interferon (IFN), CXCL10, CCL5, IL-6, IL-23, IL-1, TNF, IFN, and others. In some embodiments, a RIG-I or MDA5 ligand can stimulate DC activation including inflammasome activity. In some embodiments, a RIG-I ligand can induce tumor cells to produce IFN and CXCL10 via the IRF3 pathway. As a result of the diverse immunogenic activity of RIG-I and MDA5 ligands, anti-tumor T cells can be induced. In some embodiments, a RIG-I or mDA5 ligand can induce tumor regression in a subject, such as a human, non-human primate, or any other animal. A RIG-I ligand can additionally inhibit a Thl7 and Treg responses. In some embodiments, cell penetrating peptide complexes of this disclosure can target MDA5, a related cytoplasmic sensor for dsRNA, which shares many of the same functions as RIG-I.
[0226] In some embodiments, a RIG-I ligand may comprise a short dsRNA. The short dsRNA may have a length of from 5 base pairs to 60 base pairs, from 5 base pairs to 10 base pairs, from 7 base pairs to 10 base pairs, from 11 base pairs to 18 base pairs, from 14 base pairs to 120 base pairs, from 5 base pairs to 15 base pairs, from 15 base pairs to 25 base pairs, from 25 base pairs to 40 base pairs, from 40 base pairs to 60 base pairs, from 60 base pairs to 80 base pairs, from 80 base pairs to 100 base pairs, from 100 base pairs to 120 base pairs, from 120 base pairs to 140 base pairs, from 140 base pairs to 160 base pairs, for from 19 base pairs to 60 base pairs. For example, a short dsRNA may comprise a length of from 19 base pairs to 60 base pairs and at least one 5’ triphosphate or 5’ diphosphate. The short dsRNA may further comprise an uncapped 5’ A or G nucleotide, a 5’ triphosphate on a blunt end, a 1 nucleotide 5’ overlap, a 1 nucleotide overlap at the 5 ’ end with the triphosphate, or combinations thereof. In some embodiments, at least one 5 ’ triphosphate or 5 ’ diphosphate is located on the 5 ’ end of the sense strand of the dsRNA. The short dsRNA may comprise a mismatch, for example a mismatch that is 8 or more base pairs from the 5’ end of the dsRNA. A dsRNA RIG-I ligand may be complexed with a cellpenetrating peptide of the present disclosure via an RNA modification. In some embodiments, the RNA modification may be positioned 8 or more base pairs from the 5 ’ end of the dsRNA. Additional types of dsRNA that may function as RIG-I ligands may include dsRNA hairpin RNA (e.g., in which some ribosides are paired with a partner within the same hairpin), or any other short dsRNA comprising a 5’ triphosphate. In some embodiments, a dsRNA described herein may be a based paired region within a longer single RNA sequence or may a base paired region of two separate RNA strands.
[0227] Double stranded RNA RIG-I ligands can be made by a variety of techniques that are used to combine the sense and antisense strands of the RNAs into a double stranded form. In some embodiments, the sense and antisense strands of the dsRNA can be separately transcribed or synthesized and combined into dsRNA structures using a variety of recombinant or synthetic techniques. In other embodiments, the sense and antisense strands of the dsRNA can be transcribed or synthesized in a single RNA that contains a loop structure (hairpin) that is optionally later cleaved by an RNAse to obtain the dsRNA. A RIG-I ligand may comprise two RNA strands complexed together as a double strand, comprising a 5’ diphosphate or a 5’ triphosphate group on one or both strands, or a single RNA strand complexed together in a hairpin that is double stranded at one or more locations in the molecule. The double strand may extend throughout the sequence or there may be regions of mismatch, and there may be one or more locations of hairpin self-association within one or both strands. In addition, the ends of the double strand may be at the same blunt location, or one or the other end may overhang. Hairpins and other structures within the RNA complex can be more immunogenic and activate the RIG-I pathway at higher levels. MDA5 ligands may also exhibit all the above structural variations but may also contain no or one 5’ phosphate.
[0228] The RNA backbone or bases of a RIG-I ligand can be modified to improve in vitro and in vivo stability (e.g., serum stability, manufacturability, shelf stability) or other properties of the molecule including base pairing affinity and immune system activation. Pyrimidines can be 2'- fluoro-modified, which can increase stability to nucleases as well as increase immune system activation. The RNA backbone can be phosphorothioate-substituted (where the non-bridging oxygen is replaced with sulfur), which can increase resistance to nuclease digestion as well as altering the biodistribution and tissue retention and increasing the pharmacokinetics such as by increasing protein binding, and can also induce more immune stimulation. Methyl phosphonate modification of an RNA can also be used. 2’-Omethyl and 2’-F RNA bases can be used, which can protect against base hydrolysis and nucleases and increase the melting temperature of duplexes. The modification can also comprise a bridged nucleic acid, a morpholino nucleic acid, a PNA, an LNA, an ethyl cEt nucleic acid. Bridged, Locked, and other similar forms of Bridged Nucleic Acids (BNA, LNA, cEt) where any chemical bridge such as an N-0 linkage between the 2' oxygen and 4' carbons in ribose can be incorporated to increase resistance to exo- and endonucleases and enhance biostability. These include Bridged Nucleic Acids (BNA) where an N-0 linkage between the 2’ and 4’ carbons occur and where any chemical modification of the nitrogen (including but not limited to N-H, N-CH3, N-benzene) in the bridge can be added to increase stability RNA backbone or base modifications can be placed anywhere in the RNA sequence, at one, multiple, or all base locations. Optionally, the modifications may be distal from the end of the dsRNA complex that contains the 5’ triphosphate and interacts with the helicase. The RNA backbone or base modifications may enhance, decrease, or have no effect on the level of RIG-I activation by the peptide-RIG-I ligand complex. Optionally, phosophorothioate nucleic acids may be used at the 2-3 terminal nucleic acids of one or both sequences. Optionally, 2’F modified nucleic acids may be used at least at 5%, at least at 10%, at least at 25%, at least at 50%, at least at 75%, or at up to 100% of internal positions. In some embodiments, 2’F modified nucleic acids may be used at from 2 to 4 positions, at all internal positions, or at all positions. [0229] The RIG-I ligand may have additional modified nucleotides or bases present within the sequence, such as to allow chemical modification such as conjugation to a linker and peptide or conjugation to an additional delivery agent such as a lipid, cholesterol, or hydrocarbon chain.
Peptide Oligonucleotide Complexes
[0230] In some embodiments, a cell -penetrating peptide of the present disclosure may be complexed with an oligonucleotide. The oligonucleotide may function as an active agent, a therapeutic agent, a detectable agent, or a combination thereof. A cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) may be complexed with an oligonucleotide to form a peptide oligonucleotide complex, also referred to as a peptidenucleotide agent conjugate, a peptide oligonucleotide complex, or a peptide target-binding agent complex, may comprise a peptide complexed with a nucleotide (e g., an oligonucleotide). The peptide of the peptide oligonucleotide complex may comprise a cell-penetrating peptide, as described herein (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254). A cellpenetrating peptide of a peptide oligonucleotide complex may facilitate cell penetration of the peptide oligonucleotide complex. For example, a peptide oligonucleotide complex comprising a cell-penetrating peptide may cross a cellular membrane, enabling delivery of the peptide oligonucleotide complex to a cytoplasm or a nucleus of the cell and interaction between the nucleotide of the peptide oligonucleotide complex and various cytosolic or nuclear components (e.g., genomic DNA, an ORF, mRNA, pre-mRNA, or DNA).
[0231] The nucleotide of the peptide oligonucleotide complex may be a target-binding agent comprising single stranded DNA, single stranded RNA, double stranded DNA, double stranded RNA, or a combination thereof. As used herein, the term “nucleotide” may refer to an oligonucleotide or polynucleotide molecule or to a single nucleotide base. For example, a nucleotide of a peptide complex may comprise a DNA or RNA oligonucleotide. In some embodiments, the nucleotide may be a small interfering RNA (siRNA), a micro RNA (miRNA, or miR), an anti-miR, an antisense RNA, an antisense oligonucleotide (ASO), a complementary RNA, a complementary DNA, an interfering RNA, a small nuclear RNA (snRNA), a spliceosomal RNA, an inhibitory RNA, a nuclear RNA, an oligonucleotide complementary to a natural antisense transcript (NAT), an aptamer, a gapmer, a splice blocker ASO, or a U1 adapter. For example, a nucleotide of the peptide oligonucleotide complex may comprise a sequence of any one of any one of SEQ ID NO: 488 - SEQ ID NO: 573 or a sequence complementary to a portion of any sequence provided in SEQ ID NO: 574 - SEQ ID NO: 611 or an open reading frame listed in TABLE 6. In some embodiments, the nucleotide may be an siRNA that inhibits translation of a target mRNA by promoting degradation of the target mRNA. In some embodiments, the nucleotide may be an miRNA that inhibits translation of a target mRNA by promoting cleavage or destabilization of the target mRNA. In some embodiments, the nucleotide may be an aptamer that binds to a target protein, thereby inhibiting protein-protein interactions with the target protein, inhibiting enzymatic activity of the target protein, or activating the target protein.
[0232] Examples of structures of various peptide oligonucleotide complexes (e.g., CDP- oligonucleotide complexes containing alternative and nonconventional bases) are illustrated in FIG. 32. Examples of oligonucleotides include an aptamer, a gapmer, an anti-miR, an siRNA, a splice blocker ASO, and a U1 adapter. The peptide portion of the peptide oligonucleotide complex (e.g., a CDP of a CDP- oligonucleotide complex) can be used to guide the nucleotide sequence (e g., an oligonucleotide of a CDP- oligonucleotide complex) to a specific tissue, target, or cell, or to deliver the oligonucleotide to the cytosol or nucleus of the cell, or to cause endosomal escape of the oligonucleotide.
[0233] The peptide oligonucleotide complexes of the present disclosure may include nucleotide and nucleotide variants within the peptide oligonucleotide complex wherein the nucleotide portion is targeted to specific target molecule for modulation. Modulation of a target molecule may comprise degradation, inhibiting translation, decreasing expression, increasing expression, enhancing a binding interaction (e.g., a protein-protein interaction), or inhibiting a binding interaction (e.g., a protein-protein interaction). Disclosed herein are nucleotide sequences that may be used in the nucleotide portion of the peptide oligonucleotide complex, such as those targeting or complementary to nucleotides (e.g., DNA or RNA molecules) listed in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or to nucleotides (e.g., DNA or RNA molecules) encoding the proteins listed in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or otherwise described herein. Examples of nucleotide sequences that may be used in the nucleotide portion of the peptide oligonucleotide complex include SEQ ID NO: 488 - SEQ ID NO: 573.
[0234] As disclosed herein, nucleic acid sequences, variants, and properties of the nucleic acids that are used in the nucleic acid portion of the peptide oligonucleotide complex may be referred to as nucleic acids of the present disclosure, nucleotides of the present disclosure, or like terminology. It may be understood that such nucleic acids or nucleotides are described in the context of the peptide oligonucleotide complexes disclosed, such as a nucleotide sequence comprising single stranded (ssDNA, ssRNA), double stranded (dsDNA, dsRNA), or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter within the peptide oligonucleotide complex, with the accorded alterations, functions and uses described.
[0235] In some embodiments, the nucleotide sequence (e.g., a target binding agent capable of binding a target molecule) is single stranded (ssDNA, ssRNA), double stranded (dsDNA, dsRNA), or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or Ul Adapter. Peptides according to the present disclosure can be conjugated to, linked to, or fused to such nucleotide sequences to make a peptide oligonucleotide complex. In addition, other active agents (e.g., small molecule, protein, or peptide active agents) as described herein can be conjugated to, linked to, complexed with, or fused to such nucleotide sequences, peptides or peptide oligonucleotide complex to form peptide oligonucleotide complex conjugates.
[0236] A nucleotide (e.g., a nucleotide of a peptide oligonucleotide complex) may be fully or partially reverse complementary to all or a portion of a target molecule (e.g., a target DNA or RNA sequence). In some embodiments, a target molecule expresses or encodes a protein (e.g., an mRNA encoding a protein associated with a disease). In some embodiments, a nucleotide may be fully or partially reverse complementary to a portion of an open reading frame encoding a gene or protein of interest. In some embodiments, a nucleotide may be reverse complementary to any portion of an RNA or open reading frame encoding a transcript or protein of interest. Examples of sequences that may serve as target molecules for the target binding nucleotides described herein are provided in SEQ ID NO: 574 - SEQ ID NO: 611 along any portion of its length. In some embodiments, a target molecule may comprise a fragment of any of the sequences provided in TABLE 6 along any portion of its length. In some embodiments, a target molecule may comprise a fragment of any of the sequences provided in SEQ ID NO: 574 - SEQ ID NO: 611. In some embodiments, a target molecule may comprise a sequence with one or more T residues replaced with U or one or more U residues replaced with T. TABLE 6 - Examples of Open Reading Frame Reference Sequences
Figure imgf000104_0001
[0237] A number of technologies can be used to generate therapeutically active nucleotide sequences for use in peptide oligonucleotide complexes that include the cell-penetrating peptides (e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) disclosed herein. Several have examples of molecules in the clinic or advanced clinical development and can be employed for the nucleotide portion within the peptide oligonucleotide complexes described herein. A nucleotide of a peptide oligonucleotide complex may bind to a target molecule (e.g., a target DNA, RNA, or protein) and modulate an activity of the target molecule. In this way, the nucleotide may function as a target-binding agent, also referred to as a targeted agent. Examples of nucleotides that may function as target-binding agents include nucleotide antisense RNAs, complementary RNAs, inhibitory RNAs, interfering RNAs, nuclear RNAs, antisense oligonucleotides, microRNAs, oligonucleotides complementary to natural antisense transcripts, small interfering RNAs, small nuclear RNAs, aptamers, gapmers, anti-miRs, splice blocker antisense oligonucleotides, and Ul adapters.
[0238] Nucleotides (e g., oligonucleotides targeted to a specific sequence for its regulation) may enter into cells through complexation with a cell-penetrating peptide to form a cell-penetrating peptide oligonucleotide complex. The cell-penetrating peptide oligonucleotide complex may be delivered into a cytoplasm or nucleus of a cell. The oligonucleotide, the peptide oligonucleotide complex, or any fragment thereof may enter the cytosol and may enter the nucleus.
[0239] In one embodiment, upon entry into the nucleus, oligonucleotides can bind directly to mRNA structures and prevent the maturation (e g., capping or splicing) of the targeted sequence, modulate alternative splicing of a targeted sequence, and recruit RNaseHl to induce cleavage of a targeted sequence. In another embodiment, oligonucleotides in the cytoplasm can bind directly to the target mRNA and sterically block the ribosomal subunits from attaching and/or running along the mRNA transcript during translation hence resulting in lack of translation of the target sequence. In another embodiment, oligonucleotides can also be designed to directly bind to microRNA (miRNA) sequences or natural antisense transcripts (NATs) sequences, each of the foregoing thereby prohibiting miRNAs and NATs from inhibiting their own specific RNA targets, which ultimately leads to reduced degradation or increased translation of one or more sequences themselves targeted by the miRNA or NAT. In another embodiment, siRNA (which may be targeted to a specific sequence and regulate expression of the target sequence) may alternatively be used to bind and regulate a targeted sequence in the cytoplasm, engaging an RNA-induced silencing complex (RISC), which is a multiprotein complex that incorporates one strand of a small interfering RNA (siRNA) or micro RNA (miRNA), using the siRNA or miRNA as a template for recognizing complementary mRNA of the targeted sequence. When it finds a complementary strand, its RNase domain cleaves the targeted sequence. In another embodiment, an aptamer (e.g., a nucleotide that modulates a specific protein or other target) may alternatively be used to bind and regulate a target. An aptamer (e.g., extracellular or intracellular) may function by directly binding and modulating activity of a protein target, for example by forming aptamer-protein interactions rather than through base pairing or hybridization interactions.
[0240] For example, conventional ASO, or antisense oligonucleotides, are typically 18-30 nucleotides (nt) in length. Several ASO therapeutic strategies exist, two of which (differing in their mechanism of target RNA interference) are further described. The first ASOs are sometimes called “Gapmers” because they have a central region with DNA-based-sugar nucleotides that are often (but not always) flanked by non-DNA-sugar nucleotides with greater resistance to nucleases. The DNA region, at least 4 nt in length but typically >6, causes a DNA/RNA hybrid that engages RNase H endonuclease to cleave the target RNA. Among clinically approved gapmers are fomivirsen and mipomersen. In some embodiments, a DNA region of a gapmer may comprise from about 4 to about 30, from about 4 to about 25, from about 4 to about 20, from about 4 to about 15, from about 4 to about 10, from about 6 to about 30, from about 6 to about 25, from about 6 to about 20, from about 6 to about 15, or from about 6 to about 10 nucleotide residues. In some embodiments, a non-DNA region of a gapmer may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues 3’ of the DNA region. In some embodiments, a non-DNA region of a gapmer may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues 5’ of the DNA region. Examples of gapmers are provided in TABLE 7.
TABLE 7 - Exemplary Gapmer Sequences
Figure imgf000105_0001
Figure imgf000106_0001
[0241] The second conventional ASO simply serves to bind to the target transcript, but not induce RNase degradation, so no DNA-based-sugars are used. Instead, binding is designed to disrupt processing into mature mRNA. One such activity relies on binding to the mRNA at or near splice sites to drive particular splice isoforms in the target RNA, resulting in modulating target RNA by disrupting mRNA splicing and resulting in exon skipping. These are commonly called “splice blocking” or “splice blocker” ASOs amongst other known names. One example is eteplirsen, designed to alter splicing of DMD (dystrophin) gene in Duchenne Muscular Dystrophy patients, correcting a mutation that would otherwise create a truncated and nonfunctional dystrophin by splicing out the mutant exons and creating a different truncated (but functional) protein to appear.
[0242] Another example is siRNA molecules which specifically interact with the canonical RNAi pathway (the RISC complex) to drive cleavage or steric blocking of hybridized transcripts; cleavage-vs-blocking depends on whether the match is perfect (cleavage) or imperfect but still stable (blocking). Length is typically a double-stranded RNA where the overlapping region is 19-22 and each strand has two extra nt at their 3’ ends. Chemistry is largely RNA-based-sugars, with some DNA-based sugars at the 3’ overhangs. Clinical examples include patisiran (targets TTR) and givosiran (targets ALAS ). In some embodiments, an overlapping region of a siRNA may comprise from about 10 to about 40, from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 10 to about 22, from about 10 to about 21, from about 10 to about 20, from about 15 to about 40, from about 15 to about 35, from about 15 to about 30, from about 15 to about 25, from about 15 to about 22, from about 15 to about 21, from about 15 to about 20, from about 17 to about 40, from about 17 to about 35, from about 17 to about 30, from about 17 to about 25, from about 17 to about 22, from about 17 to about 21, from about 17 to about 20, from about 18 to about 40, from about 18 to about 35, from about 18 to about 30, from about 18 to about 25, from about 18 to about 22, from about 18 to about 21, from about 18 to about 20, from about 19 to about 40, from about 19 to about 35, from about 19 to about 30, from about 19 to about 25, from about 19 to about 22, from about 19 to about 21, or from about 19 to about 20 nucleotide residues. In some embodiments, an overhang region may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residues. Examples of siRNAs are provided in TABLE 8.
TABLE 8 - Exemplary siRNA Sequences
Figure imgf000107_0001
Figure imgf000108_0001
[0243] Another example are anti-miRs. Anti-miRs may function as steric blockers designed against miRNAs that would block a RISC complex loaded with a specific disease-associated miRNA without being subject to cleavage by the RISC complex RNase subunit. One clinical example is miravirsen, a 15-base oligo with a mixture of DNA and LNA sugars that targets miR- 122 in hepatitis C patients. An anti-miR nucleotide may be of sufficient length to anneal specifically and stably to the target miR, but the length of the sequence may vary. For example, an anti-miR may have a length of up to about 21 nt, corresponding to the maximum size loaded into RISC. In some embodiments, an anti-miR nucleotide may comprise from about 10 to about 25, from about 10 to about 23, from about 10 to about 21, from about 10 to about 20, from about 10 to about 19, from about 10 to about 18, from about 13 to about 25, from about 13 to about 23, from about 13 to about 21, from about 13 to about 20, from about 13 to about 19, from about 13 to about 18, from about 15 to about 25, from about 15 to about 23, from about 15 to about 21, from about 15 to about 20, from about 15 to about 19, from about 15 to about 18, from about 16 to about 25, from about 16 to about 23, from about 16 to about 21, from about 16 to about 20, from about 16 to about 19, or from about 16 to about 18 nucleotide residues.
TABLE 9 - Examples of Anti-miR Truncations
Figure imgf000108_0002
Figure imgf000109_0001
[0244] Another example is U1 adapters which have two parts. One anneals to the Ul-snRNA of the Ul-snRNP complex, and the other binds to the target RNA, bringing the Ul-snRNP to the polyA site and inhibiting polyadenylation; absence of a polyA tail causes the mRNA to be degraded. The Ul-binding region is at least 10 nt but up to 19 nt. Target binding region can be from about 15 nt to about 25 nt. Chemistry in early studies made heavy use of LNA and 2’-O- Methyl sugars. In some embodiments, a U1 binding region may comprise from about 10 to about 25, from about 10 to about 23, from about 10 to about 21, from about 10 to about 20, from about 10 to about 19, from about 10 to about 18, from about 13 to about 25, from about 13 to about 23, from about 13 to about 21, from about 13 to about 20, from about 13 to about 19, from about 13 to about 18, from about 15 to about 25, from about 15 to about 23, from about 15 to about 21, from about 15 to about 20, from about 15 to about 19, from about 15 to about 18, from about 16 to about 25, from about 16 to about 23, from about 16 to about 21, from about 16 to about 20, from about 16 to about 19, or from about 16 to about 18 nucleotide residues. In some embodiments, a target binding region may comprise from about 10 to about 40, from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 10 to about 22, from about 10 to about 21, from about 10 to about 20, from about 15 to about 40, from about 15 to about 35, from about 15 to about 30, from about 15 to about 25, from about 15 to about 22, from about 15 to about 21, from about 15 to about 20, from about 17 to about 40, from about 17 to about 35, from about 17 to about 30, from about 17 to about 25, from about 17 to about 22, from about 17 to about 21, from about 17 to about 20, from about 18 to about 40, from about 18 to about 35, from about 18 to about 30, from about 18 to about 25, from about 18 to about 22, from about 18 to about 21, from about 18 to about 20, from about 19 to about 40, from about 19 to about 35, from about 19 to about 30, from about 19 to about 25, from about 19 to about 22, from about 19 to about 21, or from about 19 to about 20 nucleotide residues. An exemplary nucleic acid sequence contains a U1 adapter for modulating BCL2 mRNA that is highly active against BCL2 can include: 5 ’GCCGUACAGUUCCACAAAGGGCCAGGUzL4GG4 U-3 ’ (SEQ ID NO: 504), wherein the underlined portion (GCCGUACAGUUCCACAAAGG (SEQ ID NO: 573)) corresponds to the BCL2 recognition sequence and the italicized portion (GCCAGGUAAGUAU (SEQ ID NO: 492)) corresponds to the U1 recognition sequence. [0245] Another example of a nucleotide of the present disclosure is an aptamer. Aptamers disrupt target activity using a mechanism that differs from other nucleotides described herein that form base pairing interactions with a target nucleotide. Aptamers are nucleic acids that form secondary structures (e.g., where a single strand of nucleic acid base-pairs with itself upon folding, creating loops in various locations). Aptamers may be screened for interaction with target proteins. Aptamers may have varied nucleotide chemistry and may include a mixture of conventional RNA and/or DNA sugars and modified sugars (e.g., 2’-O-Methyl (2’-O-Me) RNA or 2’-Fluoro (2’-F) RNA sugars). For example, one clinically approved aptamer, pegaptanib (a VEGF-binding aptamer), has a mixture of 2’-O-Methyl (2’-O-Me) RNA and 2’-Fluoro (2’-F) RNA sugars and regular RNA and DNA sugars. An aptamer sequence may be long enough to form a stable secondary structure (e g., through intramolecular base pairing), but the length may vary. In some embodiments, an aptamer sequence may comprise from about 20 nt to about 40 nt. For example, experiments that identified pegaptanib used oligos of 20-40 nt in length. Shorter nucleotides (e.g., sequences shorter than about 40 nt) may be advantageous, as longer oligonucleotides may complicate nucleotide synthesis or engage the interferon response pathway. In some embodiments, an aptamer may comprise from about 15 to about 60, from about 15 to about 50, from about 15 to about 40, from about 15 to about 35, from about 15 to about 30, from about 20 to about 60, from about 20 to about 50, from about 20 to about 40, from about 20 to about 35, from about 20 to about 30, from about 25 to about 60, from about 25 to about 50, from about 25 to about 40, from about 25 to about 35, or from about 25 to about 30 nucleotide residues.
[0246] Nucleotides may be designed for use in the peptide nucleotide complexes of the present disclosure. In some embodiments, nucleotides that modify processing, translation, or other RNA functions (e.g., a gapmer, splice blocker, siRNA, anti-miR, or U1 adapter), have one or more of the following properties: (a) 8-50 nt in length, but preferably 12-30 nt in length. It is understood that any length of a nucleotide (nt) can be used within the foregoing ranges; (b) cross-species homology (e g., by targeting highly-conserved motifs) is often a desirable feature but is not necessary for activity or clinical development; (c) avoidance of common SNPs in humans unless that SNP is involved in disease pathology (e.g., an allele-specific oligo) is often a desirable feature but is not necessary for activity or clinical development; (d) gene specificity (they have minimal homology to other sequences; for example, a sequence may have 3 or more mismatches to every other sequence), (e) avoid predicted secondary structures in both the oligo and the target region (there are software tools available to screen in silico for such secondary structure formation); (f) higher G/C content may be preferable, as G/C-rich sequences (e.g. CCAC, TCCC, GCCA) may be helpful for increasing affinity of the nucleotide to its target, whereas A/T-rich sequences (e.g. TAA) or runs of 4+ G (GGGG) may exhibit low or result in structural (G-quadruplex) formation. An oligonucleotide sequence can be 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity or match to the target sequence. In some situations, an oligonucleotide with 100% complementarity will result in the target RNA being degraded. In some situations, an oligonucleotide that is less than 100% complementarity may not lead to degradation of the target RNA but may prevent translation and production of the encoded protein.
[0247] In some embodiments, gapmers have one or more of the following properties: (a) 12-30 nt in length. It is understood that any length of a nucleotide (nt) can be used within the foregoing range, (b) target sites are anywhere in the pre-mRNA, including UTRs, exons, or introns (c) central DNA region: minimum of 4 contiguous DNA nucleotides, often 10 or more are used. No artificial substitutions at 2’ site (e.g. 2’-O-methyl [2’-0-ME] or 2 ’-O-m ethoxy ethyl [2’-0-M0E]) are tolerated due to requirements of RNase H recognition, (d) flanking region: can be DNA- or RNA-based-sugars. 2’ substitutions such as 2’-0-ME or 2’-0-M0E are tolerated. Linked nucleic acids (LNA) and morpholino (phosphorodiamidate morpholino oligo) chemistry are also acceptable in flanking region, (e) Backbone can be natural phosphodiester (PO) or nonnatural phosphorothioate (PS) linkages. A clinical example is fomivirsen, a 21 nt gapmer wherein the whole oligo is PS-backbone DNA. Another example is mipomersen, a 20 nt gapmer wherein the entire backbone is PS linkages, and the central region uses DNA sugar flanked by 2’-0-M0E modified RNA. For these two examples, all C bases are 5-methyl-C, though this is not a strict requirement for engagement of RNase Hl. Similarly, thiophosphorodiamidate chemistries may be used.
[0248] In some embodiments, steric blockers have one or more of the following properties; (a) as the molecule does not need to engage RNase H or any other enzyme, backbone and sugar chemistry can be more varied, (g) target sites for the nucleotide are complementary to one or more splice sites in the target RNA. A clinical example is eteplirsen, a 30 nt splice blocking ASO wherein whole oligonucleotide uses morpholino (Phosphorodiamidate morpholino oligo) chemistry. Another clinical example is nusinersen, an 18 nt ASO, whose backbone is entirely PS linked and uses 2’-0-M0E RNA chemistry. All C bases are 5-methyl-C, though this is not a strict requirement for engagement of RNase Hl. Similarly, thiophosphorodiamidate chemistries may be used. [0249] In some embodiments, siRNA have one or more of the following properties: (a) can be between 15 and 25 nt in length (between 13 to 23 nt overlap respectively), or up to 25 nt (23 nt overlap) per strand, but 21 nt (19 nt overlap) is common. It is understood that any length of a nucleotide (nt) overlap can be used within the foregoing ranges; (b) complements a sequence typically but not exclusively of 21 -nt length in the target mRNA that typically but not exclusively begins with “AA” (c) target sites are ideally found in the mature spliced mRNA as the RISC complex for RNA cleavage is primarily cytosolic; (d) preferably but not exclusively avoids sequences within 100 nt of the mRNA start site, as the transcript at start site is more likely to be occupied by RNA polymerase, (e) successful siRNA constructs typically have more G/C at 5’ end of sense strand, more A/T at 3’ end of sense strand, and are roughly 30-60% in G/C content.
[0250] In some embodiments, anti-miR (anti-miRNA) have one or more of the following properties: (a) a perfect match to target sequence (specifically the 5 ’ end of the guide strand of the miRNA); (b) length can vary and can even be greater than the length of the mature guide strand. Screening for effective anti-miR constructs may begin with the shortest sequence that achieves specificity (no off-target homology) and increase length from there to empirically determine ideal minimal length for strong miRNA inhibition; (c) 2’ sugar modifications (2’-O- Me, 2’-0-M0E, 2’-F) and LNA sugars are commonly used. Sugars can be a mixture. A clinical example of an anti-miR is miravirsen, which uses a mixture of DNA and LNA sugars (d) PS linkages in backbone are common. PS linkages may reduce affinity, but sugar modifications may increase affinity.
[0251] In some embodiments, aptamers have one or more of the following properties: (a) length of aptamers can vary widely, as there is no biological complex (e.g., RISC) they interact with to function. Although composed of nucleic acids, they are more protein-like in function (e.g., bind to a target protein, etc.). The minimum length may be determined empirically to maintain sufficient stability of intra-strand hybridization to fold into a secondary structure, the upper limit on size is limited only by pharmacology, as longer sequences have a higher risk of engaging inflammatory pathways. Aptamer screening typically begins with libraries of 20-40 nt in length (not including flanking regions required for library amplification during screening); (b) as they form interactions via secondary structure rather than base pairing interactions, there are few limitations for their base patterns, since secondary structures are not only desirable but essential to their function. Design may be empirical for each target; (c) selection is typically via Systematic Evolution of Ligands by Exponential Enrichment (SELEX): random or semirandom sequences between primer-binding flanking regions are exposed to a target of interest on a solid substrate. The pooled oligonucleotide mixture is rinsed from the substrate, leaving only sequences that interact with the target remaining, and then binding sequences are eluted and amplified by PCR. (d) Sugar modifications commonly used include 2 ’-fluoro (2’-F), 2’-0-M0E, and 2’-O-Me, though other chemistries including (but not limited to) LNA and unlocked- nucleic-acids (UNA) are also possible; (e) backbones are typically PO or PS, but other linkages such as methylphosphonate are possible. A clinical aptamer example, pegaptanib, is entirely PO backbone, but others in development use other linkages, (f) aptamer termini are typically capped with unnatural nucleotide chemistries (e.g. 3’ inverted thymidine) or biotinylated nucleotides to reduce susceptibility to nucleases; (g) because activity is not based on base-pairing, aptamers can be much more creative with chemical modifications of the bases themselves; these can include bases designed to induce covalent bonds with target proteins to permanently disable them; (h) such modifications are tested after selection of an active, high affinity aptamer, as unmodified bases are required for nucleic acid amplification during SELEX (i) if the target protein is extracellular, less considerations are necessary than for cell penetration capabilities. [0252] In some embodiments, other general design considerations aimed at enhancing pharmacokinetic (PK) properties of the nucleotide, peptide, or peptide oligonucleotide complex include one or more of the following properties: (a) building in conjugation to moieties that reduce clearance or increase cellular uptake including cholesterol or other lipids, diacylglycerol, GalNAc, palmitoyl, PEG, an RGD motif, cell penetrating peptides or moieties (e g., a cellpenetrating peptide or cell penetrating peptide as described herein). Adding cholesterol to the peptide oligonucleotide complex can improve biodistribution to the target tissue, increase cellular uptake by endocytosis, and alter the serum pharmacokinetics.
[0253] The therapeutic activity and molecular method of the peptide oligonucleotide complex may depend on which target molecule (e.g., a DNA or RNA) that the nucleic acid complements, or in the case of an aptamer, which target molecule (e.g., protein or other macromolecule) it binds. Target choice can fall into one or more non-mutually exclusive categories such as tissuetarget-based or disease-selective. Known targets have known mRNA and genomic sequences that can be used to design a variety of complementary nucleic acids for use in the peptide nucleotide complexes described herein depending on the activity (e.g., gene regulation, protein degradation, reduction of cancer cell activators) desired. Examples of targets are provided in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, and TABLE 6. For example, tissue-targeting may comprise selecting targets acting in the tissues where a cell-penetrating peptide portion of the peptide oligonucleotide complex would preferentially access. Targets for the peptide oligonucleotide complexes can include oncogenes, for example by designing the nucleic acid portion of the complex to target overexpressed genes or those for which the tumor is lacking a redundant ortholog (i.e., normal cells function by using X or Y, tumors do not express Y, so X is targeted). In addition, disease-selective targeting can be used to treat conditions where the transcript is selectively found in the diseased tissue, and preferentially accumulate there, to improve safety and reduce off-target effects.
[0254] The target-binding agent (e.g., a nucleotide of a peptide oligonucleotide complex) may be capable of binding the targets described in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or to nucleotides (e.g., DNA or RNA molecules) encoding the proteins listed in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6, or otherwise described herein. Examples of nucleotide sequences that may be used in the nucleotide portion of the peptide oligonucleotide complex include SEQ ID NO: 488 - SEQ ID NO: 573. It is understood that any oligonucleotide may be used that is complementary to a portion of the target DNA or RNA molecule. Such target binding agent may comprise a nucleotide sequence is single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter. Such oligos may be about 5 to 30 nt in length, 10 to 25 nt in length, 15 to 25 nt in length, 19 to 23 nt in length, or at least 10 nt in length, at least 15 nt in length, at least 20 nt in length, at least 25 nt in length, or at least 30 nt in length, at least 50 nt in length, at least 100 nucleotides in length across any portion of the target RNA. Examples of sequences to which such oligonucleotides may bind (e.g., are complementary to) include SEQ ID NO: 574 - SEQ ID NO: 611, or any genomic or ORF sequence referenced in TABLE 6. One of skill in the art can readily design or determine the length of the target binding agent and whether the target binding agent is complementary to the reference target RNA sequence, and can thus determine using the chemistry of RNA and DNA where such target binding agent will bind to such reference target RNA sequence for the designed length across any portion of the target RNA. Consequently, for any RNA target described herein, including for any of the targets or molecules encoding the targets described in TABLE 12, and SEQ ID NO: 574 - SEQ ID NO: 611, or any genomic or ORF sequence referenced in TABLE 6 such target binding agent of any nt length is described.
[0255] In some embodiments, a nucleotide binds to the target molecule with a melting temperature of not less than 37 °C and not more than 99 °C. In some embodiments, a nucleotide binds to the target molecule with a melting temperature of not less than 40 °C and not more than 85 °C, not less than 40 °C and not more than 65 °C, not less than 40 °C and not more than 55 °C, not less than 50 °C and not more than 85 °C, not less than 60 °C and not more than 85 °C, or not less than 55 °C and not more than 65 °C.
[0256] In some embodiments, a nucleotide binds the target molecule with an affinity of not more than 500 nM, not more than 100 nM, not more than 50 nM, not more than 10 nM, not more than 1 nM, not more than 500 pM, not more than 400 pM, not more than 300 pM, not more than 200 pM, or not more than 100 pM. In some embodiments, a nucleotide binds the target molecule with an affinity of not more than 500 nM and not less than 100 pM, not more than 100 nM and not less than 200 pM, not more than 50 nM and not less than 300 pM, not more than 10 nM and not less than 400 pM, or not more than 1 nM and not less than 500 pM.
[0257] In some embodiments, a nucleotide comprises at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 488 - SEQ ID NO: 573. In some embodiments, a nucleotide comprises a sequence of any one of SEQ ID NO: 488 - SEQ ID NO: 573, any one of SEQ ID NO: 488 - SEQ ID NO: 573 wherein U is replaced with T, or any one of SEQ ID NO: 488 - SEQ ID NO: 573 wherein T is replaced with U. In some embodiments, a nucleotide comprises no more than 1, 2, 3, 4, or 5 base changes relative to a sequence of any one of SEQ ID NO: 488 - SEQ ID NO: 573.
[0258] In some embodiments, a nucleotide is at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% reverse complementary to the target molecule. In some embodiments, a nucleotide is 100% reverse complementary to the target molecule. In some embodiments, a nucleotide comprises no more than 1, 2, 3, 4, or 5 base pair mismatches upon binding to the target molecule. In some embodiments, a nucleotide comprises at least 1, 2, 3, 4, or 5 base pair mismatches upon binding to the target molecule.
[0259] In some embodiments, a nucleotide may modulate an activity of a target molecule. In some embodiments, modulating the activity of the target molecule comprises reducing expression of the target molecule, increasing the expression of the target molecule, reducing translation of the target molecule, degrading the target molecule, reducing a level of the target molecule, modifying the processing of the target molecule, modifying the splicing of the target molecule, inhibiting processing of the target molecule, reducing a level of a protein encoded by the target molecule, or blocking an interaction with the target molecule. In some embodiments, the expression of the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%. In some embodiments, the translation of the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99 9% In some embodiments, the expression of the target molecule is reduced by a factor of at least 2, 4, 8, 10, 15, 16, 20, 32, 50, 64, 100, 128, 200, 256, 500, 512, or 1000. In some embodiments, the translation of the target molecule is reduced by a factor of at least 2, 4, 8, 10, 15, 16, 20, 32, 50, 64, 100, 128, 200, 256, 500, 512, or 1000. In some embodiments, at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the target molecule is degraded. In some embodiments, the level of the protein encoded by the target molecule is reduced by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%. In some embodiments, modifying the splicing of the target molecule increases a level of a protein encoded by the target molecule by at least 10%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, 99.5%, or 99.9%.
[0260] A peptide oligonucleotide complex of the present disclosure may comprise a nucleotide complexed with a peptide (e.g., a cell-penetrating peptide). The nucleotide may comprise single stranded DNA, single stranded RNA, double stranded DNA, double stranded RNA, or combinations thereof. In some embodiments, a nucleotide of a peptide oligonucleotide complex may be non-naturally occurring, also referred to as an “engineered nucleotide”. In some embodiments, a nucleotide may comprise a naturally occurring sequence. A nucleotide may be exogenously expressed, enzymatically synthesized in vitro, or chemically synthesized. For example, a nucleotide may be expressed in a bacterial, yeast, or mammalian cell line and purified for use in a peptide oligonucleotide complex of the present disclosure. In another example, a nucleotide may be enzymatically synthesized in vitro using an RNA or DNA polymerase. In another example, a nucleotide may be chemically synthesized on a solid support using protected nucleotides.
[0261] One example of a chemical synthesis method that may be used to prepare a nucleotide for use in a peptide oligonucleotide complex of the present disclosure is phosphoramidite synthesis. Briefly, single nucleotide residues may be sequentially added from 3’ to 5’ to the growing nucleotide chain by repeating the steps of de-blocking (detrityl ati on), coupling, capping, and oxidation. Phosphoramidite synthesis may be performed on a solid support such as controlled pore glass (CPG) or macroporous polystyrene (MPPS). Similarly, thiophosphorodiamidate may be used.
[0262] A nucleotide of a peptide oligonucleotide complex may bind to a target molecule (e.g., a target DNA, a target RNA, or a target protein). In some embodiments, binding of the oligonucleotide to the target molecule may alter an activity of the target molecule. For example, binding of an oligonucleotide (e.g., an siRNA, an miRNA, a gapmer, or a U1 adaptor) to a target mRNA or pre-mRNA may increase or decrease translation of the target mRNA or pre-mRNA. In another example, binding of a nucleotide to a target DNA may increase or decrease expression of a gene encoded by the target DNA. In another example, binding of a nucleotide to an RNA (such as a transcript, pre-RNA, unspliced RNA, nuclear RNA, complimentary sequence to a NAT, or mRNA) expressed from a target DNA such as a gene or ORF may increase or decrease expression of a gene encoded by the target DNA. In another example, binding of an oligonucleotide (e.g., an aptamer) to a target protein may increase or decrease activity (e.g., an enzymatic activity or a binding activity) of the target protein. In some embodiments, the target molecule may be associated with a disease or condition and increasing or decreasing the activity of the target molecule may treat the disease or condition.
[0263] A sequence of the oligonucleotide of a peptide oligonucleotide complex may be selected for its ability to bind to or modulate the activity of a target molecule. In some embodiments, an oligonucleotide may be reverse complementary to a target DNA or RNA molecule. For example, an siRNA oligonucleotide may be reverse complementary to a target RNA molecule. In some embodiments, am oligonucleotide may be partially reverse complementary (e.g., comprising one or more mis-matched base pairs) to a target DNA or RNA molecule. For example, an siRNA oligonucleotide may comprise a base mismatch relative to a target RNA molecule. In some embodiments, a sequence of the oligonucleotide may be selected for its annealing temperature relative to a target DNA or RNA molecule. A preferred annealing temperature may be achieved by selecting the length of the nucleotide, the degree of complementarity of the nucleotide to the target molecule, the chemistry of the nucleotides, or any combination thereof. Nucleotide sequence parameters (e.g., complementarity, annealing temperature, melting temperature, base mismatches, and binding affinity) may be calculated using any available software, such as ITD OligoAnalyzer and the like. In some embodiments, an oligonucleotide may adopt a secondary structure that binds to a target DNA, RNA, or protein molecule. For example, an aptamer may adopt a secondary structure to bind to a target protein. The aptamer sequence may be selected to adopt a secondary structure that binds to a target protein. Nucleotide secondary structure may be predicted using any available software, such as RNAfold and the like. In some embodiments, a nucleotide sequence may be determined experimentally by selecting for the ability to bind to a target molecule. For example, a nucleotide library may be contacted to a target molecule, and sequences that bind to the target molecule may be identified.
[0264] In some embodiments, a nucleotide comprises a G/C content of not less than 20% and not more than 80%. In some embodiments, a nucleotide comprises a G/C content of not less than 30% and not more than 65%. In some embodiments, the nucleotide comprises a G/C content of not less than 20%, not less than 25%, not less than 30%, not less than 35%, not less than 40%, not less than 45%, or not less than 50%. In some embodiments, the nucleotide comprises a G/C content of not more than 80%, not more than 75%, not more than 70%, not more than 65%, or not more than 50%. In some embodiments, a nucleotide comprises an A/T content or A/U content of not less than 20% and not more than 80%. In some embodiments, a nucleotide comprises an A/T content or A/U content of not less than 30% and not more than 65%. In some embodiments, the nucleotide comprises a A/U (or A/T, or combination of A/U and A/T) content of not less than 20%, not less than 25%, not less than 30%, not less than 35%, not less than 40%, not less than 45%, or not less than 50%. In some embodiments, the nucleotide comprises a A/U content (or A/T, or combination of A/U and A/T) of not more than 80%, not more than 75%, not more than 70%, not more than 65%, or not more than 50%. In some embodiments, a nucleotide has a length of no more than 1000 nt, 600 nt, 200 nt, 100 nt, 60 nt, 56 nt, 52 nt, 50 nt, 48 nt, 46 nt, 44 nt, 22 nt, 40 nt, 38 nt, 36, nt, 34 nt, 32 nt, 30 nt, or 24 nt. In some embodiments, a nucleotide has a length of from 24 to 100 nt, from 24 to 60 nt, from 24 to 50 nt, or from 36 to 50 nt. In some embodiments, a nucleotide has a length of about 42 nt.
[0265] In some embodiments, a nucleotide has a length of no more than 500 nt, 300 nt, 100 nt, 50 nt, 30 nt, 28 nt, 26 nt, 25 nt, 24 nt, 23 nt, 22 nt, 21 nt, 20 nt, 19 nt, 18, nt, 17 nt, 16 nt, 15 nt, or 12 nt. In some embodiments, a nucleotide has a length of from 12 to 50 nt, from 12 to 30 nt, from 12 to 25 nt, from 18 to 25 nt, from 18 to 25 nt, from 19 to 23 nt, or from 20 to 22 nt. In some embodiments, a nucleotide has a length of about 21 nt.
[0266] A peptide oligonucleotide complex of the present disclosure (e.g., a peptide oligonucleotide complex comprising a cell-penetrating peptide and a nucleotide) may be further conjugated, linked, or fused to an active agent in addition to the nucleotide active agent (e.g., a target-binding agent capable of binding a target molecule). Such additional active agent may be complexed, fused, linked or conjugated to one or more of the peptide, nucleotide, or linker within the peptide oligonucleotide complex. In some embodiments, the active agent may be directly or indirectly linked to the peptide of the peptide oligonucleotide complex or the nucleotide of the peptide oligonucleotide complex. A peptide nucleic acid complex further comprising an additional active agent may be referred to as a peptide-active agent conjugate or a peptide construct.
[0267] The peptide oligonucleotide complexes of the present disclosure can also be used to deliver another active agent. Peptides according to the present disclosure can be conjugated to, linked to, or fused to an agent for use in the treatment of tumors and cancers or other diseases. For example, in certain embodiments, the peptides described herein are fused or conjugated to another molecule, such as an active agent that provides an additional functional capability. A peptide or nucleotide can be fused with an active agent through expression of a vector containing the sequence of the peptide with the sequence of the active agent. In various embodiments, the sequence of the peptide and the sequence of the active agent can be expressed from the same Open Reading Frame (ORF). In various embodiments, the sequence of the peptide and the sequence of the active agent can comprise a contiguous sequence. The peptide and the active agent can each retain similar functional capabilities in the peptide construct compared with their functional capabilities when expressed separately. In certain embodiments, examples of active agents can include other peptides.
[0268] As another example, in certain embodiments, the peptides or nucleotides described herein are attached to another molecule, such as an active agent that provides a functional capability. The active agent may be any active agent (e.g., therapeutic agent, detectable agent, or binding moiety) described herein. In some embodiments, the peptide or nucleotide is covalently or non-covalently linked to an active agent, e.g., directly or via a linker. Exemplary linkers suitable for use with the embodiments herein are discussed in further detail below.
Detectable Agent Peptide Conjugates
[0269] A peptide (e.g., a cell-penetrating peptide of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) or peptide complex (e.g., comprising a cell-penetrating peptide and a cargo molecule) can be conjugated to, linked to, or fused to an agent used in imaging, research, therapeutics, theranostics, pharmaceuticals, chemotherapy, chelation therapy, targeted drug delivery, and radiotherapy. The cell-penetrating peptide may deliver the agent into a cellular space or compartment or across a cellular layer (e g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen).
[0270] The cell-penetrating peptides or peptide complexes of the present disclosure may be fused to a detectable agent that may otherwise be excluded from a cell or a cellular compartment to facilitate absorption or permeation of the detectable agent. For example, a cell -penetrating peptide of the present disclosure may facilitate absorption or permeation of a detectable agent with multiple hydrogen bond donors or hydrogen bond acceptors, a large molecular weight, or a high partition coefficient, which may lead to poor absorption or permeation of the detectable agent in the absence of the cell -penetrating peptide. In some embodiments, a cell -penetrating peptide may be conjugated to a detectable agent comprising at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors. In some embodiments, the cell-penetrating peptide may be conjugated to a detectable agent comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors. In some embodiments, the cell-penetrating peptide may be conjugated to a detectable agent comprising a molecular weight of at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, or at least 1000 Da. In some embodiments, the cell-penetrating peptide may be conjugated to a detectable agent comprising a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0. The partition coefficient may be a measure of a ratio of the solubility of the agent in a hydrophobic solvent (e.g., octanol) relative to water.
[0271] In some embodiments, a peptide is conjugated to, linked to, or fused with detectable agents, such as a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metalcontaining nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a metal, a radioisotope, a dye, radionuclide chelator, or another suitable material that can be used in imaging. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 detectable agents can be linked to a peptide. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212. In some embodiments, the near-infrared dyes are not easily quenched by biological tissues and fluids. In some embodiments, the fluorophore is a fluorescent agent emitting electromagnetic radiation at a wavelength between 650 nm and 4000 nm, such emissions being used to detect such agent. Non-limiting examples of fluorescent dyes that could be used as a conjugating molecule in the present disclosure include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, or indocyanine green (ICG). In some embodiments, near infrared dyes often include cyanine dyes (e.g., Cy7, Cy5.5, and Cy5). Additional non-limiting examples of fluorescent dyes for use as a conjugating molecule in the present disclosure include acradine orange or yellow, Alexa Fluors (e.g., Alexa Fluor 790, 750, 700, 680, 660, and 647) and any derivative thereof, 7-actinomycin D, 8- anilinonaphthalene-1 -sulfonic acid, ATTO dye and any derivative thereof, auramine-rhodamine stain and any derivative thereof, bensantrhone, bimane, 9-10-bis(phenylethynyl)anthracene, 5,12 - bis(phenylethynyl)naththacene, bisbenzimide, brainbow, calcein, carbodyfluorescein and any derivative thereof, l-chloro-9,10-bis(phenylethynyl)anthracene and any derivative thereof, DAPI, DiOC6, DyLight Fluors and any derivative thereof, epicocconone, ethidium bromide, FlAsH-EDT2, Fluo dye and any derivative thereof, FluoProbe and any derivative thereof, Fluorescein and any derivative thereof, Fura and any derivative thereof, GelGreen and any derivative thereof, GelRed and any derivative thereof, fluorescent proteins and any derivative thereof, m isoform proteins and any derivative thereof such as for example mCherry, hetamethine dye and any derivative thereof, hoeschst stain, iminocoumarin, indian yellow, indo- 1 and any derivative thereof, laurdan, lucifer yellow and any derivative thereof, luciferin and any derivative thereof, luciferase and any derivative thereof, mercocyanine and any derivative thereof, nile dyes and any derivative thereof, perylene, phloxine, phyco dye and any derivative thereof, propium iodide, pyranine, rhodamine and any derivative thereof, ribogreen, RoGFP, rubrene, stilbene and any derivative thereof, sulforhodamine and any derivative thereof, SYBR and any derivative thereof, synapto-pHluorin, tetraphenyl butadiene, tetrasodium tris, Texas Red, Titan Yellow, TSQ, umbelliferone, violanthrone, yellow fluroescent protein and YOYO-1. Other Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4', 5'-dichloro-2',7' - dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethyl- rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxy coumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.), Texas Red, Texas Red-X, SPECTRUM RED, SPECTRUM GREEN, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), ALEXA FLUOR dyes (e g., ALEXA FLUOR 350, ALEXA FLUOR 488, ALEXA FLUOR 532, ALEXA FLUOR 546, ALEXA FLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 633, ALEXA FLUOR 660, ALEXA FLUOR 680, etc ), BODIPY dyes (e.g, BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, etc ), IRDyes (e g., IRD40, IRD 700, IRD 800, etc.), and the like. Additional suitable detectable agents are described in PCT/US14/56177. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212.
[0272] In some embodiments, fluorescent biotin conjugates that can act both as a detectable label and an affinity handle can be used. Non limiting examples of commercially available fluorescent biotin conjugates include Atto 425-Biotin, Atto 488-Biotin, Atto 520-Biotin, Atto- 550 Biotin, Atto 565-Biotin, Atto 590-Biotin, Atto 610-Biotin, Atto 620-Biotin, Atto 655-Biotin, Atto 680-Biotin, Atto 700-Biotin, Atto 725-Biotin, Atto 740-Biotin, fluorescein biotin, biotin-4- fluorescein, biotin-(5-fluorescein) conjugate, and biotin-B-phycoerythrin, ALEXA FLUOR 488 biocytin, ALEXA FLUOR 546, ALEXA FLUOR 549, lucifer yellow cadaverine biotin-X, Lucifer yellow biocytin, Oregon green 488 biocytin, biotin-rhodamine and tetramethylrhodamine biocytin. In some other examples, the conjugates could include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels. In some embodiments, the peptide-active agent fusions described herein can be attached to another molecule. For example, the peptide sequence also can be attached to another active agent (e.g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar, etc.). In some embodiments, the peptide can be fused with, or covalently or non-covalently linked to an active agent. In some other examples, the conjugates can include chemiluminescent compounds, colloidal metals, luminescent compounds, enzymes, radioisotopes, and paramagnetic labels. In some embodiments, the peptide described herein can also be attached to another molecule. For example, the peptide sequence also can be attached to another active agent (e g., small molecule, peptide, polypeptide, polynucleotide, antibody, aptamer, cytokine, growth factor, neurotransmitter, an active fragment or modification of any of the preceding agents, fluorophore, radioisotope, radionuclide chelator, acyl adduct, chemical linker, or sugar). In some embodiments, the peptide can be conjugated to, linked to, or fused with, or covalently or non-covalently linked to an active agent.
[0273] Peptides can be conjugated to, linked to, or fused to a radiosensitizer or photosensitizer. Examples of radiosensitizers include but are not limited to: ABT-263, ABT-199, WEHL539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic acid base derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers can include but are not limited to: fluorescent molecules or beads that generate heat when illuminated, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metalloporphyrins, metallophthalocyanines, angelicins, chalcogenapyrrillium dyes, chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives, naphthalimides, nile blue derivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins, and cercosporins), psoralens, quinones, retinoids, rhodamines, thiophenes, verdins, xanthene dyes (e.g., eosins, erythrosins, rose bengals), dimeric and oligomeric forms of porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, this approach can allow for highly specific targeting of diseased cells (e.g., cancer cells) using both a therapeutic agent (e.g., drug) and electromagnetic energy (e.g., radiation or light) concurrently. In some embodiments, the peptide is conjugated to, linked to, fused with, or covalently or non-covalently linked to the agent, e g., directly or via a linker. Exemplary linkers suitable for use with the embodiments herein are discussed in further detail below.
[0274] In some embodiments, a cell-penetrating peptide conjugated to, linked to, or fused with a detectable agent may be used in a method of imaging a cell or tissue. A cell-penetrating peptide complex comprising a cell-penetrating peptide and a detectable agent may be contacted to a cell or tissue, and the cell or tissue may be imaged. In some embodiments, the cell or tissue may be imaged to detect the presence, absence, location, intensity, distribution of the detectable agent, or combinations thereof. In some embodiments, imaging may comprise diagnostic imaging, for example to detect the presence or absence of a disease state.
Chemical Modifications
[0275] A peptide (e.g., a cell-penetrating peptide, a cargo peptide, or a cell-penetrating peptide complex) can be chemically modified one or more of a variety of ways. In some embodiments, the peptide can be mutated to add function, delete function, or modify the in vivo behavior. For example, in some embodiments, cell -penetrating peptides of the presenting disclosure (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) may be chemically modified with a molecule that would increase the serum half-life of the peptide when administered to a subject. In some embodiments, one or more loops between the disulfide linkages of a knotted cellpenetrating peptide (e.g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) can be modified or replaced to include active elements from other peptides (such as described in Moore and Cochran, Methods in Enzymology, 503, p. 223-251, 2012). In some embodiments, loops from cell-penetrating peptides may be grafted into peptide active agents. Amino acids can also be mutated, such as to increase half-life, decrease immunogenicity, modify, add or delete binding behavior in vivo, add new targeting function, modify surface charge and hydrophobicity, or allow conjugation sites. N-methylation is one example of methylation that can occur in a peptide of the disclosure. In some embodiments, the peptide is modified by methylation on free amines. For example, full methylation may be accomplished through the use of reductive methylation with formaldehyde and sodium cyanoborohydride.
[0276] A chemical modification can, for instance, extend the half-life of a peptide or change the biodistribution or pharmacokinetic profile. A chemical modification can comprise a polymer, a poly ether, polyethylene glycol, a biopolymer, a polyamino acid, a fatty acid, a dendrimer, an Fc region, a simple saturated carbon chain such as palmitate or myristolate, an albumin binder, or albumin. A polyamino acid can include, for example, a poly amino acid sequence with repeated single amino acids (e.g., poly glycine), and a poly amino acid sequence with mixed poly amino acid sequences (e.g, gly-ala-gly-ala; SEQ ID NO: 614) that may or may not follow a pattern, or any combination of the foregoing.
[0277] The peptides of the present disclosure can be modified such that the modification increases the stability and/or the half-life of the peptides. The attachment of a hydrophobic moiety, such as to the N-terminus, the C-terminus, or an internal amino acid, can be used to extend half-life of a peptide of the present disclosure. The peptides can also be modified to increase or decrease the gut permeability or cellular permeability of the peptide. The peptide of the present disclosure can include post-translational modifications (e g., methylation and/or amidation and/or glycosylation), which can affect, e.g., serum half-life. In some embodiments, simple carbon chains (e g., by myristoylation and/or palmitylation) can be conjugated to, linked to, the fusion proteins or peptides. The simple carbon chains can render the fusion proteins or peptides easily separable from the unconjugated material. For example, methods that can be used to separate the fusion proteins or peptides from the unconjugated material include, but are not limited to, solvent extraction and reverse phase chromatography. Lipophilic moieties can extend half-life through reversible binding to serum albumin. Conjugated moieties can, e.g., be lipophilic moieties that extend half-life of the peptides through reversible binding to serum albumin. In some embodiments, the lipophilic moiety can be cholesterol or a cholesterol derivative including cholestenes, cholestanes, cholestadienes and oxysterols. In some embodiments, the peptides can be conjugated to, linked to, myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the peptides of the present disclosure can be coupled (e g., conjugated, linked, or fused) to a half-life modifying agent. Examples of half-life modifying agents can include, but is not limited to: a polymer, a polyethylene glycol (PEG), a hydroxyethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin. Additionally, conjugation of the peptide to a near infrared dye, such as Cy5.5, or to an albumin binder such as Albu-tag can extend serum half-life of any peptide as described herein. In some embodiments, immunogenicity is reduced by using minimal non-human protein sequences to extend serum half-life of the peptide.
[0278] The peptides of the present disclosure can be modified to reduce their immunogenicity. Immunogenicity can limit the utility of a therapeutic peptidic molecule, as described in FDA Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products (2014), which is incorporated by reference in its entirety. A molecule that is immunogenic and cause the formation of, or increased levels of anti-drug antibodies (ADA) can lead to complications such as toxicity, immune incompatibility, or hypersensitivity particularly in the context of human therapeutics. Consequently, it is desirable to limit immunogenicity and ADA in therapeutic applications of peptides. ADA can be neutralizing or binding but not neutralizing. The formation of ADA can reduce the efficacy of a therapeutic biologic molecule, including by causing earlier clearance of or reduced exposure to the therapeutic. The formation of ADA can reduce the safety of a therapeutic biologic molecule, such as by causing cytokine release syndrome, infusion reactions, or clearance of endogenous proteins. There are approaches to predicting and reducing the immunogenicity of therapeutics, as described in Preclinical models immunogenicity prediction of therapeutic proteins (Brinks, 2013), which is incorporated by reference in its entirety. Some peptide sequences are more likely to cause immunogenicity. Sequences may be designed for reduced immunogenicity by screening sequences and variants using experimental and in silico methods and selecting sequences that are less immunogenic. When two or more peptides or proteins are fused together, the point of linkage may create new sequences that could cause immunogenicity. The same methods can be used to facilitate design of linkages, peptides and fusions with lower immunogenicity. Peptides that are resistant to enzymatic degradation can also have reduced immunogenicity, because in some cases peptides must undergo proteolysis into shorter peptide fragments, such as 9-11 amino acids long, in order to be presented on the MHCII complex to immune cells resulting in immunogenic effects.
[0279] In some embodiments, the peptides of the present disclosure include variants with deduced immunogenicity. For example, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 42, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 195, SEQ ID NO: 198, SEQ ID NO: 311, or SEQ ID NO: 321, as described in EXAMPLE 23 and TABLE 16. In some embodiments, the first two N-terminal amino acids (GS) of SEQ ID NO: 59 - SEQ ID NO: 84, SEQ ID NO: 93 - SEQ ID NO: 101, or SEQ ID NO: 210 - SEQ ID NO: 224 serve as a spacer or linker in order to facilitate conjugation or fusion to another molecule, as well as to facilitate cleavage of the peptide from such conjugated to, linked to, or fused molecules. In some embodiments, the fusion proteins or peptides of the present disclosure can be conjugated to, linked to, or fused to other moieties that, e.g., can modify or effect changes to the properties of the peptides.
[0280] In some embodiments, a cell -penetrating peptide of the present disclosure may be modified with a tag that further promotes endosomal escape (e.g., an endosomal escape motif). For example, S19 (PFVIGAGVLGALGTGIGGI; SEQ ID NO: 359), CM18 (KWKLFKKIGAVLKVLTTG; SEQ ID NO: 360), PAS (FFLIPKG; SEQ ID NO: 361), Aureinl .2 (GLFDIIKKIAESF; SEQ ID NO: 362), or B18 (LGLLLRHLRHHSNLLANI; SEQ ID NO: 363) may be conjugated to a cell -penetrating peptide of the present disclosure.
[0281] In some embodiments, the cell-penetrating peptides of the present disclosure (e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) and complexes thereof penetrate cells with and intracellular concentration to exert a prophylactic or therapeutic effect. In some embodiments, 0.01% or more, 0.05% or more, 0.1% or more, 0.2% or more, 0.5% or more, 1% or more, 3% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, up to 100% of a molecule or cellpenetrating peptide can enter the cytosol from the extracellular space or circulation. In some embodiments, a cell-penetrating peptide may penetrate a cellular layer such that the cytosolic, nuclear, intracellular, or paracellular concentration of the peptide is at least about 0.1%, at least about 0.2% at least about 0.5%, at least about 1%, at least about 3%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about at least about 100% of the extracellular peptide concentration. Nucleotide Modifications
[0282] In some embodiments, the nucleic acid portion of a peptide oligonucleotide complex (e g., an oligonucleotide of a cell-penetrating peptide oligonucleotide complex) contains one or more bases within the nucleic acid molecule that are modified. Such modifications can occur whether the nucleic acid portion a single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter. One or more bases in a given nucleotide sequence may be modified to increase in vivo stability, to increase resistance to enzymes such as nucleases, increase protein binding including to serum proteins, increase in vivo half-life, to modify the tissue biodistribution, or to modify how the immune system responds. The phosphonate, the ribose, or the base may be modified. In some aspects, the modification comprises a phosphorothioate modification, a phosphodiester modification, a thio- phosphoramidate modification, a methyl phosphonate modification, a phosphorodi thioate modification, a methoxypropylphosphonate modification, a 5’-(E)-vinylphosphonate modification, a 5 ’methyl phosphonate modification, an (S)-5’-C-methyl with phosphate modification, a 5 ’-phosphorothioate modification, a peptide nucleic acid (PNA), a 2’-0 methyl modification, a 2’-O-methoxyethyl (2’-O-Me) modification, a 2’-fluoro (2’-F) modification, a 2’-deoxy-2’-fluoro modification, a 2’ arabino-fluor modification, a 2’-O-benyzl modification, a 2’-O-methyl-4-pyridine modification, a locked nucleic acid (LNA), an amino-LNA, a thio-LNA, an ENA, an amino ENA, a carbo-ENA, a (S)-cEt-bridged nucleic acid, an (S)-MOE, a bridged nucleic acid, a tricyclo-DNA, a morpholino nucleic acid (PMO), an unlocked nucleic acid (UNA), a glycol nucleic acid (GNA), a bridged nucleic acid (BNA), an ethyl (S)-cEt nucleic acid, a pseudouridine, a 2 ’-thiouridine, an N6’methyadenosine, a 5 ’-methylcytidine, a 5’-fluoro- 2 ’-deoxyuridine, a N’ ethylpiperidine 7 ’-EAA triazole modified adenine, an N-ethylpiperidine 6’-triazole modified adenine, a 6’-phenylpyrrolocytosine, a 2 ’,4 ’-difluorotoluyl ribonucleoside, a 5 ’nitro indole, a 5’ methyl, a 5’ phosphonate, an inverted A base, a 2’-H (deoxyribose), a 2’- OH (ribose), or any combination thereof. The oligonucleotide may be comprised entirely of a combination of 2’-O-Me and 2’-F modifications. Diastereomers or one or both stereoisomers may be used. Any of the stabilization chemistries or patterns, including STC, ESC, advanced, ESC, ADI-3, AD5, disclosed in Hu Signal Transduction and Targeted Therapy 2020,5: 101 can be used. Pyrimidines can be 2’-fluoro-modified, which can increase stability to nucleases but can also increase immune system activation. The RNA backbone can be phosphorothioate- substituted (where the non-bridging oxygen is replaced with sulfur), which can increase resistance to nuclease digestion as well as altering the biodistribution and tissue retention and increasing the pharmacokinetics such as by increasing protein binding, but can also induce more immune stimulation. Methyl phosphonate modification of an RNA can also be used. 2’-Omethyl and 2’-F RNA bases can be used, which can protect against base hydrolysis and nucleases and increase the melting temperature of duplexes. Bridged, Locked, and other similar forms of Bridged Nucleic Acids (BNA, LNA, cEt) where any chemical bridge such as an N-0 linkage between the 2’ oxygen and 4’ carbons in ribose can be incorporated to increase resistance to exo- and endonucleases and enhance biostability. These include BNA where an N-0 linkage between the 2’ and 4’ carbons occur and where any chemical modification of the nitrogen (including but not limited to N-H, N-CH3, N-benzene) in the bridge can be added to increase stability RNA backbone or base modifications can be placed anywhere in the RNA sequence, at one, multiple, or all base locations. Optionally, phosophorothioate nucleic acid linkages may be used between the 2-4 terminal nucleic acids of one or both sequences. Optionally 2’F modified nucleic acids may be used at least at 2-4 positions, at least 5%, at least 10% at least 25% of internal positions, at least 50%, at least 75%, or up to 100% of internal positions, all internal positions or all positions. Optionally, one or more of 2’F base, an LNA base, a BNA base, an ENA base, a 2’O-MOE base, a morpholino base, a 2’0Me base, a 5 ’-Me base, a (S)-cEt base or combinations thereof may be used at least at 2-4 positions, at least 5%, at least 10% at least 25% of internal positions, at least 50%, at least 75%, or up to 100% of internal positions, all internal positions or all positions.
[0283] Modified bases can be used to increase in the in vivo half-life of the oligonucleotide. They can allow the oligonucleotide to remaining intact in the serum, endosome, cytosol, or nucleus, including for days, weeks, or months. This can allow ongoing activity, including if the oligonucleotide is slowly released from the endosome over days, weeks, or months within a given cell (such as described in Brown et al., Nucleic Acids Research, 2020, pl 1827-11844). [0284] In some embodiments, a nucleotide comprises at least one phosphorothioate linkage. In some embodiments, a peptide oligonucleotide complex comprises from 1 to 12 phosphorothioate linkages. In some embodiments, a nucleotide comprises at least one thiophosphoroamidate linkage. In some embodiments, a nucleotide comprises from 1 to 12 thiophosphoroamidate linkages. In some embodiments, a nucleotide comprises at least one modified base. In some embodiments, at least modified base comprises a 2’F base, an LNA base, a BNA base, an ENA base, a 2’O-MOE base, a 5’-Me base, a (S)-cEt base, a 2’OMe base, a morpholino base, or combinations thereof.
Peptide Linkers
[0285] The cell-penetrating peptides of the presented disclosure (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) can be linked to a cargo peptide (e.g., an active agent peptide, a target-binding peptide, a therapeutic peptide, a detectable peptide, or a cystine-dense peptide) in numerous ways. For example, a cell -penetrating peptide can be conjugated to a cargo peptide via a peptide linker to form a cell-penetrating peptide fusion. In some embodiments, a peptide linker does not disturb the independent folding of peptide domains (e.g., a cystine-dense peptide). In some embodiments, a peptide linker does not negatively impact manufacturability (synthetic or recombinant) of the peptide complex (e g., the cell -penetrating peptide complex). In some embodiments, a peptide linker does not impair post-synthesis chemical alteration (e.g. conjugation of a fluorophore or albumin-binding chemical group) of the peptide fusion.
[0286] In some embodiments, a peptide linker can connect the C-terminus of a first peptide (e.g., a cell-penetrating peptide or a cargo peptide) to the N-terminus of a second peptide (e.g., a cell-penetrating peptide or a cargo peptide). In some embodiments, a peptide linker can connect the C-terminus of the second peptide (e.g., a cell-penetrating peptide or a cargo peptide) to the N-terminus of a third peptide (e g., a cell -penetrating peptide or a cargo peptide). For example, a linker (e.g., any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485) can connect the C-terminus of a cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) to the N-terminus of a cargo peptide to form a cellpenetrating peptide fusion (e g., SEQ ID NO: 299 - SEQ ID NO: 324, SEQ ID NO: 486, or SEQ ID NO: 487). In another example, a linker (e g., any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485) can connect the C-terminus of a cargo peptide to the N- terminus of a cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) to form a cell-penetrating peptide fusion.
[0287] In some embodiments, a peptide linker can comprise a Tau-theraphotoxin-Hsla, also known as DkTx (double-knot toxin), extracted from a native knottin-knottin dimer from Haplopelma schmidti (e.g., SEQ ID NO: 266). The linker can lack structural features that would interfere with dimerizing independently functional CDPs (e.g., a cell-penetrating CDP and a target-binding CDP). In some embodiments, a linker can comprise a glycine-serine (Gly-Ser or GS) linker (e.g., SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485). Gly-Ser linkers can have minimal chemical reactivity and can impart flexibility to the linker. Serines can increase the solubility of the linker or the peptide complex, as the hydroxyl on the side chain is hydrophilic. In some embodiments, a linker can be derived from a peptide that separates the Fc from the Fv domains in a heavy chain of human immunoglobulin G (e.g., SEQ ID NO: 267). In some embodiments, a linker derived from a peptide from the heavy chain of human IgG can comprise a cysteine to serine mutation relative to the native IgG peptide. [0288] In some embodiments, peptides of the present disclosure can be dimerized using an immunoglobulin heavy chain Fc domain. These Fc domains can be used to dimerize functional domains (e.g., a cell-penetrating peptide and a cargo peptide), either based on antibodies or other otherwise soluble functional domains. In some embodiments, dimerization can be homodimeric via Fc sequences. In some embodiments, dimerization can be heterodimeric by mutating the Fc domain to generate a “knob-in-hole” format where one Fc CH3 domain contains novel residues (knob) designed to fit into a cavity (hole) on the other Fc CH3 domain. A first peptide domain (e.g., a cell-penetrating peptide) can be coupled to the knob, and a second peptide domain (e.g., a TfR-binding peptide or target-binding peptide) can be coupled to the hole. Knob+knob dimers can be highly energetically unfavorable. A purification tag can be added to the “knob” side to remove hole+hole dimers and select for knob+hole dimers.
[0289] The peptides of the present disclosure (e.g., the cell-penetrating peptides) can be linked to another peptide (e.g., a cargo peptide) at the N-terminus or C-terminus. In some embodiments, one or more peptides can be linked or fused via a peptide linker (e.g., a peptide linker comprising a sequence of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485). For example, a cell -penetrating peptide can be fused to a cargo peptide via a peptide linker of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485. A peptide linker can have a length of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, or about 50 amino acid residues. A peptide linker can have a length of from about 2 to about 5, from about 2 to about 10, from about 2 to about 20, from about 3 to about 5, from about 3 to about 10, from about 3 to about 15, from about 3 to about 20, from about 3 to about 25, from about 5 to about 10, from about 5 to about 15, from about 5 to about 20, from about 5 to about 25, from about 10 to about 15, from about 10 to about 20, from about 10 to about 25, from about 15 to about 20, from about 15 to about 25, from about 20 to about 25, from about 20 to about 30, from about 20 to about 35, from about 20 to about 40, from about 20 to about 45, from about 20 to about 50, from about 3 to about 50, from about 3 to about 40, from about 3 to about 30, from about 10 to about 40, from about 10 to about 30, from about 50 to about 100, from about 100 to about 200, from about 200 to about 300, from about 300 to about 400, from about 400 to about 500, or from about 500 to about 600 amino acid residues.
[0290] In some embodiments, a first peptide (e.g., a cell -penetrating peptide) and a second peptide (e.g., a cargo peptide) can be connected by a flexible peptide linker. A flexible linker can provide rotational freedom between the first peptide and the second peptide and can allow the first peptide and the second peptide to perform their respective functions with minimal strain. In some embodiments, a peptide linker can have a persistence length of no more than 6 A, no more than 7 A, no more than 8 A, no more than 9 A, no more than 10 A, no more than 12 A, no more than 15 A, no more than 20 A, no more than 25 A, no more than 30 A, no more than 40 A, or no more than 50 A. In some embodiments, a peptide linker can have a persistence length of from about 4 A to about 100 A, from about 4 A to about 50 A, from about 4 A to about 20 A, from about 4 A to about 10 A, from about 10 A to about 20 A, from about 20 A to about 30 A, from about 30 A to about 50 A, or from about 50 A to about 100 A. The persistence length of the linker can be a measure of the flexibility of the peptide linker and can be quantified as the peptide length over which correlations in the direction of the tangent are lost.
[0291] In some embodiments, a peptide linker can be selected based on a desired linker length, hydrodynamic radius, chromatographic mobility, posttranslational modification propensity, or combinations thereof. In some embodiments, a linker separating two or more functional domains of a peptide complex (e.g., separating a cell -penetrating peptide and a cargo peptide) can comprise a large, stable, globular domain, for example to reduce a propensity for glomerular filtration. In some embodiments, a linker separating two or more functional domains of a peptide complex (e.g., separating a cell-penetrating peptide and a cargo peptide) can comprise a small, flexible linker, for example to reduce the hydrodynamic radius of the complex for use in tight spaces like dense-core tumor stroma. In some embodiments, a peptide linker can support independent folding of the two or more functional domains and may not inhibit functions of the respective functional domains.
[0292] In some embodiments, a peptide can be appended to the N-terminus of any peptide of the present disclosure following an N-terminal GS dipeptide and preceding, for example, a GGGS (SEQ ID NO: 264) spacer. In some embodiments, a peptide (e.g., a target-binding peptide) can be appended to either the N-terminus or C-terminus of any peptide disclosed herein (e.g., a cell- penetrating peptide) using a peptide linker such as GxSy (SEQ ID NO: 256) peptide linker, wherein x and y can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises (GS)x (SEQ ID NO: 258), wherein x can be any whole number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the peptide linker comprises GGSSG (SEQ ID NO: 259), GGGGG (SEQ ID NO: 260), GSGSGSGS (SEQ ID NO: 261), GSGG (SEQ ID NO: 262), GGGGS (SEQ ID NO: 263), GGGS (SEQ ID NO: 264), GGS (SEQ ID NO: 265), GGGSGGGSGGGS (SEQ ID NO: 255), or a variant or fragment thereof. Additionally, KKYKPYVPVTTN (SEQ ID NO: 266) from DkTx, and EPKSSDKTHT (SEQ ID NO: 267) from human IgG3 can be used as a peptide linker. In some embodiments, the peptide linker comprises GGGSGGSGGGS (SEQ ID NO: 292). In some embodiments, the peptide linker comprises a linker of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485. Examples of peptide linkers compatible with the cell-penetrating peptides and cell-penetrating peptide fusions of the present disclosure are provided in TABLE 10. It is understood that any of the foregoing linkers or a variant or fragment thereof can be used with any number of repeats or any combinations thereof. It is also understood that other peptide linkers in the art or a variant or fragment thereof can be used with any number of repeats or any combinations thereof.
TABLE 10 - Exemplary Peptide Linkers
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Linkers
[0293] Peptides according to the present disclosure (e.g., cell -penetrating peptides) can be attached to another moiety (e.g., an active agent or an detectable agent), such as a small molecule, a second peptide, a protein, an antibody, an antibody fragment, an aptamer, polypeptide, polynucleotide, a fluorophore, a radioisotope, a radionuclide chelator, a polymer, a biopolymer, a fatty acid, an acyl adduct, a chemical linker, or sugar or other active agent or detectable agent described herein through a linker, or directly in the absence of a linker. In the absence of a linker, for example, an active agent or a detectable agent can be conjugated to, linked to, or fused to the N-terminus or the C-terminus of a peptide to create an active agent or detectable agent fusion peptide. In other embodiments, the link can be made by a peptide fusion via reductive alkylation. In some embodiments, a cleavable linker is used for in vivo delivery of the peptide, such as a linker that can be cleaved or degraded upon entry in a cell, endosome, or a nucleus. In some embodiments, in vivo delivery of a peptide requires a small linker that does not interfere with penetration of a cell or localization to a nucleus of a cell. A linker can also be used to covalently attach a peptide as described herein to another moiety or molecule having a separate function, such a targeting, cytotoxic, therapeutic, homing, imaging, or diagnostic functions.
[0294] A peptide can be directly attached to another molecule by a covalent attachment. For example, the peptide is attached to a terminus of the amino acid sequence of a larger polypeptide or peptide molecule, or is attached to a side chain, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, or glutamic acid residue. The attachment can be via an amide bond, an ester bond, an ether bond, a carbamate bond, a carbon-nitrogen bond, a triazole, a macrocycle, an oxime bond, a hydrazone bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond. In some embodiments, similar regions of the disclosed peptide(s) itself (such as a terminus of the amino acid sequence, an amino acid side chain, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, or glutamic acid residue, via an amide bond, an ester bond, an ether bond, a carbamate bond, a carbon-nitrogen bond, a triazole, a macrocycle, an oxime bond, a hydrazone bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond, or linker as described herein) can be used to link other molecules.
[0295] Attachment via a linker can involve incorporation of a linker moiety between the other molecule and the peptide. The peptide and the other molecule can both be covalently attached to the linker. The linker can be cleavable, labile, non-cleavable, stable, stable self-immolating, hydrophilic, or hydrophobic. As used herein, the term “non-cleavable” or “stable” (such as used in association with an amide, cyclic, or carbamate linker or as otherwise as described herein) is often used by a skilled artisan to distinguish a relatively stable structure from one that is more labile or “cleavable” (e.g., as used in association with cleavable linkers that may be dissociated or cleaved structurally by enzymes, proteases, self-immolation, pH, reduction, hydrolysis, certain physiologic conditions, or as otherwise described herein). It is understood that “non- cleavable” or “stable” linkers offer stability against cleavage or other dissociation as compared to “cleavable” linkers, and the term is not intended to be considered an absolute non-cleavable or non-dissociative structure under any conditions. Consequently, as used herein, a “non- cleavable” linker is also referred to as a “stable” linker. The linker can have at least two functional groups with one bonded to the peptide, the other bonded to the other molecule, and a linking portion between the two functional groups. Some example linkers are described in Jain, N., Pharm Res. 32(11): 3526-40 (2015), Doronina, S O , Bioconj Chem. 19(10): 1960-3 (2008), Pillow, TH., J Med Chem. 57(19): 7890-9 (2014), Dorywalksa, M., Bioconj Chem. 26(4): 650- 9 (2015), Kellogg, B.A., Bioconj Chem. 22(4): 717-27 (2011), and Zhao, R.Y., J Med Chem. 54(10): 3606-23 (2011).
[0296] Non-limiting examples of the functional groups for attachment can include functional groups capable of forming an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond. Non-limiting examples of functional groups capable of forming such bonds can include amino groups; carboxyl groups; hydroxyl groups; aldehyde groups; azide groups; alkyne and alkene groups; ketones; hydrazides; acid halides such as acid fluorides, chlorides, bromides, and iodides; acid anhydrides, including symmetrical, mixed, and cyclic anhydrides; carbonates; carbonyl functionalities bonded to leaving groups such as cyano, succinimidyl, and JV-hydroxysuccinimidyl; hydroxyl groups; sulfhydryl groups; and molecules possessing, for example, alkyl, alkenyl, alkynyl, allylic, or benzylic leaving groups, such as halides, mesylates, tosylates, triflates, epoxides, phosphate esters, sulfate esters, and besylates. [0297] Non-limiting examples of the linking portion can include alkylene, alkenylene, alkynylene, polyether, such as polyethylene glycol (PEG), hydroxy carboxylic acids, polyester, polyamide, polyamino acids, polypeptides, cleavable peptides, valine-citrulline, aminobenzylcarbamates, D-amino acids, and polyamine, any of which being unsubstituted or substituted with any number of substituents, such as halogens, hydroxyl groups, sulfhydryl groups, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, urethane groups, epoxides, and ester groups. [0298] A peptide and drug complexed, conjugated, or fused via a linker is described with the formula Peptide-A-B-C-Drug, wherein the linker is A-B-C. A can be a stable amide link, is an amine on the peptide and the linker and can be achieved via a tetrafluorophenyl (TFP) ester or an NHS ester. B can be (-CH2-)X- or a short PEG (-CEECFEO-jx (x is 1-10), and C can be the ester bond to the hydroxyl or carboxylic acid on the drug. In some embodiments, C can refer to the “cleavable” or “stable” part of the linker. In other embodiments, A can also be the “cleavable” part. In some embodiments, A can be amide, carbamate, thioether via maleimide or bromoacetamide, triazole, oxime, or oxacarboline. The cleaved active agent or drug can retain the chemical structure of the active agent before cleavage or can be modified as a result of cleavage. Moreover, depending on the desired therapeutic properties of the peptide-drug conjugate, such active agent can be active while linked to the peptide, remain active after cleavage or become inactivated, be inactive while linked to the peptide, or it can be activated upon cleavage.
[0299] In some embodiments, peptide complexes have stable linkers. A peptide of the disclosure can be expressed recombinantly or chemically synthesized. The peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide linkage or a carbamate linkage. The peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide bond using standard 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or dicylcohexylcarbodiimide (DCC) based chemistry or thionyl chloride or phosphorous chloride-based bioconjugation chemistries. A stable linker may or may not be cleaved in buffer over extended periods of time (e.g., hours, days, or weeks). A stable linker may or may not be cleaved in body fluids such as plasma or synovial fluid over extended periods of time (e.g., hours, days, or weeks). A stable linker, may or may not be cleaved after exposure to enzymes, reactive oxygen species, other chemicals or enzymes that can be present in cells (e.g., macrophages), cellular compartments (e.g., endosomes and lysosomes), inflamed areas of the body (e g., inflamed joints), tissues or body compartments. A stable linker may be cleaved by unknown mechanisms. A stable linker may or may not be cleaved in vivo but remains an active agent after peptide conjugation.
[0300] A peptide and drug complexed, conjugated, or fused via a linker can be described with the formula Peptide-A-B-C-Drug, wherein the linker is A-B-C. A can be a stable amide link such as that formed by reacting an amine on the peptide with a linker containing a tetrafluorophenyl (TFP) ester or an NHS ester. A can also be a stable carbamate linker such as that formed by reacting an amine on the peptide with an imidazole carbamate active intermediate formed by reaction of CDI with a hydroxyl on the linker. A can also be a stable secondary amine linkage such as that formed by reductive alkylation of the amine on the peptide with an aldehyde or ketone group on the linker. A can also be a stable thioether linker formed using a maleimide or bromoacetamide in the linker with a thiol in the peptide, a triazole linker, a stable oxime linker, or a oxacarboline linker. B can be (-CH2-)X- or a short PEG (-CH2CH2O-)X (x is 0-20) or other spacers or no spacer. C can be an amide bond formed with an amine or a carboxylic acid on the drug, a thioether formed between a maleimide on the linker and a sulfhydroyl on the drug, a secondary or tertiary amine, a carbamate, or other stable bonds. Any linker chemistry described in “Current ADC Linker Chemistry,” Jain et al., Pharm Res, 2015 DOI 10. 1007/sl 1095-015-1657-7 can be used.
[0301] The resulting peptide complexes can be administered to a human or animal subcutaneously, intravenously, orally, or injected directly into a joint to treat disease. The peptide is not specifically cleaved from the detectable agent or active agent via a targeted mechanism. The peptide can be degraded by mechanisms such as catabolism, releasing a drug that is modified or not modified form its native form (Antibody-Drug Conjugates: Design, Formulation, and Physicochemical Stability, Singh, Luisi, and Pak. Pharm Res (2015) 32:3541- 3571). The peptide drug conjugate exerts its pharmacological activity while still intact, or while partially or fully degraded, metabolized, or catabolized.
[0302] In some embodiments, peptide complexes can have cleavable linkers. In some embodiments, a peptide and drug can be complexed, conjugated, or fused via a linker and can be described with the formula Peptide-A-B-C-Drug, wherein the linker is A-B-C. In some embodiments, A can be a stable amide link such as that formed by reacting an amine on the peptide with a linker containing a tetrafluorophenyl (TFP) ester or an NHS ester. In certain embodiments, A can also be a stable carbamate linker that is formed by an amine reaction on the peptide with an imidazole carbamate active intermediate formed by reaction of CDI with a hydroxyl on the linker. In other embodiments, A can also be a stable secondary amine linkage such as that formed by reductive alkylation of the amine on the peptide with an aldehyde or ketone group on the linker. In some embodiments, A can also be a stable thioether linker formed using a maleimide or bromoacetamide in the linker with a thiol in the peptide, a triazole linker, a stable oxime linker, or an oxacarboline linker. B can be (-CH2-)X- or a short PEG (-CEECFLO-jx (x is 0-20) or other spacers or no spacer. C can be an ester bond to the hydroxyl or carboxylic acid on the drug, or a carbonate, hydrazone, or acylhydrazone, designed for hydrolytic cleavage. The hydrolytic rate of cleavage can be varied by varying the local environment around the bond, including carbon length (-CH2-)x, steric hindrance (including adjacent side groups such as methyl, ethyl, cyclic), hydrophilicity or hydrophobicity. In some embodiments, peptide complexes can have a linear or cyclic ester linkage, which can include or do not include side chains such as methyl or ethyl groups. A linear ester linkage can be more susceptible to cleavage (such as by hydrolysis, an enzyme such as esterase, or other chemical reaction) than a cyclic ester due to steric hindrance or hydrophobicity /hydrophilicity effects. Likewise, side chains such as methyl or ethyl groups on the linear ester linkage can optionally make the linkage less susceptible to cleavage than without the side chains. In some embodiments, hydrolysis rate can be affected by local pH, such as lower pH in certain compartments of the body or of the cell such as endosomes and lysosomes or diseased tissues. In some embodiments, C can also be a pH sensitive group such as a hydrazone or oxime linkage. In other embodiments, C can be a disulfide bond designed to be released by reduction, such as by glutathione. In other embodiments, (or A-B-C) can be a peptidic linkage design for cleavabe by enzymes. Optionally, a self-immolating group such as pABC can be included to cause release of a free unmodified drug upon cleavage (Antibody-Drug Conjugates: Design, Formulation, and Physicochemical Stability, Singh, Luisi, and Pak. Pharm Res (2015) 32:3541-3571). The linker can be cleaved by enzymes such as esterases, matrix metalloproteinases, cathepsins such as cathepsin B, glucuronidases, a protease, or thrombin. Alternatively, the bond designed for cleavage can be at A, rather than C, and C can be a stable bond or a cleavable bond. An alternative design can be to have stable linkers (such as amide or carbamate) at A and C and have a cleavable linker in B, such as a disulfide bond. The rate of reduction can be modulated by local effects such as steric hindrance from methyl or ethyl groups or modulating hydrophobicity /hydrophilicity. In some embodiments, peptide complexes can have an ester carbonyl linkage, a long hydrocarbon linker, or carbamate linker, each of which can include hydrophilic groups, such as alcohols, acids, or ethers, or include a hydrocarbon side chain or other moiety that tunes the rate of cleavage. For example, the rate of hydrolysis can be faster with hydrophilic groups, such as alcohols, acids, or ethers, near an ester carbonyl. In another example, hydrophobic groups present as side chains or as a longer hydrocarbon linker can slow the cleavage rate of the ester. Likewise, cleavage of a carbamate group can also be tuned by hindrance, hydrophobicity, and the like. In another example, using a less labile linking group, such as a carbamate rather than an ester, can slow the cleavage rate of the linker.
[0303] In some cases, a linker can comprise a triazole group, such as any one of the heterocyclic compounds with molecular formula C2H3N3, having a five-membered ring of two carbon atoms and three nitrogen atoms, optionally with a hydrogen atom bonded to N at any position in the ring, such as:
Figure imgf000140_0001
r example, a 1, 2, 3-Triazole
(such as \H\, 2, 3-Triazole, 27/1 ,2, 3-Triazole, or l-methyl-4,5,6,7,8,9-hexahydro-lH- cycloocta[d][l,2,3]triazole) or a 1,2,4-Triazole (such as 1771,2,4-Triazole or 4/71 ,2,4-Triazolc), or any other triazole including those with multiple additional appended rings.
[0304] Additional non- limiting examples of linkers include linear or non-cyclic linkers such as:
Figure imgf000140_0002
ach n is independently 0 to about 1,000; 1 to about 1,000; 0 to about 500; 1 to about 500; 0 to about 250; 1 to about 250; 0 to about 200; 1 to about 200; 0 to about 150; 1 to about 150; 0 to about 100; 1 to about 100; 0 to about 50; 1 to about 50; 0 to about 40; 1 to about 40; 0 to about 30; 1 to about 30; 0 to about 25; 1 to about 25; 0 to about 20; 1 to about 20; 0 to about 15; 1 to about 15; 0 to about 10; 1 to about 10; 0 to about 5; or 1 to about 5. In some embodiments, each n is independently 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50. In some embodiments, m is 1 to about 1,000; 1 to about 500; 1 to about 250; 1 to about 200; 1 to about 150; 1 to about 100; 1 to about 50; 1 to about 40; 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; or 1 to about 5. In some embodiments, m is 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50, or any linker as disclosed in Jain, N., Pharm Res. 32(11): 3526-40 (2015) or Ducry, L., Antibody Drug Conjugates (2013).
[0305] In some cases, a linker can comprise a cyclic group, such as an organic nonaromatic or aromatic ring, optionally with 3-10 carbons in the ring, optionally built from a carboxylic acid,
Figure imgf000141_0001
optionally be used to form a carbamate linkage. In some cases, a carbamate linkage can be more resistant to cleavage, such as by hydrolysis, enzymes such as esterases, or other chemical reactions, than an ester linkage.
[0306] In some cases, a linker can comprise a cyclic carboxylic acid, for example a cyclic di carboxylic acid, for example one of the following groups: 1,4-cyclohexane dicarboxylic acid, 1,2-cyclohexane dicarboxylic acid, or 1,3 -cyclohexane dicarboxylic acid, 1,1- cyclopentanediacetic acid,
Figure imgf000141_0002
stereoisomer thereof. For example, the linker can comprise one of the following groups.
Figure imgf000141_0003
used to form an ester linkage. In some cases, a cyclic ester linkage can be more sterically resistant to cleavage, such as by hydrolysis by water, enzymes such as esterases, or other chemical reactions, than a noncyclic or linear ester linkage. [0307] In some cases, a linker can comprise an aromatic dicarboxylic acid, for example terephthalic acid, isophthalic acid, phthalic acid
Figure imgf000142_0001
substituted analog thereof.
[0308] In some cases, a linker can comprise a natural or non-natural amino acid, for example
O
Figure imgf000142_0002
N I I cysteine, 2 or a substituted analog or a stereoisomer thereof. In some instances, a linker can comprise alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Vai); or any plurality or combination thereof. In some embodiments, the non-natural amino acid can comprise one or more functional groups, e.g., alkene or alkyne, that can be used as functional handles.
[0309] In some cases, a linker can comprise one of the following groups:
Figure imgf000142_0003
nl = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 n2 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or a substituted analog or a stereoisomer thereof. In some instances, the linker is selected from one of the following groups:
Figure imgf000143_0001
substituted analog or a stereoisomer thereof.
[0310] In some cases, a linker can comprise one of the following groups:
Figure imgf000143_0002
nl = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 n2 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or a substituted analog or a stereoisomer thereof. In some instances, the linker is selected from one of the following groups:
Figure imgf000143_0003
or a substituted analog or a stereoisomer thereof.
[0311] In some cases, a substituted analog or a stereoisomer is a structural analog of a compound disclosed herein, for which one or more hydrogen atoms of the compound can be substituted by one or more groups of halo (e.g., Cl, F, Br), alkyl (e.g., methyl, ethyl, propyl), alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocycloalkyl, or any combination thereof. In some cases, a stereoisomer can be an enantiomer, a diastereomer, a cis or trans stereoisomer, a E or Z stereoisomer, or a R or S stereoisomer.
[0312] Non-limiting examples of linear linkers include;
Figure imgf000143_0004
Figure imgf000144_0001
H H H
Figure imgf000144_0002
; ; wherein each nl, or n2 or m is independently 0 to about 1,000; 1 to about 1,000; 0 to about 500; 1 to about 500; 0 to about 250; 1 to about 250; 0 to about 200; 1 to about 200; 0 to about 150; 1 to about 150; 0 to about 100; 1 to about 100; 0 to about 50; 1 to about 50; 0 to about 40; 1 to about 40; 0 to about 30; 1 to about 30; 0 to about 25; 1 to about 25; 0 to about 20; 1 to about 20; 0 to about 15; 1 to about 15; 0 to about 10; 1 to about 10; 0 to about 5; or 1 to about 5. In some embodiments, each n is independently 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50. In some embodiments, m is 1 to about 1,000; 1 to about 500; 1 to about 250; 1 to about 200; 1 to about 150; 1 to about 100; 1 to about 50; 1 to about 40; 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; or 1 to about 5. In some embodiments, m is 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50. In some instances, the linker can comprise a linear dicarboxylic acid, e.g., one of the following groups: succinic acid, 2,3 -dimethylsuccinic acid, glutaric acid, adipic acid, 2,5 -dimethyladipic acid,
Figure imgf000144_0003
or a substituted analog or a stereoisomer thereof. In some cases, the linker can be used to form a carbamate linkage. In some embodiments, the carbamate linkage can be more resistant to cleavage, such as by hydrolysis, enzymes such as esterases, or other chemical reactions, than an ester linkage In some cases, the linker can be used to form a linear ester linkage. In some embodiments, the linear ester linkage can be more susceptible to cleavage, such as by hydrolysis, enzymes such as esterases, or other chemical reactions, than a cyclic ester or carbamate linkage. Side chains such as methyl groups on the linear ester linkage can optionally make the linkage less susceptible to cleavage than without the side chains.
[0313] In some cases a linker can be a succinic linker, and a targeting agent (e.g., a single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or U1 Adapter) or other active agent or detectable agent can be attached to a peptide via an ester bond or an amide bond with two methylene carbons in between. In other cases, a linker can be any linker with both a hydroxyl group and a carboxylic acid, such as hydroxy hexanoic acid or lactic acid.
[0314] In some cases, a nucleotide (e.g., a single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or Ul Adapter), an active agent, or a detectable agent can be attached to a peptide using any one or more of the linkers shown below in TABLE 11. In some embodiments, a peptide, an additional active agent, or a detectable agent can be attached to a nucleotide any one or more of the linkers shown below in TABLE 11
TABLE 11 - Exemplary Linkers for Use in Peptide Conjugates or Complexes
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
[0315] In some cases, an active agent is attached to a linker wherein a nucleophilic functional group (e.g., a hydroxyl group) of the active agent molecule acts as the nucleophile and replaces a leaving group on the linker moiety, thereby attaching it to the linker.
[0316] In other cases, an active agent is attached to a linker wherein a nucleophilic functional group (e.g., thiol group, amine group, etc.) of the linker replaces a leaving group on the active agent, thereby attaching it to the linker. Such leaving group (or functional group that may be converted into a leaving group) may be a primary alcohol to form a thioether bond, thereby attaching it to the linker. A primary alcohol can be converted into a leaving group such as a mesylate, a tosylate, or a nosylate in order to accelerate the nucleophilic substitution reaction. [0317] The peptide complexes of the present disclosure (e.g., cell-penetrating peptide complexes) can comprise a cargo molecule (e g., a target-binding agent, an active agent, a therapeutic agent, or a detectable agent), a linker, and/or a peptide of the present disclosure. A general connectivity between these three components can be active agent-linker-peptide, such that the linker is attached to both the active agent and the peptide. In many cases, the peptide is attached to a linker via an amide bond. Amide bonds can be relatively stable (e.g., in vivo) compared to other bonds described herein, such as esters, carbonates, etc. The amide bond between the peptide and the linker may thus provide advantageous properties due to its in vivo stability of the active agent is sought to be cleaved from a peptide-active agent-conjugate without the linker being attached to the active agent after such in vivo cleavage. Thus, in various cases, an active agent is attached to the linker-peptide moiety via linkages such as ester, carbonate, carbamate, etc., wherein the peptide or active agent is attached to the linker via an amide bond. This can allow for selective cleavage of the active agent-linker bond (as opposed to the linker-peptide bond) allowing the active agent to be released without a linker moiety attached to it after cleavage. The use of such different active agent-linker bonds or linkages can allow the modulation of active agent release in vivo, e.g., in order to achieve a therapeutic function while minimizing off-target effects (e.g., reduction in drug release during circulation). [0318] The linker can be a cleavable or a stable linker. The use of a cleavable linker permits release of the conjugated moiety (e.g., a nucleotide targeting agent, a therapeutic agent, a detectable agent, or a combination thereof) from the peptide, e.g., after targeting to the target tissue or cell or subcellular compartment or after endocytosis. In some cases, the linker is enzyme cleavable, e.g., a valine-citrulline linker that can be cleavable by cathepsin, or an ester linker that can be cleavable by esterase. In some embodiments, the linker contains a selfimmolating portion. In other embodiments, the linker includes one or more cleavage sites for a specific protease, such as a cleavage site for matrix metalloproteases (MMPs), thrombin, urokinase-type plasminogen activator, or cathepsin (e.g., cathepsin K).
[0319] Thus, in some cases, a peptide-active agent complexes of the present disclosure can comprise one or more, about two or more, about three or more, about five or more, about ten or more, or about 15 or more amino acids that can form an amino acid sequence cleavable by an enzyme. Such enzymes can include proteinases. A peptide-active agent complex can comprise an amino acid sequence that can be cleaved by a Cathepsin, a Chymotrypsin, an Elastase, a Subtilisin, a Thrombin I, or a Urokinas, or any combination thereof.
[0320] Alternatively or in combination, the cleavable linker can be cleaved, dissociated, or broken by other mechanisms, such as via pH, reduction, or hydrolysis. Hydrolysis can occur directly due to water reaction, or be facilitated by an enzyme, or be facilitated by presence of other chemical species. A hydrolytically labile linker, (amongst other cleavable linkers described herein) can be advantageous in terms of releasing active agents from the peptide. For example, an active agent in a conjugate form with the peptide may not be active, but upon release from the conjugate after targeting to the target tissue or cell or subcellular compartment, the active agent is active. The cleaved active agent may retain the chemical structure of the active agent before cleavage or may be modified. In some embodiments, a stable linker may optionally not cleave in buffer over extended periods of time (e.g., hours, days, or weeks). In some embodiments, a stable linker may optionally not cleave in body fluids such as plasma or synovial fluid over extended periods of time (e.g., hours, days, or weeks). In some embodiments, a stable linker optionally may cleave, such as after exposure to enzymes, reactive oxygen species, other chemicals or enzymes that may be present in cells (such as macrophages), cellular compartments (such as endosomes and lysosomes), inflamed areas of the body (such as inflamed joints), or tissues or body compartments. In some embodiments, a stable linker may optionally not cleave in vivo but present an active agent that is still active when conjugated to, linked to, or fused to the peptide.
The rate of hydrolysis of the linker (e.g., a linker of a peptide conjugate) can be tuned. For example, the rate of hydrolysis of linkers with unhindered esters may be faster compared to the hydrolysis of linkers with bulky groups next an ester carbonyl. A bulky group can be a methyl group, an ethyl group, a phenyl group, a ring, or an isopropyl group, or any group that provides steric bulk. In another example, the rate of hydrolysis can be faster with hydrophilic groups, such as alcohols, acids, or ethers, or near an ester carbonyl. In another example, hydrophobic groups present as side chains or by having a longer hydrocarbon linker can slow cleavage of the ester. In some embodiments, cleavage of a carbamate group can also be tuned by hindrance, hydrophobicity, and the like. In another example, using a less labile linker, such as a carbamate rather than an ester, can slow the cleavage rate of the linker. In some cases, the steric bulk can be provided by the drug itself, such as by ketorolac when conjugated via its carboxylic acid. The rate of hydrolysis of the linker can be tuned according to the residency time of the conjugate in the target tissue or cell or subcellular compartment, according to how quickly the peptide accumulates in the target tissue or cell or subcellular compartment, or according to the desired time frame for exposure to the active agent in the target tissue or cell or subcellular compartment. For example, when a peptide is cleared from the target tissue or cell or subcellular compartment relatively quickly, the linker can be tuned to rapidly hydrolyze. In contrast, for example, when a peptide has a longer residence time in the target tissue or cell or subcellular compartment, a slower hydrolysis rate can allow for extended delivery of an active agent. This can be important when the peptide is used to deliver a drug to the target tissue or cell or subcellular compartment (e.g., a tumor cell or a tumor tissue). “Programmed hydrolysis in designing paclitaxel prodrug for nanocarrier assembly” Sci Rep 2015, 5, 12023 Fu et al., provides an example of modified hydrolysis rates. In some embodiments, rates of cleavage can vary by species, body compartment, and disease state. For instance, cleavage by esterases may be more rapid in rat or mouse plasma than in human plasma, such as due to different levels of carboxyesterases. In some embodiments, a linker may be tuned for different cleavage rates for similar cleavage rates in different species.
[0321] In some cases, a linker can be a succinic linker, and a drug can be attached to a peptide via an ester bond or an amide bond with two methylene carbons in between. In other cases, a linker can be any linker with both a hydroxyl group and a carboxylic acid, such as hydroxy hexanoic acid or lactic acid.
[0322] In some embodiments, the linker can release the active agent in an unmodified form. In other embodiments, the active agent can be released with chemical modification. In still other embodiments, catabolism can release the active agent still linked to parts of the linker and/or peptide. In some embodiments, peptide complexes have stable linkers. A peptide of the disclosure can be expressed recombinantly or chemically synthesized. The peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide linkage or a carbamate linkage. The peptide can be complexed, conjugated, or fused to a detectable agent or an active agent via a stable linker, such as an amide bond using standard l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or dicylcohexylcarbodiimide (DCC) based chemistry or thionyl chloride or phosphorous chloride-based bioconjugation chemistries. A stable linker may or may not be cleaved in buffer over extended periods of time (e.g., hours, days, or weeks). A stable linker may or may not be cleaved in a cellular, intracellular, or paracellular space over extended periods of time (e.g., hours, days, or weeks). A stable linker, may or may not be cleaved after exposure to enzymes, reactive oxygen species, other chemicals or enzymes that can be present in cells (e.g., cancer cells, pancreatic cells, liver cells, colon cells, smooth muscle cells, ovarian cells, breast cells, lung cells, brain cells, skin cells, ocular cells, blood cells, lymph cells, immune system cells, reproductive cells, reproductive organ cells, prostate cells, fibroblasts, kidney cells, adenocarcinoma cells, glioma stem cells, or tumor cells,), cellular, paracellular, or intracellular compartments (e.g., cytosols, nuclei, or nanolumen), cells, tissues or body compartments. A stable linker may be cleaved by unknown mechanisms. A stable linker may or may not be cleaved in vivo but remains an active agent after peptide conjugation.
[0323] The linker can be a stable linker or a cleavable linker. In some embodiments, the stable linker can slowly release the conjugated moiety by an exchange of the conjugated moiety onto the free thiols on serum albumin. In some embodiments, the use of a cleavable linker can permit release of the conjugated moiety (e.g., a therapeutic agent) from the peptide, e.g., after administration to a subject in need thereof. In other embodiments, the use of a cleavable linker can permit the release of the conjugated therapeutic from the peptide. In some cases, the linker is enzyme cleavable, e.g., a valine-citrulline linker. In some embodiments, the linker contains a self-immolating portion. In other embodiments, the linker includes one or more cleavage sites for a specific protease, such as a cleavage site for matrix metalloproteases (MMPs), thrombin, cathepsins, peptidases, or beta-glucuronidase. Alternatively or in combination, the linker is cleavable by other mechanisms, such as via pH, reduction, or hydrolysis.
[0324] The rate of hydrolysis or reduction of the linker can be fine-tuned or modified depending on an application. For example, the rate of hydrolysis of linkers with unhindered esters can be faster compared to the hydrolysis of linkers with bulky groups next to an ester carbonyl. A bulky group can be a methyl group, an ethyl group, a phenyl group, a ring, or an isopropyl group, or any group that provides steric bulk. In some cases, the steric bulk can be provided by the drug itself, such as by ketorolac when conjugated, linked, or fused via its carboxylic acid. The rate of hydrolysis of the linker can be tuned according to the residency time of the conjugate or fusion in the target location. For example, when a peptide is cleared from a tumor, or the brain, relatively quickly, the linker can be tuned to rapidly hydrolyze. When a peptide has a longer residence time in the target location, a slower hydrolysis rate would allow for extended delivery of an active agent. “Programmed hydrolysis in designing paclitaxel prodrug for nanocarrier assembly” Sci Rep 2015, 5, 12023 Fu et al., provides an example of modified hydrolysis rates. [0325] The rate of hydrolysis of the linker (e.g., a linker of a peptide conjugate) can be measured. Such measurements can include determining free active agent in plasma, serum, or synovial fluid, or other fluid or tissue of a subject in vivo and/or by incubating a linker or a peptide conjugate comprising a linker of the present disclosure with a buffer (e.g., PBS) or blood plasma from a subject (e.g., rat plasma, human plasma, etc.) or synovial fluid or other fluids or tissues ex vivo. The methods for measuring hydrolysis rates can include taking samples during incubation or after administration and determine free active agent, free peptide, or any other parameter indicate of hydrolysis, including also measuring total peptide, total active agent, or conjugated active agent-peptide. The results of such measurements can then be used to determine a hydrolysis half-life of a given linker or peptide conjugate comprising the linker. A hydrolysis half-life of a linker can differ depending on the plasma or fluid or species or other conditions used to determine such half-life. This can be due to certain enzymes or other compounds present in a certain plasma (e.g., rat plasma). For instance, different fluids (such as plasma or synovial fluid) can contain different amounts of enzymes such as esterases, and these levels of these compounds can also vary depending on species (such as rat versus human) as well as disease state (such as normal versus arthritic). [0326] The complexes of the present disclosure can be described as having a modular structure comprising various components, wherein each of the components (e.g., peptide, linker, active agent and/or detectable agent) can be selected dependently or independently of any other component. For example, a targeted degradation peptide complex for targeted degradation of a target peptide can comprise a cell-penetrating peptide of the present disclosure (e.g., those having the amino acid sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 254), a targetbinding peptide (e.g., any one of SEQ ID NO: 293 - SEQ ID NO: 298 or SEQ ID NO: 364 - SEQ ID NO: 407), a linker (e.g., any linker described in TABLE 10 or TABLE 11, SEQ ID NO: 255 - SEQ ID NO: 292, SEQ ID NO: 458 - SEQ ID NO: 485, or otherwise described), and a ubiquitin ligase-binding agent (e.g., thalidomide, pomalidomide, lenalidomide, methyl bestatin, bestatin, nutlin-3, or VHL ligand 1). The linker, for example, can be selected and/or modified to achieve a certain active agent release (e.g., a certain release rate) via a certain mechanism (e.g., via hydrolysis, such as enzyme and/or pH-dependent hydrolysis) at the target site (e.g., in the brain) and/or to minimize systemic exposure to the active agent. During the testing of a conjugate any one or more of the components of the conjugate can be modified and/or altered to achieve certain in vivo properties of the conjugate, e.g., pharmacokinetic (e.g., clearance time, bioavailability, uptake and retention in various organs) and/or pharmacodynamic (e.g., target engagement) properties. Thus, the conjugates of the present disclosure can be modulated to prevent, treat, and/or diagnose a variety of diseases and conditions, while reducing side effects (e.g., side effects that occur if such active agents are administered alone (i.e., not conjugated to a peptide)).
[0327] In some embodiments, the non-natural amino acid can comprise one or more functional groups, e.g., alkene or alkyne, that can be used as functional handles. For example, a multiple bond of such functional groups can be used to add one or more molecules to the conjugate. The one or more molecules can be added using various synthetic strategies, some of which may include addition and/or substitution chemistries. For example, an addition reaction using a multiple bond can comprise the use of hydrobromic acid, wherein the bromine can act as a leaving group and thus be substituted with various moieties, e.g., active agents, detectable agents, agents that can modify or alter the pharmacokinetic (e.g., plasma half-life, retention and/or uptake in central nervous system (CNS) or elsewhere) and/or pharmacodynamic (e.g., hydrolysis rate such as an enzymatic hydrolysis rate) properties of the conjugate.
[0328] In some embodiments, a conjugate as described herein comprises one or more nonnatural amino acid and/or one or more linkers. Such one or more non-natural amino acid and/or one or more linkers can comprise one or more functional groups, e g., alkene or alkyne (e.g., non-terminal alkenes and alkynes), which can be used as functional handles. For example, a multiple bond of such functional groups can be used to add one or more molecules to the conjugate. The one or more molecules can be added using various synthetic strategies, some of which may include addition and/or substitution chemistries, cycloadditions, etc. For example, an addition reaction using a multiple bond can comprise the use of hydrogen bromide (e g., via hydrohalogenation reactions), wherein the bromide substituent, once attached, can act as a leaving group and thus be substituted with various moieties comprising a nucleophilic functional groups, e.g., active agents, detectable agents, agents. As another example, a multiple bond can be used as a functional handle in a cycloaddition reaction. Cycloaddition reactions can comprise 1,3-dipolar cycloadditions, [2+2] -cycloadditions (e.g., photocatalyzed), Di els- Alder reactions, Huisgen cycloadditions, nitrone -olefin cycloadditions, etc. Such cycloaddition reactions can be used to attached various functional groups, functional moieties, active agents, detectable agents, and so forth to the conjugate. For example, a 1,3-dipolar cycloaddition reaction can be used to attach a molecule to a conjugate, wherein the molecule comprises a 1,3-dipole that can react with, e.g., an alkyne to form a 5 -membered ring, thereby attaching said molecule to the conjugate.
[0329] The addition of such agents or molecules (e.g., via nucleophilic or electrophilic addition followed by nucleophilic substitution) can have various application. For example, attaching such molecule or agent can modify or alter the pharmacokinetic (e g., plasma halflife, retention and/or uptake in CNS or biodistribution) and/or pharmacodynamic (e.g., hydrolysis rate such as an enzymatic hydrolysis rate) properties of the conjugate. Attaching such molecule or agent can also alter (e.g., increase) the depot effect of a conjugate, or provide functionality for in vivo tracking, e.g., using fluorescence or other types of detectable agents. [0330] In some embodiments, a conjugate of the present disclosure can comprise a linker comprising one or more of the following groups:
Figure imgf000154_0001
substituted analog or a stereoisomer thereof, wherein each nl and n2 is independently a value from 1 to 10. Such a group can be used as a handle to attach one or more molecules to a conjugate, e.g., to alter the pharmacokinetic (e.g., plasma half-life, retention and/or uptake in central nervous system (CNS) or elsewhere) and/or pharmacodyna via nucleophilic or electrophilic addition followed by nucleophilic substitution mic properties of the conjugate. Functionalization of such a group can occur using one or more multiple bonds (e.g., double bonds, triple bonds, etc.) of the groups. Such functionalization can comprise addition and/or substitution chemistries. For example, a functional group of a linker, such as a double bond, can be converted into a single bond (e g., via an addition reaction such as a nucleophilic addition reaction), wherein one or both of the carbon atoms of the newly formed single bond can have a leaving group (e.g., a bromine) attached to them. Such a leaving group can then be used (e.g., via nucleophilic substitution reaction) to attach a specific molecule (e.g., an active agent, a detectable agent, etc.) to that carbon atom(s) of the linker.
[0331] As another example, a multiple bond can be used as a functional handle in a cycloaddition reaction. Cycloaddition reactions can comprise 1,3 -dipolar cycloadditions, [2+2]-cycloadditions (e.g., photocatalyzed), Diels-Alder reactions, Huisgen cycloadditions, nitrone-olefin cycloadditions, etc. Such cycloaddition reactions can be used to attached various functional groups, functional moieties, active agents, detectable agents, and so forth to the conjugate. For example, a 1,3 -dipolar cycloaddition reaction can be used to attach a molecule to a conjugate, wherein the molecule comprises a 1,3-dipole that can react with, e.g., an alkyne to form a 5-membered ring, thereby attaching said molecule (e.g., active agent, detectable agent, etc.) to the conjugate. In some cases, molecules may be attached to a conjugate to e.g., modulate the half-life, increase the depot effect, or provide new functionality of a conjugate, such as fluorescence for tracking.
Peptide Stability
[0332] A peptide of the present disclosure can be stable in various biological or physiological conditions, such as the pH or reducing environments inside a cell, in the cytosol, in a cell nucleus, lysosome, or endosome. For example, any cell-penetrating cystine-dense peptide of SEQ ID NO: 1 - SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 18 - SEQ ID NO: 20, 30 - SEQ ID NO: 40, SEQ ID NO: 59 - SEQ ID NO: 61, SEQ ID NO: 66 - SEQ ID NO: 69 - SEQ ID NO: 71 - SEQ ID NO: 81, SEQ ID NO: 85 - SEQ ID NO: 100, SEQ ID NO: 197, SEQ ID NO: 200, SEQ ID NO: 202 - SEQ ID NO: 209, SEQ ID NO: 212, SEQ ID NO: 215, SEQ ID NO: 217 - SEQ ID NO: 224, or SEQ ID NO: 408 - SEQ ID NO: 457 can exhibit resistance to reducing agents, heat, denaturation, proteases, oxidative conditions, or acidic conditions. In some embodiments, a cell-penetrating peptide of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 may be conjugated to a peptide (e.g., a cystine-dense peptide) that can exhibit resistance to reducing agents, heat, denaturation, proteases, oxidative conditions, or acidic conditions, for example a cell -penetrating peptide complex of SEQ ID NO: 299 - SEQ ID NO: 308 or SEQ ID NO: 312 - SEQ ID NO: 321.
[0333] In some cases, biologic molecules (such as peptides and proteins) can provide therapeutic functions, but such therapeutic functions are decreased or impeded by instability caused by the in vivo environment. (Moroz et al. Adv Drug Deliv Rev 101 : 108-21 (2016), Mitragotri et al. Nat Rev Drug Discov 13(9):655-72 (2014), Bruno et al. Ther Deliv (11): 1443- 67 (2013), Sinha et al. CritRev Ther Drug Carrier Syst. 24(l):63-92 (2007), Hamman et al. BioDrugs 19(3): 165-77 (2005)). For instance, the GI tract can contain a region of low pH (e.g. pH ~1), a reducing environment, or a protease-rich environment that can degrade peptides and proteins. Proteolytic activity in other areas of the body, such as the mouth, eye, lung, intranasal cavity, joint, skin, vaginal tract, mucous membranes, and serum, can also be an obstacle to the delivery of functionally active peptides and polypeptides. Additionally, the half-life of peptides in serum can be very short, in part due to proteases, such that the peptide can be degraded too quickly to have a lasting therapeutic effect when administering reasonable dosing regimens. Likewise, proteolytic activity in cellular compartments such as lysosomes and reduction activity in lysosomes and the cytosol can degrade peptides and proteins such that they may be unable to provide a therapeutic function on intracellular targets. Therefore, peptides that are resistant to reducing agents, proteases, and low pH may be able to provide enhanced therapeutic effects or enhance the therapeutic efficacy of co-formulated or conjugated, linked, or fused active agents in vivo.
[0334] Additionally, oral delivery of drugs can be desirable in order to target certain areas of the body (e.g., disease in the GI tract such as colon cancer, irritable bowel disorder, infections, metabolic disorders, and constipation) despite the obstacles to the delivery of functionally active peptides and polypeptides presented by this method of administration. For example, oral delivery of drugs can increase compliance by providing a dosage form that is more convenient for patients to take as compared to parenteral delivery. Oral delivery can be useful in treatment regimens that have a large therapeutic window. Therefore, peptides that are resistant to reducing agents, proteases, and low pH can allow for oral delivery of peptides without nullifying their therapeutic function.
[0335] Peptide Resistance to Reducing Agents. Cell-penetrating peptides or peptide complexes of this disclosure can contain one or more cysteines, which can participate in disulfide bridges that can be integral to preserving the folded state of the peptide. Exposure of peptides to biological environments with reducing agents can result in unfolding of the peptide and loss of functionality and bioactivity. For example, glutathione (GSH) is a reducing agent that can be present in many areas of the body and in cells and can reduce disulfide bonds. As another example, a peptide can become reduced during trafficking of a peptide across the gastrointestinal epithelium after oral administration. A peptide can become reduced upon exposure to various parts of the GI tract. The GI tract can be a reducing environment, which can inhibit the ability of therapeutic molecules with disulfide bonds to have optimal therapeutic efficacy, due to reduction of the disulfide bonds. A peptide can also be reduced upon entry into a cell, such as after internalization by endosomes or lysosomes or into the cytosol, or other cellular compartments. Reduction of the disulfide bonds and unfolding of the peptide can lead to loss of functionality or affect key pharmacokinetic parameters such as bioavailability, peak plasma concentration, bioactivity, and half-life. Reduction of the disulfide bonds can also lead to loss of functionality due to increased susceptibility of the peptide to subsequent degradation by proteases, resulting in rapid loss of intact peptide after administration. In some embodiments, a peptide that is resistant to reduction can remain intact and can impart a functional activity for a longer period of time in various compartments of the body and in cells, as compared to a peptide that is more readily reduced.
[0336] In certain embodiments, the peptides of this disclosure can be analyzed for the characteristic of resistance to reducing agents to identify stable peptides. In some embodiments, the peptides of this disclosure can remain intact after being exposed to different molarities of reducing agents such as 0.00001 M - 0.0001 M, 0.0001 M - 0.001 M, 0.001 M - 0.01 M, 0.01 M - 0.05 M, 0.05 M - 0.1 M, or 0.1 M to 0.2 M for 15 minutes or more. In some embodiments, the reducing agent used to determine peptide stability can be dithiothreitol (DTT), Tris(2- carboxyethyl)phosphine HC1 (TCEP), 2-Mercaptoethanol, (reduced) glutathione (GSH), or any combination thereof. In some embodiments, at least 5%- 10%, at least 10%-20%, at least 20%- 30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%- 80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to a reducing agent. In some embodiments, peptides are completely resistant to GSH reducing conditions and are partially resistant to degradation in DTT reducing conditions. In some embodiments, peptides described herein can withstand or are resistant to degradation in physiological reducing conditions.
[0337] Peptide Resistance to Proteases. The stability of peptides of this disclosure can be determined by resistance to degradation by proteases. Proteases, also referred to as peptidases or proteinases, are enzymes that can degrade peptides and proteins by breaking bonds between adjacent amino acids. Families of proteases with specificity for targeting specific amino acids can include serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, and asparagine proteases. Additionally, metalloproteases, matrix metalloproteases, elastase, carboxypeptidases, Cytochrome P450 enzymes, and cathepsins can also digest peptides and proteins. Proteases can be present at high concentration in blood, in mucous membranes, lungs, skin, the GI tract, the mouth, nose, eye, and in compartments of the cell. Misregulation of proteases can also be present in various diseases such as rheumatoid arthritis and other immune disorders. Degradation by proteases can reduce bioavailability, biodistribution, half-life, and bioactivity of therapeutic molecules such that they are unable to perform their therapeutic function. In some embodiments, peptides that are resistant to proteases can better provide therapeutic activity at reasonably tolerated concentrations in vivo.
[0338] In some embodiments, peptides of this disclosure can resist degradation by any class of protease. In certain embodiments, peptides of this disclosure resist degradation by pepsin (which can be found in the stomach), trypsin (which can be found in the duodenum), serum proteases, or any combination thereof. In some embodiments, the proteases used to determine peptide stability can be pepsin, trypsin, chymotrypsin, or any combination thereof. In certain embodiments, peptides of this disclosure can resist degradation by lung proteases (e.g., serine, cysteinyl, and aspartyl proteases, metalloproteases, neutrophil elastase, alpha-1 antitrypsin, secretory leucoprotease inhibitor, and elafin), or any combination thereof. In some embodiments, at least 5%- 10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to a protease.
[0339] Peptide Stability in Acidic Conditions. Peptides of this disclosure can be administered in biological environments that are acidic. For example, after oral administration, peptides can experience acidic environmental conditions in the gastric fluids of the stomach and gastrointestinal (GI) tract. The pH of the stomach can range from about 1-4 and the pH of the GI tract ranges from acidic to normal physiological pH descending from the upper GI tract to the colon. In addition, the vagina, late endosomes, and lysosomes can also have acidic pH values, such as less than pH 7. These acidic conditions can lead to denaturation of peptides and proteins into unfolded states. Unfolding of peptides and proteins can lead to increased susceptibility to subsequent digestion by other enzymes as well as loss of biological activity of the peptide. In certain embodiments, the peptides of this disclosure can resist denaturation and degradation in acidic conditions and in buffers, which simulate acidic conditions. In certain embodiments, peptides of this disclosure can resist denaturation or degradation in buffer with a pH less than 1, a pH less than 2, a pH less than 3, a pH less than 4, a pH less than 5, a pH less than 6, a pH less than 7, or a pH less than 8. In some embodiments, peptides of this disclosure remain intact at a pH of 1-3. In certain embodiments, at least 5%- 10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to a buffer with a pH less than 1, a pH less than 2, a pH less than 3, a pH less than 4, a pH less than 5, a pH less than 6, a pH less than 7, or a pH less than 8. In other embodiments, at least 5%-10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to a buffer with a pH of 1-3. In other embodiments, the peptides of this disclosure can be resistant to denaturation or degradation in simulated gastric fluid (pH 1-2). In some embodiments, at least 5%-l 0%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to simulated gastric fluid. In some embodiments, low pH solutions such as simulated gastric fluid can be used to determine peptide stability.
[0340] In some embodiments, the peptides described herein are resistant to degradation in vivo, in the serum of a subject, or inside a cell. In some embodiments, the peptides are stable at physiological pH ranges, such as about pH 7, about pH 7.5, between about pH 5 to 7.5, between about 6.5 to 7.5, between about pH 5 to 8, or between about pH 5 to 7. In some embodiments, the peptides described herein are stable in acidic conditions, such as less than or equal to about pH 5, less than or equal to about pH 3, or within a range from about 3 to about 5. In some embodiments, the peptides are stable in conditions of an endosome or lysosome, or inside a nucleus.
[0341] Peptide Stability at High Temperatures. Peptides of this disclosure can be administered in biological environments with high temperatures. For example, after oral administration, peptides can experience high temperatures in the body. Body temperature can range from 36°C to 40°C. High temperatures can lead to denaturation of peptides and proteins into unfolded states. Unfolding of peptides and proteins can lead to increased susceptibility to subsequent digestion by other enzymes as well as loss of biological activity of the peptide. In some embodiments, a peptide of this disclosure can remain intact at temperatures from 25°C to 100°C. High temperatures can lead to faster degradation of peptides. Stability at a higher temperature can allow for storage of the peptide in tropical environments or areas where access to refrigeration is limited. In certain embodiments, 5%-100% of the peptide can remain intact after exposure to 25°C for 6 months to 5 years. 5%-100% of a peptide can remain intact after exposure to 70°C for 15 minutes to 1 hour. 5%-100% of a peptide can remain intact after exposure to 100°C for 15 minutes to 1 hour. In other embodiments, at least 5%-10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to 25°C for at least 6 months to 5 years. In other embodiments, at least 5%- 10%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%- 60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to 70°C for 15 minutes to 1 hour. In other embodiments, at least 5%-l 0%, at least 10%-20%, at least 20%-30%, at least 30%-40%, at least 40%-50%, at least 50%-60%, at least 60%-70%, at least 70%-80%, at least 80%-90%, or at least 90%-100% of the peptide remains intact after exposure to 100°C for 15 minutes to 1 hour.
Methods of Manufacture
[0342] Various expression vector/host systems can be utilized for the recombinant expression of peptides described herein. Non-limiting examples of such systems include microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a nucleic acid sequence encoding peptides or peptide fusion proteins/ chimeric proteins described herein, yeast transformed with recombinant yeast expression vectors containing the aforementioned nucleic acid sequence, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the aforementioned nucleic acid sequence, plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)), or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the aforementioned nucleic acid sequence, or animal cell systems infected with recombinant virus expression vectors (e.g., adenovirus, vaccinia virus, lentivirus) including cell lines engineered to contain multiple copies of the aforementioned nucleic acid sequence, either stably amplified (e.g., CHO/dhfr, CHO/glutamine synthetase) or unstably amplified in double-minute chromosomes (e.g., murine cell lines) Disulfide bond formation and folding of the peptide could occur during expression or after expression or both.
[0343] A host cell can be adapted to express one or more peptides described herein. The host cells can be prokaryotic, eukaryotic, or insect cells. In some cases, host cells are capable of modulating the expression of the inserted sequences or modifying and processing the gene or protein product in the specific fashion desired. For example, expression from certain promoters can be elevated in the presence of certain inducers (e.g., zinc and cadmium ions for metallothionine promoters). In some cases, modifications (e.g., phosphorylation) and processing (e.g., cleavage) of peptide products can be important for the function of the peptide. Host cells can have characteristic and specific mechanisms for the post-translational processing and modification of a peptide. In some cases, the host cells used to express the peptides secrete minimal amounts of proteolytic enzymes.
[0344] In the case of cell- or viral -based samples, organisms can be treated prior to purification to preserve and/or release a target polypeptide. In some embodiments, the cells are fixed using a fixing agent. In some embodiments, the cells are lysed. The cellular material can be treated in a manner that does not disrupt a significant proportion of cells, but which removes proteins from the surface of the cellular material, and/or from the interstices between cells. For example, cellular material can be soaked in a liquid buffer, or, in the case of plant material, can be subjected to a vacuum, in order to remove proteins located in the intercellular spaces and/or in the plant cell wall. If the cellular material is a microorganism, proteins can be extracted from the microorganism culture medium. Alternatively, the peptides can be packed in inclusion bodies. The inclusion bodies can further be separated from the cellular components in the medium. In some embodiments, the cells are not disrupted. A cellular or viral peptide that is presented by a cell or virus can be used for the attachment and/or purification of intact cells or viral particles. In addition to recombinant systems, peptides can also be synthesized in a cell-free system prior to extraction using a variety of known techniques employed in protein and peptide synthesis.
[0345] In some cases, a host cell produces a peptide that has an attachment point for a drug. An attachment point could comprise a lysine residue, an N-terminus, a cysteine residue, a cysteine disulfide bond, a glutamic acid or aspartic acid residue, a C-terminus, or a non-natural amino acid. The peptide could also be produced synthetically, such as by solid-phase peptide synthesis, or solution-phase peptide synthesis. Peptide synthesis can be performed by fluorenylmethyloxycarbonyl (Fmoc) chemistry or by butyloxycarbonyl (Boc) chemistry. The peptide could be folded (formation of disulfide bonds) during synthesis or after synthesis or both. Peptide fragments could be produced synthetically or recombinantly. Peptide fragments can be then be joined together enzymatically or synthetically.
[0346] In other aspects, the peptides of the present disclosure can be prepared by conventional solid phase chemical synthesis techniques, for example according to the Fmoc solid phase peptide synthesis method (“Fmoc solid phase peptide synthesis, a practical approach,” edited by W. C. Chan and P. D. White, Oxford University Press, 2000). The peptides of the present disclosure can be prepared by solution phase peptide synthesis. The peptides can be folded by one cystine at a time, two at a time, three at a time, four at a time, or all at a time, either in solution or on the resin. The peptide made by manufactured as two or more fragments that are subsequently ligated or joined together.
[0347] In some embodiments, sequences from a peptide library generated from computational design can be synthesized using expression vectors or solid phase or solution phase peptide synthesis methods. For example, cell-penetrating peptides or cell-penetrating peptide fusions can be cloned into a secreted, soluble protein production/expression vector and purified, as per Bandaranayake et al., 2011. Purification methods include, but are not limited to, affinity purification columns, ion exchange (cation and/or anion columns), reversed-phase, hydrophobic interaction, and size exchange columns. SDS-PAGE followed by Coomassie staining and reverse phase HPLC can be used to analyze a sample of the purified protein. Protein concentrations were determined by UV spectral absorption and/or amino acid analysis.
[0348] In some embodiments, the peptides of this disclosure can be more stable during manufacturing. For example, peptides of this disclosure can be more stable during recombinant expression and purification, resulting in lower rates of degradation by proteases that are present in the manufacturing process, a higher purity of peptide, a higher yield of peptide, or any combination thereof. In some embodiments, the peptides can also be more stable to degradation at high temperatures and low temperatures during manufacturing, storage, and distribution. For example, in some embodiments peptides of this disclosure can be stable at 25°C. In other embodiments, peptides of this disclosure can be stable at 70°C or higher than 70°C. In some embodiments, peptides of this disclosure can be stable at 100°C or higher than 100°C.
Peptide Screening Methods
[0349] Improved cell -penetrating peptides may be identified using various screening methods to select for peptides with cell-penetrating properties. In some embodiments, a peptide library (e g., a library comprising amino acid substitution variants of a parent peptide) may be screened using cell-based assays to quantify cytosolic or nuclear access of individual members of the peptide library In some embodiments, a single peptide may be screened at a time using cell -based assays to quantify cytosolic or nuclear access. Such cell-based assays can utilize interactions between a tag protein (e.g., a SNAP -tag, a Halo-tag protein, a split GFP, or a biotin binding protein) and a small molecule substrate (e.g., a BG-GLA-NHS SNAP substrate, a chloroalkane substrate, or a biotin molecule) to identify peptides able to penetrate a cellular layer (e.g., a cell membrane or a nuclear membrane). Each peptide of a peptide library comprising cellpenetrating peptide candidates can be labeled with the small molecule substrate. The labeled peptide library can be incubated with cells expressing the tag protein in a cellular location of interest (e.g., in the cytosol or in the nucleus). Labeled peptides that are able to penetrate the cellular layer and reach the location of interest can bind to the expressed tag protein. Peptides that penetrated the cellular layer can be identified in a number of ways. For example, peptides may be identified by adding small molecule substrate labeled with a fluorescent molecule to the cells. Cells with high concentrations of available tag protein, indicative of low peptide pentration, will bind higher levels of fluorescently labeled substrate, and cells with lower concentrations of available tag protein, indicative of higher peptide penetration, will bind lower levels of fluorescently labeled substrate. Cells can be fluorescently imaged to quantify cellpenetration, where cells contacted with high cell -penetrating peptides have lower fluorescence and cells contacted with low cell-penetrating peptides have higher fluorescence. In another example, peptides can be identified by purifying the tag protein (e.g., by immunoprecipitation) along with bound peptides, and the peptides can be identified by mass spectrometry. Identified peptides can penetrate cell membranes.
[0350] The SNAP penetration assay (SNAPP A) uses many reagents that are commonly referred to herein in a shorthand manner to aid in understanding. As used herein “SNAP-tag protein” is also referred to as “SNAP-tag”. The SNAP-tag can be expressed in a variety of eukaryotic cells (e.g., NIH3T3, HEK293, HeLa, CHO, COS cells and the like). The SNAP-tag is introduced into cells via transfection of a plasmid vector such as for SNAP-tag protein (e.g., pSNAPf) or H2B- tagged SNAP-tag protein (e.g., pSNAPf-H2B) resulting in a cell line expressing the SNAP-tag (e.g., commonly referred to as “NI3T3 pSNAPf’, “HEK293 pSNAPf’, “HeLa pSNAPf’, generically as “pSNAPf cells” or “pSNAPf-H2B cells”, and the like). As used herein a “SNAP substrate”, for example a reactive benzylguanine reagent (e.g., BG-GLA-NHS) can be used as a substrate to “tag” test peptides, is also referred to as a “BG-substrate”. The BG-substrate (BG- GLA-NHS) as used herein is a reactive benzylguanine reagent chemical species used in a chemical reaction that reacts with a test reagent such as a peptide to generate a benzylguanine- containing product, for example a BG-peptide as described below. It is understood that a variety of BG-substrates may be used as a reactive moiety, and one or more reactive benzylguanine reagent chemical agents (e.g., such agents with various properties, fluorescence, radiolabel, for imaging, other measurable aspects) may be created and used to “tag” a test peptide, for example, for use in assessing cell penetration. As used herein a “SNAP substrate-tagged peptide”, or peptide that has been reacted with or “tagged” with a reactive benzylguanine reagent (e.g., BG- GLA-NHS), is also referred to as a “BG-peptide”. BG-peptide is typically a test peptide used with cells expressing the SNAP-tag to assess e.g., cytosolic and nuclear cell penetration of the BG-peptide in the SNAP assay. It is worth noting that the reactive BG-substrate (BG-GLA- NHS), when hydrolyzed, is modified to a non-reactive form, BG-GLA-OH, for use as a positive control and for normalization in the SNAP assay. As used herein a “SNAP substrate-tagged fluorophore”, or fluorophore that has been reacted with or “tagged” with a benzylguanine reagent (e.g., BG-GLA-NHS), are also referred to as a “BG-fluorophore”. An exemplary BG- fluorophore is the “SNAP-Cell TMR-Star” reagent from New England Biolabs. The BG- fluorophore can diffuse across the cell membrane to bind any unoccupied SNAP -tag in the cell, resulting in fluorescence, and the fluorescence of the cells can be visualized after washing away any free BG-fluorophore that has not bound to unoccupied SNAP -tag. When the cells expressing SNAP -tag are exposed to a BG-peptide and then exposed to the BG-fluorophore, a decrease in fluorescence relative to the PBS control indicates that the SNAP -tag is bound to the BG-peptide (rather than the BG-fluorophore), indicating that the BG-peptide has penetrated the cell. The level of fluorescence is in inverse relationship, wherein adding a cell penetrant BG-fluorophore and then washing away unbound BG- fluorophore, and then detecting fluorescence, the absence of which, relative to the negative control (PBS), is indicative of the SNAP -tag covalently bound to the BG-peptide. It is understood that a “SNAP substrate” is a general term that can describe a variety of substrates that are used in the SNAPPA as an enzymatic substrate for the SNAP -tag. For example, “SNAP substrate” can be used to describe whether a reactive benzylguanine- containing reagent (such as a BG-peptide or BG-fluorophore) will react to the SNAP -tag protein in the SNAP assay, serving as a substrate to the SNAP -tag itself. Consequently, it is understood that a “SNAP substrate” is non-limiting and can be an enzymatic substrate for the SNAP -tag in the SNAP assay. That is, each of the BG-GLA-NHS (BG- substrate), and its hydrolyzed form BG-GLA-OH, BG-peptide, and BG-fluorophore can serve as a substrate (SNAP substrate) for the SNAP-tag in the SNAP assay.
[0351] In some embodiments, a cell-based assay to identify cell-penetrating peptides may comprise contacting a library of BG-peptide variants to cells expressing a SNAP-tag in either the cytosol or the nucleus. BG-peptides capable of penetrating the cell membrane may covalently bind to SNAP-tag expressed in the cytosol, and BG-peptides capable of penetrating the cell membrane and the nuclear envelope may covalently bind to SNAP-tag expressed in the nucleus. In some embodiments, peptides that enter the cell may be subsequently identified by contacting the cells with a BG-fluorophore that can permeate the cell membrane and nuclear envelope and bind to any remaining SNAP-tag that is not bound to a BG-peptide. In some embodiments, peptides that enter the cell may be identified by identifying which cells have a lower level of fluorescence, indicating a higher level of cell penetration. In some embodiments, peptides that enter the cell may be identified by immunoprecipitating the SNAP-tag and any bound BG-peptides and performing mass spectrometry to identify the bound peptides. In some embodiments, these screening methods may be performed as high throughput assays, for example, in microarrays.
[0352] A valuable aspect of a SNAP penetration assay is that can measure delivery of a peptide to the cytosol or nucleus, rather than solely measuring its uptake into the cell. Other assays exist that merely measure general uptake into the cell, such as by labeling a peptide with a fluorophore and measuring total cellular fluorescence. However, such general uptake assays may measure peptide that is only present in the endosome or lysosome, rather than distinguish peptide that has entered the cytosol. Peptides may be taken up by endocytosis but remain trapped and never enter the cytosol which makes general uptake assays not very effective in identifying cytosolic or nuclear penetrating peptides, as described in Depray, et al (Bioconjug Chem, 2019 Apr 17; 30(4): 1006-1027). In contrast, the SNAP penetration assay can assess delivery to the cytosol or nucleus. Additionally, SNAP penetration assay may avoid possible false positives from surface-bound peptides, which may result from using fluorophore labels combined with alternative methods such as fluorescence activated cell sorting (FACS). The SNAP penetration assay uses SNAP -tag comprising a 20 kDa mutant of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (SNAP-tag) which reacts specifically and rapidly with benzylguanine (BG) derivatives (BG-substrate) (e.g., such BG-substrate conjugated to a peptide (BG-peptide) as a synthetic probe, test peptide, or substrate for the SNAP-tag in the SNAPPA assay, and similarly such BG-substrate can be attached to a small molecule that is known to enter cells for use as a positive control in SNAPPA), leading to irreversible covalent labeling of the SNAP-tag with the synthetic probe, as described in Keppler, et al (Nat. Biotechnol. 21, 86 (2003)). Unoccupied SNAP-tag binding cites may be detected by subsequently incubating with a BG-fluorophore to covalently bind SNAP -tags not bound by BG-peptides. Once covalently labeled, the labeling reaction is measured by detecting fluorescence, the absence of which, relative to the negative control, is indicative of the SNAP-tag covalently bound to the BG- peptide. The small size of the SNAP-tag and relatively neutral charge may be favorable for the SNAP penetration assay. Formation of a covalent bond during the SNAP reaction may facilitate recovery and identification of bound species, for example using mass spectrometry.
[0353] Such BG derivatives are also referred to herein as BG-substrate or SNAP substrates. Such SNAP substrates are chemically inert towards other proteins, avoiding nonspecific labeling in cellular applications. In SNAPPA, a BG-substrate is conjugated to the peptide being tested for cell penetration, creating a BG-peptide. SNAP-tag is expressed in the cell itself, either without organelle targeting or with nuclear localization by way of histone H2B fusion. BG-GLA resembles the enzyme’s native substrate, an alkylated guanine nucleotide. The enzyme’s mutations, combined with the chemical structure of BG-GLA, can cause the BG-GLA to be covalently bound to the SNAP protein. By this means, species covalently attached to the BG- GLA, for example via NHS reaction, are also covalently attached to the SNAP -tag, blocking it from further reactions. The BG-peptide, for example generated by mixing BG-GLA-NHS with peptides of interest resulting in the BG-GLA to covalently bond with solvent-accessible primary amines in the peptide, can be added to the cell culture media, and if it is cell penetrant, it can enter the cytosol and become bound to a SNAP protein there. If a peptide is sufficiently cell penetrant, the SNAP -tag in the cells may become saturated, and subsequently-dosed fluorescently-labeled BG-GLA, which can penetrate cells due to its small size, may have no SNAP protein with which to react. If, however, the BG-GLA is attached to a peptide without cell penetration capabilities, then the attached BG-GLA may not come into contact with the SNAP protein upon cell media incubation. This leaves the SNAP protein un-saturated and may react with subsequently-dosed BG- fluorophore, rendering the cells fluorescent.
[0354] Generation of pSNAPf and pSNAPf-H2B cell lines. In some embodiments, plasmid vector for SNAP -tag (pSNAPf) and H2B-tagged SNAP -tag (pSNAPf-H2B) can be purchased from commercial vendors. Plasmids can be sequence validated and purified by Endofree Maxiprep before transfection into cell lines, such as NIH3T3, HeLa or HEK293 cells using Lipofectamine 2000. Following a period of recovery, selection for positive transfectants can be initiated using geneticin over a period of 2-3 weeks. The pool of stable transfectants can be expanded and banked in liquid nitrogen.
[0355] SNAP conjugation and purification of SNAP-CDPs. In some embodiments, peptides can be produced and purified, as described in Bandaranayake et al., Nucleic Acids Res. 2011 Nov; 39(21): el43. For SNAP substrate conjugation, lyophilized peptides can be resuspended in PBS to an approximate 1 mg/mL concentration. SNAP substrate can be attached to reactive amine groups on peptides via NHS-ester reaction. Benzylguanine-NHS (BG-GLA-NHS) can be obtained from a commercial vendor and resuspended at 10 pg/uL in anhydrous DMSO. One molar equivalent of BG-GLA-NHS can be added to resuspended peptide and reaction can be allowed to proceed with stirring at room temperature for one hour. Samples of starting material and crude reaction mixture can be analyzed by HPLC/MS to assess reaction progress. For peptides with only one reactive amine group, a total of three molar equivalents can be added over the course of three hours. For peptides with multiple reactive amine groups, reactions can be halted when the presence of multi-SNAP substrate-tagged species becomes apparent. A molar reaction or reaction time can be chosen to target a single SNAP substrate to be conjugated per peptide. The reaction can occur with the N-terminus of the peptide. The reaction can occur with one or more lysine residues of a peptide. Additionally, if precipitation is noted, addition of BG- GLA-NHS can be halted.
[0356] Crude reactions comprised of unreacted peptide, BG-peptide and hydrolyzed BG-GLA- NHS (e.g., BG-GLA-OH) can be purified using size exclusion chromatography (SEC) using either a Superdex Peptide or Superdex 75 column, depending on reaction volume. Following column equilibration in PBS, BG-peptides can be separated and eluted over 1.5 column volumes. Fractions with notable 280 nm/214 nm/200 nm absorbance can be pooled and concentrated through a 3 kDa molecular weight cut off spin filter. Individual BG-peptides may elute in distinct volumes, often around 1 column volume. BG-peptides can be verified using HPLC/MS BG-peptide concentration can be assessed using the bicinchoninic acid assay.
[0357] SNAP penetration assay (SNAPP A). In some embodiments, BG-peptides and controls can be diluted in standard cell growth media (DMEM +10% FBS +pen/strep). 2 pM hydrolyzed BG-GLA-NHS (hydrolyzed to BG-GLA-OH) can be used as a positive control for cell penetration, while PBS can serve as a negative control. An additional mock conjugation/purification of BG-GLA-NHS can also serve as a negative control for purification. pSNAPf and pSNAPf-H2B cell lines can be plated on collagen-coated 96-well plates, 20,000 cells/well, and can be allowed to recover in growth media overnight. Plates can be rinsed with PBS and incubated with diluted BG-peptides and controls in growth media for 2 hours at 37° C. The cells can then be incubated with labelling media containing 600 nM SNAP-Cell TMR-Star and 1 pg/mL Hoescht 33342 in growth media for 15 minutes at 37° C. In some embodiments, the labelling media may contain a SNAP substrate conjugated to a rhodamine dye, an Alexa fluor, an Atto dye, a cyanine dye, or a fluorescein dye. The labelling media can be removed, and cells can be incubated with fresh growth media for 30 minutes at 37° C as a washout phase for excess SNAP-Cell TMR-Star dye. The media can be replaced with phenol red-free OptiMEM for imaging.
[0358] Imaging can be performed on a Molecular Devices ImageXpress Nano, utilizing DAPI and Texas Red filters. For scoring, cells can be segmented using ImageXpress software to generate counts for nuclei and average cytoplasmic or nuclear intensity of SNAP-Cell TMR- Star. The average intensity can then be normalized using the positive control of 2 pM hydrolyzed BG-GLA-NHS (BG-GLA-OH) as 100% cell penetration and PBS as 0% penetration to determine normalized SNAP -tag occupancy. The degree of cell penetration is inversely proportional to the level of fluorescence in the cells. [0359] Endocytotic profiling via SNAPPA. In some embodiments, pSNAPf cells can be preincubated with a variety of endocytosis inhibitors to assess mechanisms responsible for uptake of BG-peptides. Macropinocytosis can be inhibited using 50 pM ethylisopropyl amiloride (EIP A), which blocks Na+/H+ exchangers and prevents formation of macropinosomes. Clathrin-mediated endocytosis can be inhibited using either 20 pM nocodazole, which inhibits microtubule polymerization, 3 pM cytochalasin D, which interferes with actin polymerization, or 80 pM dynasore, an inhibitor of dynamin GTPase. Endosomal acidification or lysosomal maturation can be inhibited using 50 nM bafilomycin A, which inhibits the vacuolar ATPase complex, or 50 pM chloroquine, which diffuses into endosomes as a weak base. pSNAPf cells can be preincubated with the specified concentration of inhibitor in growth media for 1 hour prior to SNAPPA, which can be performed as described herein.
[0360] High-throughput screening for cell-penetrating peptides. In some embodiments, cells expressing a labeled SNAP -tag in either the cytoplasm or the nucleus can be cultured in a microplate format. In some embodiments, the labeled SNAP -tag is labeled with a green fluorescent protein, a yellow florescent protein, a red fluorescent protein, a blue fluorescent protein, or a cyan fluorescent protein. Candidate cell-penetrating peptides can be tagged with SNAP substrate and purified using the method described herein. Each BG-candidate peptide can be added to the cells expressing labeled SNAP -tag in a well of the microplate. Peptides capable of cell penetration may cross the cell membrane and covalently bind to the labeled SNAP -tag expressed in the cytoplasm or the nucleus. Following incubation, excess BG-candidate peptides that did not enter the cells can be washed off. Cells can be pooled and lysed, and labeled SNAP- tag that reacted with cell penetrant BG-peptides can be purified by immunoprecipitation via the label. Candidate peptides that entered the cell can be immunoprecipitated with the SNAP -tag. Cell -penetrating peptides can be identified by mass spectrometry.
[0361] SDPR Protease Resistance Testing. In some embodiments, SDPR protease resistance testing can be used with sequencing-grade enzymes, including Trypsin and Chymotrypsin. Trypsin and trypsin inhibitor can be used for HPLC analysis.
[0362] Next Generation Sequencing. In some embodiments, screens for cell penetration can be assessed by Illumina sequencing. Such sequencing method involves collecting cell pellets (1.5E6, 3 technical replicates) resupended in 50 pL Terra Direct PCRMix (from Clontech) and amplified for 16 cycles using the original cloning primers. Up to four aliquots can be diluted 16- fold into 60 pL Phusion DNA Polymerase reactions and amplified using distinct Illumina primers, containing adaptor sequences for flow cell adherence. Forward primers can include a 6 bp barcode for multiplexing. Illumina HiSeq 2500 in rapid mode can be used to run samples, which Bowtie2 software can be used for mapping, and Excel (Microsoft) and MATLAB (MathWorks) used for data analysis.
Pharmaceutical Compositions
[0363] A pharmaceutical composition of the disclosure can be a combination of any peptide as described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, antioxidants, solubilizers, buffers, osmolytes, salts, surfactants, amino acids, encapsulating agents, bulking agents, cryoprotectants, and/or excipients. The pharmaceutical composition facilitates administration of a peptide described herein to an organism. In some cases, the pharmaceutical composition comprises factors that extend half-life of the peptide and/or help the peptide to penetrate the target cells. [0364] Pharmaceutical compositions can be administered in therapeutically-effective amounts as pharmaceutical compositions by various forms and routes including, for example, intravenous, subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, optic, nasal, oral, sublingual, inhalation, dermal, intrathecal, intravitreal, intratumoral, intranasal, and topical administration. A pharmaceutical composition can be administered in a local or systemic manner, for example, via injection of the peptide described herein directly into an organ, optionally in a depot.
[0365] Parenteral injections can be formulated for bolus injection or continuous infusion. The pharmaceutical compositions can be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of a peptide described herein in water-soluble form. Suspensions of peptide-antibody complexes described herein can be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension can also contain suitable stabilizers or agents which increase the solubility and/or reduce the aggregation of such peptide-antibody complexes described herein to allow for the preparation of highly concentrated solutions.
[0366] The cell-penetrating peptides described herein can be used to increase cell penetration of a cargo molecule (e g., a therapeutic agent) and deliver the cargo molecule to a cellular compartment. In some embodiments, protein transfection agents, direct cytosolic expression of the peptide, or electrophoration of the peptide can be used to increase cell penetration. In some embodiments, other excipients can be formulated with a peptide in order to increase the cell penetration of the peptide, such as those approaches described in “Protein and Peptide Drug Delivery: Oral Approaches” Indian J. Pharm. Sci., Shaji and Patole, v70(3) 269-277, 2008. Any combination of these formulations or approaches can be used to increase cell penetration of a peptide as described herein.
[0367] Alternatively, the peptide described herein can be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In some embodiments, a purified peptide is administered intravenously. A peptide described herein can be administered to a subject, home, target, migrate to, or be directed to cancerous cell, a tumor, or a cell with dysregulated HIPPO pathway. In some embodiments, a peptide can be conjugated to, linked to, or fused to another peptide that provides a targeting function to a specific target cell type in the central nervous system or across the blood brain barrier.
[0368] A peptide of the disclosure can be applied directly to an organ, or an organ tissue or cells, such as brain or brain tissue or cells, during a surgical procedure. The recombinant peptide described herein can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, and ointments. Such pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
[0369] In practicing the methods of treatment or use provided herein, therapeutically effective amounts of a peptide described herein can be administered in pharmaceutical compositions to a subject suffering from a condition that affects the immune system. In some embodiments, the subject is a mammal such as a human or a primate. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.
[0370] In some embodiments, a peptide is cloned into a viral or non-viral expression vector. Such expression vector can be packaged in a viral particle, a virion, or a non-viral carrier or delivery mechanism, which is administered to patients in the form of gene therapy. In other embodiments, patient cells are extracted and modified to express a cell-penetrating peptide ex vivo before the modified cells are returned back to the patient in the form of a cell-based therapy, such that the modified cells will express the peptide once transplanted back in the patient.
[0371] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulation can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising a peptide described herein can be manufactured, for example, by expressing the peptide in a recombinant system, purifying the peptide, lyophilizing the peptide, mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or compression processes. The pharmaceutical compositions can include at least one pharmaceutically acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically acceptable salt form.
[0372] Methods for the preparation of peptide described herein comprising the compounds described herein include formulating peptide described herein with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.
[0373] Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.
[0374] Pharmaceutical compositions can also include permeation or absorption enhancers (Aungst et al. AAPS J. 14(l):10-8. (2012) and Moroz et al. Adv Drug Deliv Rev 101: 108-21. (2016)). Permeation enhancers can facilitate uptake of molecules from the GI tract into systemic circulation. Permeation enhancers can include salts of medium chain fatty acids, sodium caprate, sodium caprylate, N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC), N-(5- chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), hydrophilic aromatic alcohols such as phenoxyethanol, benzyl alcohol, and phenyl alcohol, chitosan, alkyl glycosides, dodecyl-2-N,N- dimethylamino propionate (DDAIPP), chelators of divalent cations including EDTA, EGTA, and citric acid, sodium alkyl sulfate, sodium salicylate, lecithin-based, or bile salt-derived agents such as deoxy cholates,
[0375] Compositions can also include protease inhibitors including soybean trypsin inhibitor, aprotinin, sodium glycocholate, camostat mesilate, vacitracin, or cyclopentadecalactone, Use of Peptides in Treatments
[0376] The cell-penetrating peptides described herein (e.g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) may be conjugated to, linked to, or fused with a therapeutic agent for use in a method of treatment. The cell-penetrating peptides or cell-penetrating peptide complexes may be used to deliver a therapeutic agent that may otherwise be excluded from a cellular compartment across a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) an into a target cell. Delivery of the therapeutic agent using the cell -penetrating peptides described herein may increase access to a target (e.g., a target protein or nucleotide sequence) by the therapeutic agent. In some embodiments, the method includes administering an effective amount of a cell -penetrating peptide-therapeutic agent conjugate as described herein to a subject in need thereof. In some embodiments, a cell-penetrating peptide-therapeutic agent conjugate may be administered to the subject in need thereof to treat a disease or a condition in the subject.
[0377] In some embodiments, a therapeutic agent that may be delivered into a cell by a cellpenetrating peptide of the present disclosure may include an antibody, an antibody fragment, an Fc domain, a single chain Fv, an intrabody, or a nanobody. In some embodiments, the therapeutic agent may be a cystine-dense peptide, an affibody, a B-hairpin, an avimer, an adnectin, a stapled peptide, a nannofittin, a kunitz domain, a fynomer, or a bicyclic peptide. In some embodiments, the therapeutic agent may be an anti -cancer agent and may be delivered into a cancer cell. In some embodiments, the therapeutic agent may be a peptide (e.g., a cystine- dense peptide), a transcription factor, an RNA, a Cas enzyme or other CRISPR component, an immunomodulating agent, or a hormone. For example, an active agent, such as a cystine-dense peptide, that binds to a transcription factor (such as a forkhead box class O transcription factor (FOXO), Nuclear factor E2 -related factor 2 (NRF2), runt-related transcription factor 1 (RUNX1), methyl CpG binding protein 2 transcription factor (MECP2), maturity onset diabetes of the young transcription factor (MODY), forkhead box P3 (FOXP3), p53, p65, signal transducer and activator of transcription (STAT), homeodomain transcription factor (HOX), or SOX9 transcription factor) may be conjugated to a cell -penetrating peptide and delivered to a nucleus of a cell to alter the expression level of a target gene. In some embodiments, a cellpenetrating peptide is conjugated to NRF2 to carry NRF2 to the nucleus, facilitate transcription of antioxi dative genes, and mediate recovery from acetaminophen-induced hepatotoxicity. In some embodiments, a cell-penetrating peptide is conjugated to RUNX1 to carry RUNX1 to the nucleus, regulate gene expression, and mediate cartilage deposition. In some embodiments, a cell-penetrating peptide complex of the present disclosure may target AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, -catenin, IKK, TEAD, NF-KB, PKC, AP-1, Jun, Fos, REL, a transcription factor, Ras, Rho, Ran, Rab, Arf, androgen receptor, ikaros, aiolos, nuclear receptors, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-3, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, or tau. ADP- Ribosylation Factor (Arf) GTPases are part of the Ras superfamily used in regulation of vesicular cellular trafficking, lipid modification, cytokinesis and cell adhesion. Arf GTPases are tightly regulated by specific guanine nucleotide exchange factors (GEFs) with a conserved Sec7 domain, and GTPase-activating proteins (GAPs) with a conserved zinc finger domain. Like all Ras superfamily members, the GEFs switch on/activate Arfs while the GAPS switch off/inactivate them. Such proteins have been implicated in several essential cellular functions, like cell spreading and migration. These functions are used by cancer cells to disseminate and invade the tissues surrounding the primary tumor, leading to the formation of metastases.
Disrupting such ADP-Ribosylation Factor (Arf) GTPases, guanine nucleotide exchange factors (GEFs) with a conserved Sec7 domain, and GTPase-activating proteins (GAPs), using the cellpenetrating peptide of the invention can be used in cancer therapy, including metastatic phases of cancer. In a further example, one or more CRISPR components (e g., guide RNA, a tracrRNA, a crRNA, or a Cas nuclease) may be conjugated to a cell-penetrating peptide and delivered to a nucleus of a cell to genetically modify a target nucleic acid sequence. In a further example, an immunomodulating agent may be conjugated to a cell-penetrating peptide and delivered into a cell to modulate or inhibit an immune response. In another example, a therapeutic agent for treatment of neurodegeneration may be conjugated to a cell-penetrating peptide and delivered across the blood brain barrier to treat a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS)). In some embodiments, the therapeutic agent may facilitate degradation of excess proteins in a target cell upon delivery to the target cell by the cell-penetrating peptide. [0378] In some embodiments, a method of treating a condition may comprise targeted degradation of a target molecule, such as a target protein. In some embodiments the condition is associated with increased activity, increased expression, or novel activity brought on by gene amplification, missense mutation, nonsense mutation, aberrant splicing, or fusion protein brought on by chromosomal translocation of the target molecule. A method of treatment may comprise administering to a subject a cell-penetrating peptide complex comprising a cellpenetrating peptide linked or fused to a cargo molecule comprising a target-binding peptide (e.g., a target-binding CDP) and a ubiquitin ligase-binding molecule. A method of treatment can also comprise administering to a subject a cell-penetrating peptide complex comprising a cellpenetrating peptide linked or fused to a cargo molecule comprising a target-binding peptide (e.g., a target-binding CDP) and a molecule that binds any part of the ubiquitin-proteasome system (UPS) or cytosolic protein degradation machinery, such as the 26S proteosome, or any component thereof such as the 19S ubiquitin recognition proteins Rpn-13 and Rpn-10. Binding to the UPS could be through an inhibitor, such as KDT-11, which inhibits Rpn-13. Upon administration to a subject, the peptide complex may cross a cellular layer of a cell (e.g., a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, or bone marrow cell) to access the target and the ubiquitin ligase in a cellular compartment (e.g., the cytosol or the nucleus) or intercellular compartment (e.g., a nanolumen, intercellular space, or paracellular space). Upon gaining access to the target and the ubiquitin ligase by way of the cell-penetrating peptide, the cargo molecule may bind to the target and the ubiquitin ligase, forming a ternary complex. The ubiquitin ligase may ubiquitinate the target, labeling it for proteasomal degradation, thereby treating the condition. A method of treatment may comprise administering to a subject a cell -penetrating peptide complex comprising a cell-penetrating peptide linked or fused to a cargo molecule comprising a PROTAC, a molecular glue, a targetbinding peptide, or other cargos that cause targeted protein degradation. In some embodiments, the cargo molecule may comprise an IMiD, a Boc3Arg, an adamantyl group, or a carborane. Upon administration to a subject, the peptide complex may cross a cellular layer of a cell, such as by entering the cytosol, and bind, label, target, traffic, or otherwise direct a protein or other target for degradation, such as by the ubiquitin-proteosome system.
[0379] In some embodiments, a method of treating a condition may comprise inhibition of a target molecule. In some embodiments the condition is associated with increased activity, increased expression, or novel activity brought on by gene amplification, missense mutation, nonsense mutation, aberrant splicing, or fusion protein brought on by chromosomal translocation of the target molecule, such as a target protein. Inhibiting the target molecule may comprise inhibiting a conformational change, inhibiting an enzymatic activity, inhibiting ligand binding, inhibiting a protein-protein interaction, or inhibiting a protein-nucleic acid interaction of the target molecule. A method of treating a condition may comprise administering to a subject a cell-penetrating peptide complex comprising a cell -penetrating peptide linked or fused to a cargo molecule that binds to and inhibits a target. The cargo molecule may comprise a target-binding peptide (e.g., a target-binding CDP). Upon administration to a subject, the peptide complex may cross a cellular layer of a cell (e g , a cancer cell, pancreatic cell, liver cell, colon cell, ovarian cell, breast cell, lung cell, spleen cell, or bone marrow cell) to access the target in a cellular compartment (e g., the cytosol or the nucleus) or intercellular compartment (e g., a nanolumen, intercellular space, or paracellular space). Upon gaining access to the target by way of the cellpenetrating peptide, the cargo molecule may bind to and inhibit the target, thereby treating the condition.
[0380] The term “effective amount,” as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. Compositions containing such agents or compounds can be administered for prophylactic, enhancing, and/or therapeutic treatments. An appropriate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study.
[0381] The methods, compositions, and kits of this disclosure may comprise a method to prevent, treat, arrest, reverse, or ameliorate the symptoms of a condition. The treatment may comprise treating a subject (e.g., an individual, a domestic animal, a wild animal, or a lab animal afflicted with a disease or condition) with a peptide of the disclosure. The disease may be a cancer or tumor. In treating the disease, the peptide may contact the tumor or cancerous cells. The subject may be a human. Subjects can be humans; non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants, and fetuses in utero.
[0382] Treatment may be provided to the subject before clinical onset of disease. Treatment may be provided to the subject after clinical onset of disease. Treatment may be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years or more after clinical onset of the disease. Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years or more after clinical onset of disease. Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may also include treating a human in a clinical trial. A treatment can comprise administering to a subject a pharmaceutical composition, such as one or more of the pharmaceutical compositions described throughout the disclosure. A treatment can comprise a once daily dosing. A treatment can comprise delivering a peptide of the disclosure to a subject, either intravenously, subcutaneously, intramuscularly, by inhalation, dermally, topically, by intra-articular injection, orally, sublingually, intrathecally, intravitreally, transdermally, intranasally, via a peritoneal route, directly into a tumor, e.g., injection directly into a tumor, directly into the brain, e.g., via and intracerebral ventricle route, or directly onto a joint, e.g., via topical, intra-articular injection route. A treatment can comprise administering a peptide-active agent conjugate to a subject, either intravenously, subcutaneously, intramuscularly, by inhalation, by intra-articular injection, dermally, topically, orally, intrathecally, intravitreally, transdermally, intransally, parenterally, orally, via a peritoneal route, nasally, sublingually, or directly onto cancerous tissues.
[0383] Improving delivery of therapeutic agents into cellular compartments using the cell penetrating peptides of the present disclosure can have implications in a number of diseases, condition, or disorders, including disorders associated to dysregulated cell growth, cell proliferation, angiogenesis, organogenesis, tumor progression, and/or metastasis. Compositions comprising any one of the cell-penetrating peptides described herein conjugated to a therapeutic agent (e.g., an anti-cancer agent), or a pharmaceutical composition thereof, can be used in a method of treating a cancer, tumor progression, and/or dysregulated cell growth. Exemplary diseases, disorder, or condition include: multiple myeloma, plastic anemia, myelodysplasia, and related bone marrow failure syndromes, myeloproliferative diseases, acute and chronic myeloid leukemia, malignancies of lymphoid cells, hematologic malignancies, plasma cell disorders, skeletal muscle disorder, myopathy, muscular dystrophy (e.g., Becker muscular dystrophy, Duchenne muscular dystrophy, Emery -Dreifuss muscular dystrophy, Facioscapulohumoeral muscular dystrophy, Myotonia congentia, and myotonic dystrophy), Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis (ALS), and chronic obstructive pulmonary disorder. In some embodiments, compositions/peptides disclosed herein are used to treat dysregulated cell growth, cancer, tumor, and/or metastasis associated with any of the following cell, tissue, or organ types: brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cell, pancreatic, colon, stomach, cervix, breast, endometrial, prostate, testicle, ovarian, skin, head and neck, esophageal, oral tissue, and bone marrow. In further embodiments, compositions/peptides disclosed herein are used to treat any of the following: osteosarcoma, hepatocellular carcinoma, malignant mesothelioma, schwannoma, meningioma, renal carcinoma, cholangiocarcinoma, bile duct hamartoma, soft tissue carcinoma, ovarian carcinoma, colonic adenoma, T cell acute lymphoblastic leukaemia, gastrointestinal hyperplasia, fibrosarcoma, pancreatic ductal metaplasia, squamous cell carcinoma, kaposis sarcoma, and HIV-induced non-Hodgkin’s lymphoma.
[0384] In some embodiments, the peptides of this disclosure (e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) conjugated to a therapeutic agent can be used to access and treat these disorders by delivering the therapeutic agent across a cellular layer (e.g., a cell membrane, a nuclear envelope, an intercellular space, a paracellular space, an endosomal membrane, a lysosomal membrane, other subcellular compartment membrane, a blood brain barrier, or a nanolumen) to a cellular compartment. Delivery of a therapeutic agent using a cell -penetrating peptide of the present disclosure may increase the efficacy of the therapeutic agent by increasing access of the therapeutic agent to intracellular targets (e g., cytosolic proteins or target nucleic acid sequences) relative to the therapeutic agent alone.
[0385] The cell penetrating peptides of this disclosure (e g., SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, and SEQ ID NO: 195 - SEQ ID NO: 254) and complexes described herein can deliver a cargo molecule to the cytosol or the nucleus of a cell. In some embodiments, the cargo molecule is delivered to the cytosol to achieve a cytosolic concentration of at least 1 nM, at least 5 nM, at least 10 nM, at least 25 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1000 nM, at least 1100 nM, at least 1200 nM, at least 1300 nM, at least 1400 nM, at least 1500 nM, at least 1600 nM, at least 1700 nM, at least 1800 nM, or at least 2000 nM. In some embodiments, the cargo molecule is delivered to the cytosol to achieve a cytosolic concentration of from about 1 nM to about 50 nM, from about 10 nM to about 100 nM, from about 50 nm to about 100 nm, from about 50 nm to about 200 nm, from about 50 nm to about 500 nm, from about 50 nm to about 700 nm, from about 50 nm to about 1000 nm, from about 50 nm to about 1200 nm, from about 50 nm to about 1500 nm, from about 50 nm to about 2000 nm, from about 100 nm to about 200 nm, from about 100 nm to about 500 nm, from about 100 nm to about 700 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 1200 nm, from about 100 nm to about 1500 nm, from about 100 nm to about 2000 nm, from about 500 nm to about 700 nm, from about 500 nm to about 1000 nm, from about 500 nm to about 1200 nm, from about 500 nm to about 1500 nm, from about 500 nm to about 2000 nm, from about 1000 nm to about 1200 nm, from about 1000 nm to about 1500 nm, from about 1000 nm to about 2000 nm, or from about 1500 nm to about 2000 nm. In some embodiments, the cargo molecule is delivered to the nucleus to achieve a nuclear concentration of at least 1 nM, at least 5 nM, at least 10 nM, at least 25 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1000 nM, at least 1100 nM, at least 1200 nM, at least 1300 nM, at least 1400 nM, at least 1500 nM, at least 1600 nM, at least 1700 nM, at least 1800 nM, or at least 2000 nM. In some embodiments, the cargo molecule is delivered to the nucleus to achieve a nuclear concentration of from about 1 nM to about 50 nM, from about 10 nM to about 100 nM, from about 50 nm to about 100 nm, from about 50 nm to about 200 nm, from about 50 nm to about 500 nm, from about 50 nm to about 700 nm, from about 50 nm to about 1000 nm, from about 50 nm to about 1200 nm, from about 50 nm to about 1500 nm, from about 50 nm to about 2000 nm, from about 100 nm to about 200 nm, from about 100 nm to about 500 nm, from about 100 nm to about 700 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 1200 nm, from about 100 nm to about 1500 nm, from about 100 nm to about 2000 nm, from about 500 nm to about 700 nm, from about 500 nm to about 1000 nm, from about 500 nm to about 1200 nm, from about 500 nm to about 1500 nm, from about 500 nm to about 2000 nm, from about 1000 nm to about 1200 nm, from about 1000 nm to about 1500 nm, from about 1000 nm to about 2000 nm, or from about 1500 nm to about 2000 nm. The targeted moiety may also be delivered to the to the cytosol, the nucleus, or the cytosol and the nucleus in varying concentrations as described herein.
Nucleotide and Oligo Delivery
[0386] A peptide (e.g., a cell-penetrating peptide) may be linked, conjugated, complexed with, or fused to a nucleotide via various chemistries resulting in peptide oligonucleotide complexes that may form either a cleavable or stable linkage to deliver the oligonucleotide to a cell. For example, in some embodiments, a cell-penetrating peptide may delivery an oligonucleotide to the cytoplasm or nucleus, or both, of a cell. Some of the nucleotides within the peptide oligonucleotide complex can function within the nucleus of a cell, including gapmers, ASO splice blockers, and U1 adapters. Others function within the cytosol, including siRNA and anti- miRs. Aptamers are unique in that they do not function through hybridization or base paring interactions with nucleic acid targets. Instead, aptamers form secondary structures to bind to proteins or other macromolecules. Aptamers may function wherever the target protein or macromolecule is located.
[0387] The nucleotide portion of the peptide oligonucleotide complexes described herein may target specific RNAs (e.g., mRNAs or pre-mRNAs) from genes expressed in cancer and other diseases. For example, the nucleotide sequence in the complex may be complementary to any target provided in SEQ ID NO: 574 - SEQ ID NO: 611, TABLE 12, or TABLE 6. The nucleotide sequence in the complex may be complementary to the target RNA, or in the case of an aptamer, may bind a target protein or other macromolecule. The a nucleotide sequence may be single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, splice blocker ASO, or Ul Adapter.
[0388] In some embodiments, a target of the nucleotide in a peptide oligonucleotide complex may be a gastrointestinal target, such as a gene with pro-inflammatory, extracellular matrixmodifying, or immune cell recruitment functionality. Peptide oligonucleotide complexes described herein (e.g., a peptide oligonucleotide complex comprising a cell-penetrating peptide and a nucleotide that binds a gene target mRNA) that target gastrointestinal gene targets may be used to treat various gastrointestinal disorders, including inflammatory bowel disease (IBD), ulcerative colitis, and Crohn’s disease.
[0389] In some embodiments, a target of the nucleotide in a peptide oligonucleotide complex may be a cancer target, such as a gene involved in oncogenic signaling, anti-apoptotic genes, pro-inflammatory signaling genes, protein homeostasis genes, developmental regulatory genes, or adapter protein genes that initiate downstream cell growth signaling. For example, targeting an over-expressed growth factor like HERZ can be challenging, but HER2 and other RTK (e.g., EGFR, ERBB3) signaling depends on adapter proteins like Grb2 to initiate cell growth signaling downstream. Knockdown of Grb2 can halt signaling in a way that is difficult to mutationally compensate as Grb2 loss is epistatic to HER2 activity. Cancer cells are typically under low levels of proteotoxic stress, as they are growing so quickly that their protein folding machinery struggles to keep up, so targeting protein homeostasis genes, such as heat shock proteins (HSPs), hypoxia-sensing proteins (e g., HIF), and upregulators of the heat shock response, may reduce proteotoxic stress by helping to fold or stabilize proteins during folding. In some embodiments, a pro-inflammatory cytokine may be delivered via an mRNA in a peptide oligonucleotide complex, or an antisense construct targeting an anti-inflammatory signal may be delivered. Delivery of a pro-inflammatory signal or reduction of an anti-inflammatory signal may help to recruit B cells, T cells, macrophages, or other immune infiltrates to a tumor microenvironment. Peptide oligonucleotide complexes described herein (e.g., a peptide oligonucleotide complex comprising a cell-penetrating peptide and a nucleotide that binds a gene target mRNA) that target cancer gene targets may be used to treat various cancers, including solid tumors. Developmental regulators, such as transcription factors involved in early cell fate and pluripotency, and chromatin remodeling enzymes, may be targeted to specifically harm dedifferentiated cells which may be present in advanced tumors and associated with a more mobile and/or mitotic cell state. A peptide oligonucleotide construct targeting a cancer target may treat or prevent cancer by reducing oncogenic signaling, reducing target over-expression, reducing oncogenic antisense activity (e g., miRNAs targeting tumor suppressors), and/or eliminating the source of the oncogenic signaling cascade.
[0390] Examples of gene targets (e.g., gastrointestinal, or cancer gene targets) are provided in
TABLE 12
TABLE 12 - Examples of Disease-Specific Gene Targets
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
[0391] In some embodiments, an oligonucleotide may target a gene for downregulation. For example, PvRBSA, PvRBP2b, PfEMPl, pfmdrl, pfgchl, GPX4, SLC7al l, alpha-synuclein, PD-L1, NUP98-KDM5A, NTRK1, JAK2, K-/N-RAS, the JAK-STAT pathway, the Hedgehog pathway, the PI3K/AKT pathway, the RAF/MEK/ERK pathway, the mTOR pathway, HD AC, MDM2, LSD1, CALR, PKC, NF-KB, HSP90, HIV Tat, TNF-a, CCR2, CCR5, TAR (tat), RRE (rev), vpr, U5 leader, Nef, Gag, Vif, Env, IL lb, IL6, TNFa, IFNg, LRRK2, or Myostatin may be targeted for downregulation.
[0392] An example of an antagomir that may be complexed with a cell-penetrating peptide to target a gene includes cobomarsen. An example of an aptamer that may be complexed with a cell-penetrating peptide to target a gene includes pegaptanib. Examples of gapmers that may be complexed with a cell-penetrating peptide to target a gene include fomivirsen, mipomersen, inotersen, volanesorsen, tofersen, tominersen, pelacarsen, alicaforsen, apatorsen, and trabedersen. Examples of siRNAs that may be complexed with a cell-penetrating peptide to target a gene include patisiran, vutrisiran, revusiran, fitusiran, lumasiran, givosiran, and inclisiran. Examples of splice blockers that may be complexed with a cell-penetrating peptide to target a gene include nusinersen, eteplirsen, golodirsen, viltolarsen, casimersen, and sepofarsen. An example of a translation blocker that may be complexed with a cell-penetrating peptide to target a gene includes prexigebersen.
[0393] Any targets for the nucleic acid portion of the peptide oligonucleotide complex described herein can be used in conjunction with a U1 adapter to degrade targeted mRNAs. The target recognition (or complementary nucleic acid to the target mRNA) portion directs the peptide oligonucleotide complex to the targeted mRNA selected for degradation, while the U1 portion prevents the addition of polyA to the mRNA resulting in degradation of the targeted mRNA. U1 adapters can comprise any nucleotide sequence complementary to the ssRNA component of the U1 small nuclear ribonucleoprotein (U1 snRNP). In some embodiments, the U1 adapter sequences engage the U1 snRNP near its poly A site. In some embodiments, the length of the U1 adapter is 15 to 25 nt in length, or about 20 nt in length. In some embodiments, the U1 adapter is above 40% in its G/C content. Exemplary U1 adapters are shown in TABLE 13, in conjunction with a target nucleic acid “target recognition” portion which comprises a nucleotide sequence is single stranded (ssDNA, ssRNA) or double stranded (dsDNA, dsRNA) or a combination of single and double stranded (for example with a mismatched sequence, hairpin or other structure), an antisense RNA, complementary RNA, inhibitory RNA, interfering RNA, nuclear RNA, antisense oligonucleotide (ASO), microRNA (miRNA), an oligonucleotide complementary to a natural antisense transcripts (NATs) sequences, siRNA, snRNA, aptamer, gapmer, anti-miR, or splice blocker ASO. The 10-19 nt U1 Adapter is italicized.
TABLE 13 - Examples of Target Recognition Constructs with U1 Adapters
Figure imgf000183_0001
Figure imgf000184_0001
[0394] Exemplary U1 adapters include: UCCCCUGCCAGGUAAGUAU (SEQ ID NO: 488); CCCUGCCAGGUAAGUAU (SEQ ID NO: 489); CUGCCAGGUAAGUAU (SEQ ID NO: 490); UGCCAGGUAAGUAU (SEQ ID NO: 491); GCCAGGUAAGUAU (SEQ ID NO: 492); CCAGGUAAGUAU (SEQ ID NO: 493); CAGGUAAGUAU (SEQ ID NO: 494); and CAGGUAAGUA (SEQ ID NO: 495).
Peptide Kit
[0395] In one aspect, peptides described herein can be provided as a kit. In another embodiment, peptide complexes described herein can be provided as a kit. In another embodiment, a kit comprises amino acids encoding a peptide described herein, a vector, a host organism, and an instruction manual. In some embodiments, a kit includes written instructions on the use or administration of the peptides.
EXAMPLES
[0396] The following examples are included to further describe some aspects of the present disclosure, and should not be used to limit the scope of the invention.
EXAMPLE 1
Manufacture of Peptides
[0397] This example describes the manufacture of the peptides described herein. Peptides derived from proteins were generated in mammalian cell culture using a published methodology. (A D Bandaranayke, C. Correnti, B Y Ryu, M. Brault, R.K. Strong, D. Rawlings. 2011. Nucleic Acids Research. (39)21, el43).
[0398] The peptide sequence was reverse-translated into DNA, synthesized, and cloned in-frame with siderocalin using standard molecular biology techniques (M R. Green, Joseph Sambrook. Molecular Cloning. 2012 Cold Spring Harbor Press). The resulting construct was packaged into a lentivirus, transduced into HEK-293 cells, expanded, isolated by immobilized metal affinity chromatography (IMAC), cleaved with tobacco etch virus protease, and purified to homogeneity by reverse-phase chromatography. Following purification, each peptide was lyophilized and stored frozen. EXAMPLE 2
Peptide Expression Using a Mammalian Expression System
[0399] This example describes expression of the peptides using a mammalian expression system. Peptides were expressed according to the methods described in Bandaranayake et al., Nucleic Acids Res. 2011 Nov; 39(21): el 43. Peptides were cleaved from siderocalin using tobacco etch virus protease and purified by FPLC on a size exclusion chromatography (SEC) column using an isocratic elution over four column volumes in IX DPBS. Peptides were then stored at 4°C.
EXAMPLE 3
Generation of pSNAPf and pSNAPf-H2B Cell Lines
[0400] This example describes generation of pSNAPf and pSNAPf-H2B cell lines. A plasmid vector for SNAP -tag (pSNAPf) was obtained from New England Biolabs and histone H2B- tagged SNAP -tag (pSNAPf-H2B) was obtained from Addgene. Plasmids were sequence validated and purified by Endofree Maxiprep before transfection into NIH3T3, HeLa or HEK293 cells using Lipofectamine 2000. Following a period of recovery, selection for positive transfectants was initiated using geneticin over a period of 2-3 weeks. The pool of stable transfectants was expanded and banked in liquid nitrogen.
EXAMPLE 4
SNAP Conjugation and Purification of BG-Substrate and BG-Peptides and generation of BG-substrate Based Positive Control Reagents
[0401] This example describes conjugation and purification of BG-peptides and generation of BG-substrate positive control reagents. Peptides were produced and purified as described in EXAMPLE 1. For SNAP substrate conjugation, lyophilized peptides were resuspended in PBS to an approximate 1 mg/mL concentration. A benzylguanine SNAP substrate (BG-GLA-NHS, also referred to as BG-substrate) was attached to reactive amine groups on peptides viaNHS- ester reaction. Reactive amine groups in peptides may include the N-terminus and may include one or more lysine residues. FIG. 1A schematically illustrates the SNAP substrate tagging reaction between a benzylguanine NHS-ester and a reactive amine group of a peptide to generate a SNAP substrate-tagged peptide (also referred to as BG-peptide). Benzylguanine-NHS (BG- GLA-NHS) was obtained from New England Biolabs and resuspended at 10 pg/uL in anhydrous DMSO. One molar equivalent of BG-GLA-NHS per mole of peptide was added to the resuspended peptide and the reaction was allowed to proceed while stirring at room temperature for one hour. Samples of starting material and crude reaction mixture were analyzed by HPLC/MS to assess reaction progress. Additional BG-GLA-NHS was added as needed. Generally, for peptides with only one reactive amine group, a total of three molar equivalents of BG-GLA-NHS was added over the course of three hours. For peptides with multiple reactive amine groups, reactions were halted when the presence of multi-BG-species became apparent, with of goal of producing a reaction product that was primarily a single SNAP substrate tag per peptide. Additionally, if precipitation was noted, addition of BG-GLA-NHS was halted.
[0402] Crude reactions containing unreacted peptide, BG-peptide and hydrolyzed BG-GLA- NHS (hydrolyzed to BG-GLA-OH) were purified using size exclusion chromatography (SEC) using either a Superdex Peptide or Superdex 75 column, depending on reaction volume.
Following column equilibration in PBS, BG-peptides were separated and eluted over 1.5 column volumes. Fractions with notable 280 nm/214 nm/200 nm absorbance were pooled and concentrated through a 3 kDa molecular weight cut off spin filter. Individual BG-peptides eluted in distinct volumes, though they generally occurred around 1 column volume. BG-peptides were thus purified and separated from any residual BG-GLA-OH. BG-peptides were verified using HPLC/MS. BG-peptide concentration was then assessed using the bicinchoninic acid assay. [0403] Representative images of RP-HPLC and SEC associated with a typical SNAP substrate conjugation reaction and purification process are shown in FIG. 2. Fhe first image (top) shows multiple elution peaks from an HPLC run on the crude reaction mixture representing SNAP substrate BG-GLA-NHS, hydrolyzed BG-GLA-NHS (hydrolyzed to BG-GLA-OH), unconjugated KR CTX (SEQ ID NO: 71) and KR CTX conjugated to SNAP substrate (BG- KR CTX, BG-SEQ ID NO: 71), showing absorbance at 214 nm. Peaks were validated by mass calculated by mass spectrometry (MS). The crude reaction mixture was then run through a size exclusion column (SEC). The SEC absorbance trace is shown in the second image. Details of the individual SEC peaks are shown in the bottom images. Fractions were pooled and concentrated before verifying identity using HPLC/MS.
[0404] Hydrolyzed BG-GLA-NHS, which is BG-GLA-OH, was created by incubating BG- GLA-NHS in PBS, pH 7.4, at room temperature for at least 10 minutes. BG-GLA-OH was used in assays as a positive control.
[0405] BG-GLA-NHS contains a reactive NHS ester, which can react with amine-containing molecules and with hydroxyl-containing molecules. BG-GLA-NHS can react with amines present in peptides to form a BG-peptide conjugate. BG-GLA-NHS can also react with water, becoming hydrolyzed, yielding BG-GLA-OH, which no longer reacts with amine or hydroxyl groups. If BG-GLA-NHS is added to an aqueous buffer, it will typically hydrolyze to form BG- GLA-OH. If BG-GLA-NHS is added to an amine-containing peptide within an aqueous buffer, some of it may react to form BG -peptide and some of it may hydrolyze to form BG-GLA-OH.
EXAMPLE 5
SNAP Penetration Assay (SNAPPA) to Identify Cell-Penetrating Peptides
[0406] This example describes a SNAP penetration assay (SNAPPA) to identify cell-penetrating peptides. The SNAP penetration assay is illustrated schematically in FIG. IB. Cell-penetrating peptide candidates were labeled with a benzylguanine SNAP substrate as described in EXAMPLE 4. The BG-peptides were incubated with cells expressing a SNAP-tag-protein, which will covalently bind to BG-peptides that enter the cell and reach the cytosol or nucleus where the SNAP-tag-protein is expressed. Cells expressing the SNAP-tag-protein were generated as described in EXAMPLE 3. BG-peptides and controls were diluted in standard cell growth media (DMEM +10% FBS +pen/strep). FBS means fetal bovine serum. 2 pM hydrolyzed BG-GLA-NHS (hydrolyzed to BG-GLA-OH) was used as a positive control for cell penetration, while PBS served as a negative control. As a small molecule, BG-GLA-OH is able to diffuse into cells and reach the cytosol, thus representing a positive control of the level of SNAPPA signal obtained when a molecule is cell penetrant. Additionally, an additional mock conjugation/purification of BG-GLA-NHS also served as a quality control for purification. The mock reaction was performed by adding 1 molar equivalent (250 pM BG-GLA-NHS) to PBS and incubating at room temperature for 1 hour, which allows hydrolysis of BG-GLA-NHS. This was repeated until the concentration reached 750 pM. This mixture of PBS and BG-GLA-NHS (which is hydrolyzed to BG-GLA-OH) was then run on SEC and equivalent fractions where BG-peptides have been found were pooled and concentrated as would be for BG-peptide production. This served as a control to demonstrate adequate purification of free BG-GLA-OH. pSNAPf and pSNAPf-H2B cell lines were plated on collagen-coated 96-well plates, 20,000 cells/well, and allowed to recover in standard cell growth media overnight. Plates were rinsed with PBS and incubated with diluted BG-peptides and controls in standard cell growth media for 2 hours at 37° C. Peptides capable of cell penetration entered the cytosol and reached the SNAP- tag and then covalently bound to the SNAP-tag-protein expressed in the cells (FIG. IB, left path), while peptides that did not penetrate the cell did not bind to the SNAP-tag-protein (FIG. IB, right path).
[0407] The cells were then incubated with a BG-fluorophore (SNAP-Cell TMR-Star dye; or SNAP-dye) that, as a small molecule, diffuses across the cell membrane and can access the cytosol and nucleus. Cells were incubated with labelling media comprised of 600nM SNAP-Cell TMR-Star (BG-fluorophore), which labels unbound SNAP-tag-protein and is excited at 562 nm excitation, and 1 pg/mL Hoescht 33342, which labels nuclei and is excited at 377 nm excitation, in growth media for 15 minutes at 37° C Free SNAP-tag-protein in the cell bound to the BG- fluorophore. SNAP-tag-protein that was already bound to benzylguanine-peptides (BG-peptide) did not react with the BG-fluorophore. The labelling media was removed, and cells were incubated with fresh growth media for 30 minutes at 37° C as a washout phase for excess SNAP-Cell TMR-Star dye. Excess BG-fluorophore was washed away, and the remaining fluorescence, detected by exciting at 562 nm, was inversely related to the amount of peptide that penetrated the cell membrane. Molecules in cells with unbound SNAP-tag-protein exhibited fluorescence from the reaction between the SNAP-tag-protein and BG-fluorophore. Molecules in cells with SNAP-tag bound to BG-peptide did not exhibit fluorescence as the BG-fluorophore did not bind SNAP-tag-protein and was washed away. The media was replaced one last time with phenol red-free OptiMEM for imaging. Imaging occurred on the Molecular Devices ImageXpress Nano, utilizing DAPI and Texas Red filters. For scoring, cells were segmented using ImageXpress software to generate counts for nuclei and average cytoplasmic or nuclear intensity of SNAP-Cell TMR-Star dye. The average intensity was then normalized using the positive control of 2 pM hydrolyzed BG-GLA-NHS (BG-GLA-OH) as 100% cell penetration and PBS as 0% penetration to determine normalized SNAP-tag-protein occupancy.
[0408] Representative images of various BG-peptides comprising cystine-dense peptides after SNAPPA in HeLa cells stably transfected with pSNAPf captured on ImageXpress Nano are shown in FIG. 3. Eight BG-peptides comprising cystine-dense peptides that were tested include: KR_KTxl5.8 (BG-SEQ ID NO: 74), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG-SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59). Bright fluorescence was observed in the negative control sample (“PBS”) and very low fluorescence observed in the BG-GLA-OH positive control sample incubated with 2 pM BG- GLA-OH. The tested BG-peptides comprising cystine-dense peptides were all incubated at 10 pM with SNAP-tag-expressing cells for 2 hours. KR IpTxa (SEQ ID NO: 59) and KR HwTx- IV (SEQ ID NO: 66) demonstrated uptake comparable to that of the positive control, 2 pM BG- GLA-OH.
[0409] The cells exposed to PBS in the absence of a SNAP substrate-tagged peptide showed high fluorescence, indicating that the SNAP-tag was free and available to bind with a high level of BG-fluorophore, resulting in high fluorescence in the cells. The cells exposed to BG-GLA- OH showed low fluorescence, indicating that the SNAP-tag was bound by BG-GLA-OH, and thus did not bind the BG-fluorophore. The low level of fluorescence indicated that the majority of SNAP -tag was likely bound by BG-GLA-OH under these conditions, suggesting that they hydrolyzed SNAP substrate was capable of penetrating the cell membrane and substantially occupying the expressed SNAP -tag. To quantify the percent penetration for BG-peptides applied to cells, fluorescence level of the cells exposed to PBS was used for normalization (0% penetration) and cellular fluorescence levels of the cells exposed to BG-GLA-OH were used for normalization (100% penetration). Various BG-peptides comprising cystine-dense peptides were tested and fluorescence levels measured to assess whether they penetrated the cells. The cells exposed to various BG-peptides comprising cystine-dense peptides showed varying levels of fluorescence, indicating various levels of penetration by those peptides. BG-KR_IpTxa (BG- SEQ ID NO: 59) showed a higher level of cell penetration and BG-KR_KTxl5.8 (BG-SEQ ID NO: 74) showed a lower level of penetration, as shown in FIG. 3.
EXAMPLE 6
Assaying Cytosolic and Nuclear Access of Cell-Penetrating Peptides using SNAPPA [0410] This example describes assaying cytosolic and nuclear access of cell-penetrating peptides using SNAPPA. A SNAP penetration assay (SNAPPA) as described in EXAMPLE 5 and illustrated in FIG. 1 was used to measure SNAP -tag occupancy by various BG-peptides comprising cystine-dense peptides in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytosol or nucleus, as measured by fluorescence, where the higher relative occupancy is indicative of higher cell penetrance. SNAP -tag expressing cells, expressing either pSNAPf to quantify cytosolic access or pSNAPf-H2B to quantify nuclear access, were generated as described in EXAMPLE 3. Hydrolyzed SNAP reagent BG-GLA-OH was used as a positive control and set to 100% (1.0 fraction) penetration or protein occupancy. The cell penetrance of BG-peptides comprising cystine-dense peptides was normalized to the BG-GLA-OH positive control. The SNAPPA measured the relative percentage penetrance of various BG-peptides comprising cystine-dense peptides tested in the various cell lines
[0411] To quantify cytosolic access, BG-peptides comprising cystine-dense peptides were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously. Eight BG-peptides comprising cystine-dense peptides that were assayed for cytosolic penetration include: KR_KTxl5.8 (BG-SEQ ID NO: 74,), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG- SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59). Uptake was noticeably high for BG- MCa varl (n=4), BG-KR_HwTx-IV (n=3) and BG-KR_IpTxa (n=17) in all three cell lines, as shown in FIG. 4A. Uptake of BG-KR_Txl5.8 (n=3), BG-KR_KTx22 (n=3), BG-Tx677 (n=3), BG-KR CTI (n=3) and BG-KR CTX (n=l) occurred at lower moderate levels. The data indicated the different BG-peptides comprising cystine-dense peptides penetrated cells to reach the cytosol at various levels in the various cell lines, where the highest overall penetration levels in this assay graph were seen for the peptides KR IpTxa (SEQ ID NO: 59), KR_HwTx-IV (SEQ ID NO: 66), and MCa_varl (SEQ ID NO: 67), and various moderate levels of penetration were seen for KR CTX (SEQ ID NO: 71), KR CTI (SEQ ID NO: 72), Tx677 (SEQ ID NO: 93), KR_KTx2.2 (SEQ ID NO: 73), and KR_KTxl5.8 (SEQ ID NO: 74).
[0412] To quantify nuclear access, BG-peptides comprising cystine-dense peptides were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf-H2B, which encodes for nuclear expression of the SNAP -tag. Eight BG-peptides comprising cystine- dense peptides were assayed for nuclear penetration including: KR_KTxl5.8 (BG-SEQ ID NO: 74), KR_KTx2.2 (BG-SEQ ID NO: 73), Tx677 (BG-SEQ ID NO: 93), KR CTI (BG-SEQ ID NO: 72), KR CTX (BG-SEQ ID NO: 71), KR MCa (BG-SEQ ID NO: 67), KR_HwTx-IV, (BG-SEQ ID NO: 66), and KR IpTxa (BG-SEQ ID NO: 59). Uptake was noticeably high for BG-MCa_varl (SEQ ID NO: 67), BG-KR_HwTx-IV (SEQ ID NO: 66) and BG-KR_IpTxa (SEQ ID NO: 59) in all three cell lines, as shown in FIG. 4B. Uptake of BG-KR_Txl5.8 (BG- SEQ ID NO: 74), BG-KR_KTx2.2 (BG-SEQ ID NO: 73), BG-Tx677 (BG-SEQ ID NO: 93), BG-KR CTI (BG-SEQ ID NO: 72) and BG-KR CTX (BG-SEQ ID NO: 71) occurred at lower moderate levels. The data indicated the different BG-peptides comprising cystine-dense peptides penetrated cells to reach the nucleus at various levels in the various cell lines, where the highest overall penetration levels in this assay graph were seen for the peptides KR IpTxa (SEQ ID NO: 59), KR_HwTx-IV (SEQ ID NO: 66), and MCa_varl (SEQ ID NO: 67), and various moderate levels of penetration were seen for KR CTX (SEQ ID NO: 71), KR CTI (SEQ ID NO: 72), Tx677 (SEQ ID NO: 93), KR KTx22 (SEQ ID NO: 73), and KR KTxl5.8 (SEQ ID NO: 74).
EXAMPLE 7
Assaying Cytosolic and Nuclear Access of Cell-Penetrating Peptide Complexes [0413] This example describes assaying cytosolic and nuclear access of cell-penetrating peptide complexes using SNAPPA. Peptides identified using SNAPPA, as described in EXAMPLE 5, were conjugated by recombinant fusion to an additional cystine-dense peptide with lower cell penetration to determine if the cell-penetrating peptide could carry an additional peptide into the cytosol or nucleus. Cytosolic and nuclear access of the cell-penetrating peptide complexes was assayed by SNAPPA in three different SNAP -tag expressing cell lines. SNAP-tag expressing cells, expressing either pSNAPf to quantify cytosolic penetration or pSNAPf-H2B to quantify nuclear penetration, were generated as described in EXAMPLE 3.
[0414] To quantify cytosolic access, BG-peptides comprising cystine-dense peptide complexes were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP-tag ubiquitously. To quantify nuclear access, BG-peptides comprising cystine-dense peptide complexes were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf-H2B and expressing the SNAP-tag in the nucleus. Cystine-dense peptide complexes were constructed by linking a cell-penetrating cystine-dense peptide to an additional cystine-dense peptide with lower cell penetration using a (GGGS)3 linker (GGGSGGGSGGGS, SEQ ID NO: 255). Cell-penetrating peptides MCa (SEQ ID NO: 197) and KR IpTxa (SEQ ID NO: 1, corresponding to SEQ ID NO: 59 without an N- terminal GS) were tested, and low-cell-penetrating cystine-dense peptides KTx3.10 (AQEPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ; SEQ ID NO: 294, corresponding to SEQ ID NO: 296 without an N-terminal GS) and elafin (GVPINVKCRGSRDCLDPCKKAGMRFGKCINSKCHCTP; SEQ ID NO: 293, corresponding to SEQ ID NO: 297 without an N-terminal GS) were conjugated by recombinant fusion to the cell-penetrating peptides, and tagged with SNAP substrate as described in EXAMPLE 4. The peptide complexes that were tested in this assay are show in FIG. 5.
[0415] An assay to measure cytosolic access of cell-penetrating peptide complexes is shown in FIG. 6A. As a comparator to assess the basal level of penetration, uptake of BG-cargo peptides was measured in the absence of conjugation to a cell-penetrating peptide. BG-KTx3.10 (GSGVPINVKCRGSRDCLDPCKKAGMRFGKCINSKCHCTP; SEQ ID NO: 296, n=6) and BG-elafm (GSAQEPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ; SEQ ID NO: 297, n=6) showed lower levels of cytosolic access. Peptide complexes incorporating the MCa cell-penetrating peptide, BG-MCa-KTx3.10 (BG-SEQ ID NO: 312, n=4) and BG-MCa-elafin (BG-SEQ ID NO: 316, n=4), showed significantly increased uptake into the cytoplasm as compared to the cargo peptides alone. Peptide complexes incorporating the KR IpTxa cell-penetrating peptide, BG-KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313, n=3) and BG-KR IpTxa-elafm (BG-SEQ ID NO: 317, n=3) also showed high levels of cytosolic access as compared to the cargo peptides alone. BG-KR IpTxa (BG-SEQ ID NO: 59) was tested as a comparator to assess the level of penetration of the cell-penetrating peptide in the absence of conjugation to a cargo peptide. BG-KR_IpTxa (BG-SEQ ID NO: 59) showed comparable high cytosolic access as the positive control BG-GLA-OH, to which all data was normalized The data showed that conjugating MCa or KR_IpTxa to KTx3.10 or to elafin greatly increased the level of KTx3.10 or elafin that reached the cytosol of each of the three different cell lines The data also showed that KR_IpTxa reached the cytosol at levels comparable to the cell penetrant small molecule BG-GLA-OH.
[0416] The same BG-peptide complexes comprising cell-penetrating peptide were tested at 10 pM, 3 pM, and 1 pM using SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously. Titration of the BG-peptide complexes cell-penetrating peptide demonstrated that peptide complexes incorporating the IpTxa cellpenetrating peptide (FIG. 7D and FIG. 7B) and the peptide complexes incorporating the MCa cell-penetrating peptide (FIG. 7C and FIG. 7A) were capable of delivering SNAP substrate- peptides comprising cargo peptides to all 3 cell lines at varying concentrations, though the level delivered varied with concentration, cell line, and cell-penetrating peptide. The levels are normalized to cell exposed to 10 pM BG-GLA-OH, but the relative level of peptide penetrating versus the level of SNAP tag in the cell or versus the level of peptide applied to the cell is not determined, thus the data provides a qualitative comparison between different peptide constructs. At lower concentrations in some cell lines, the peptide complexes incorporating the IpTxa cell-penetrating peptide (SEQ ID NO: 1) exhibited higher cytosolic delivery than the peptide complexes incorporating the MCa cell-penetrating peptide (SEQ ID NO: 197). Together, the data demonstrate a dose-dependent and cell-type dependent effect of the various peptide complexes on SNAP tag occupancy.
[0417] An assay to measure nuclear access of cell-penetrating peptide complexes is shown in FIG. 6B. As a comparator to assess the basal level of penetration of the lower penetrating peptides, uptake of BG-cargo peptides was measured in the absence of conjugation to a cellpenetrating peptide. BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) showed lower levels of nuclear access. Peptide complexes incorporating the MCa cellpenetrating peptide, BG-MCa-KTx3.10 (BG-SEQ ID NO: 312) and BG-MCa-elafin (BG-SEQ ID NO: 316), showed significantly increased uptake into the nucleus as compared to the lower penetrating peptides alone. Peptide complexes incorporating the KR IpTxa cell-penetrating peptide (SEQ ID NO: 1), BG-KR_IpTxa-KTx3.10 (BG-SEQ ID NO: 313) and BG-KR_IpTxa- elafin (BG-SEQ ID NO: 317) also showed higher levels of nuclear access as compared to the lower penetrating peptides alone. BG-KR IpTxa (BG-SEQ ID NO: 59) was tested as a comparator to assess the level of penetration of the cell-penetrating peptide in the absence of conjugation to a cargo peptide. BG-KR_IpTxa (BG-SEQ ID NO: 59) showed comparable high cytosolic access as the positive control BG-GLA-OH, to which all data was normalized. The data showed that conjugating MCa (SEQ ID NO: 212) or KR IpTxa (SEQ ID NO: 59) to KTx3.10 (SEQ ID NO: 293) or to elafin (SEQ ID NO: 294) greatly increased the level of KTx3.10 or elafin that reached the nucleus of each of the three different cell lines. The data also showed that KR IpTxa (SEQ ID NO: 59) reached the nucleus at levels comparable to the cell penetrant small molecule BG-GLA-OH.
[0418] The same BG-peptide complexes comprising cell-penetrating peptide were tested at 10 pM, 3 pM, and 1 pM using SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf-H2B and expressing the SNAP -tag in the nucleus. Again, both peptide complexes incorporating the IpTxa cell-penetrating peptide (SEQ ID NO: 1, FIG. 8D and FIG. 8B) and peptide complexes incorporating the MCa cell-penetrating peptide (SEQ ID NO: 197, FIG. 8C and FIG. 8A) were capable of delivering cargo peptides to all 3 cell lines at varying concentrations, though the level delivered varied with concentration, cell line, and cellpenetrating peptide. The levels are normalized to cell exposed to 10 pM BG-GLA-OH, but the relative level of peptide penetrating versus the level of SNAP -tag in the cell or versus the level of peptide applied to the cell is not determined, thus the data provides a qualitative comparison between different peptide constructs. At some lower concentrations in some cell lines, the peptide complexes incorporating the IpTxa cell-penetrating peptide (SEQ ID NO: 1) exhibited higher cytosolic delivery than the peptide complexes incorporating the MCa cell-penetrating peptide (SEQ ID NO: 197), but in other cell lines or concentrations, the peptides conjugates incorporating the MCa cell-penetrating peptide (SEQ ID NO: 197) showed comparable or higher levels of deliver than those incorporating the IpTxa cell-penetrating peptide (SEQ ID NO: 1). [0419] Additional cell-penetrating peptide peptides to deliver KTx3.10 (SEQ ID NO: 293) and elafin (SEQ ID NO: 294) to the cytoplasm or nucleus were tested by SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf or pSNAPf-H2B, respectively. One alternative strategy included protein engineering an existing loop from a MCa peptide into KTx3.10 (SEQ ID NO: 293) via recombinant fusion to generate cell-penetrating peptide fusions such as the following SNAP substrate-peptides: BG-MCa(loop)-KTx3.10 (BG-SEQ ID NO: 314), and to elafin (SEQ ID NO: 294) to generate: BG-MCa(loop)-elafin (BG-SEQ ID NO: 318). A second alternative strategy included fusion with a C3A_MCa(l-9) (SEQ ID NO: 70) short cell-penetrating peptide to generate cell-penetrating peptide fusions: BG-C3A_MCa(l-9)- KTx3.10 (BG-SEQ ID NO: 315) and BG-C3A MCa(l-9)-elafin (BG-SEQ ID NO: 319). The peptide complexes that were tested in this assay are show in FIG. 9. [0420] FIG. 10A shows results of a SNAP penetration assay to measure cytosolic access of the cell-penetrating peptide complexes illustrated in FIG. 9 in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the cytoplasm Cytoplasmic access of cell-penetrating peptide complexes BG-MCa(loop)-KTx3.10 (BG-SEQ ID NO: 314), BG-MCa(loop)-elafin (BG-SEQ ID NO: 318), BG-C3A_MCa(l-9)-KTx3.10 (BG-SEQ ID NO: 315), and BG-C3A_MCa(l-9)- elafm (BG-SEQ ID NO: 319) was measured. BG-cargo peptides without additional cellpenetrating peptides BG-KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration. Data was normalized to positive control BG-GLA-OH. The level of cargo peptide delivered to the cytosol varied with different cell lines, species of cargo peptide, and species of cell-penetrating peptide.
[0421] FIG. 10B shows results of a SNAP penetration assay to measure nuclear access of the cell-penetrating peptide complexes illustrated in FIG. 9 in NIH3T3, HeLa and HEK293 cells expressing SNAP -tag in the nucleus. Nuclear access of cell-penetrating peptide complexes BG- MCa(loop)-KTx3.10 (BG-SEQ ID NO: 314), BG-MCa(loop)-elafin (BG-SEQ ID NO: 318), BG-C3A_MCa(l-9)-KTx3.10 (BG-SEQ ID NO: 315), and BG-C3A_MCa(l-9)-elafin (BG-SEQ ID NO: 319) was measured. BG-cargo peptides without additional cell-penetrating peptides BG- KTx3.10 (BG-SEQ ID NO: 296) and BG-elafin (BG-SEQ ID NO: 297) were used as comparators to assess the basal level of penetration. The level of cargo peptide delivered to the cytosol varied with different cell lines, species of cargo peptide, and species of cell-penetrating peptide.
EXAMPLE 8
Cell Penetration of TEAD-Binding Cystine-Dense Peptide Complexes [0422] This example describes cell penetration of TEAD-binding cystine-dense peptide complexes. A TEAD-binding cystine-dense peptide (PDEYIERAKECCKKQDIQCCLRIFDESKDPNVMLICLFCW; SEQ ID NO: 295, corresponding to SEQ ID NO: 298 without an N-terminal GS) was conjugated to various cellpenetrating peptides to determine whether the cell-penetrating peptides were capable of facilitating cytosolic or nuclear localization of the TEAD-binding peptides. The tested peptide complexes are illustrated in FIG. 11.
[0423] BG-peptides comprising cell-penetrating cystine-dense peptide fusions containing the TEAD-binding peptide were tested using the SNAPPA in NIH3T3, HeLa and HEK293 cells. Cells were stably transfected with pSNAPf expressing the SNAP-tag ubiquitously to test cytosolic access. Cell-penetrating cystine-dense peptide fusions were constructed by linking a short N-terminal sequence derived from either MCa (C3A_MCa(l-9), SEQ ID NO: 210) or Had (C5A_Had(l-l 1), SEQ ID NO: 82) and fused to the N-terminus of the TEAD-binding peptide (GSPDEYIERAKECCKKQDIQCCLRIFDESKDPNVMLICLFCW; SEQ ID NO: 298) to generate a C3A_MCa(l-9)-TEAD-binder fusion (SEQ ID NO: 320) or a C5A_Had(l-l 1)- TEAD-binder fusion (SEQ ID NO: 321). Peptide fusions were labeled with SNAP substrate as described in EXAMPLE 4. The TEAD-binding cystine-dense peptide without an additional cell-penetrating peptide tag was tested as a comparator to assess the basal level of penetration. Cytosolic access of BG-TEAD-binder (BG-SEQ ID NO: 298) was low (n=8), but cytosolic access of BG-C3A_MCa(l-9)-TEAD-binder (BG-SEQ ID NO: 320, n=4) and BG-C5A_Had(l- 1 l)-TEAD-binder (BG-SEQ ID NO: 321, n=3) was significantly increased relative to the TEAD-binding cystine-dense peptide without a cell-penetrating peptide, as shown in FIG. 12A. Penetration of BG-KR IPTxa (BG-SEQ ID NO: 59) was also tested and BG-GLA-OH was included as a positive control for normalization. The data showed that conjugating C3A_MCa(l- 9) (SEQ ID NO: 195) or C5A_Had(l-l 1) (SEQ ID NO: 41) to the TEAD-binding peptide greatly increased the level of the TEAD-binding peptide that reaches the cytosol of each of three different cell lines.
[0424] The same BG-peptides complexes comprising cell-penetrating cystine-dense peptide fusions containing the TEAD-binding peptide were tested as with cytosolic access, but in cells stably transfected with pSNAPf-H2B expressing the SNAP -tag in the nucleus to test nuclear access. Nuclear access of BG-TEAD-binder (BG-SEQ ID NO: 298) was low, but nuclear access of BG-C3A_MCa(l-9)-TEAD-binder (BG-SEQ ID NO: 320) and BG-C5A_Had(l-l 1)-TEAD- binder (BG-SEQ ID NO: 321) was significantly increased relative to the TEAD-binding cystine- dense peptide without a cell-penetrating peptide, as shown in FIG. 12B. Penetration of the BG- peptides comprising BG-KR_IPTxa (BG-SEQ ID NO: 59) was also tested and BG-GLA-OH was included as a positive control for normalization. The data showed that conjugating C3A MCa(l-9) (SEQ ID NO: 195) or C5A Had(l-11) (SEQ ID NO: 41) to the TEAD-binding peptide greatly increased the level of the TEAD-binding peptide that reached the nucleus of each of three different cell lines. The data also showed that KR IpTxa (SEQ ID NO: 59) reached the nucleus at levels comparable to the cell penetrant small molecule BG-GLA-OH.
[0425] In another assay, nuclear access of the same BG-peptides fusions comprising a TEAD- binding cystine-dense peptide shown in FIG. 11 was tested in various primary glioma stem cells (GSC) transiently transfected with pSNAPf-H2B and expressing the SNAP -tag in the nucleus. Significant increase in nuclear access of the BG-peptide comprising a TEAD-binding cystine- dense peptide was observed in all four cell lines when fused to C3A_MCa(l-9) (SEQ ID NO: 320, n=l) or C5A_Had(l-l 1) (SEQ ID NO: 321, n=l) compared to the TEAD-binding cystine- dense peptide without a cell-penetrating peptide tag (SEQ ID NO: 298), as shown in FIG. 13 following a 2 hour incubation period “Mock” denotes a mock conjugation and purification. Nuclear uptake continued to increase after 5 hours as compared to the nuclear uptake after 2 hours. The data showed that conjugating C3A_MCa(l-9) (SEQ ID NO: 195) or C5A_Had(l-l 1) (SEQ ID NO: 41) to the TEAD-binding peptide greatly increased the level of TEAD-binding peptide that reached the nucleus of four different glioma cell lines.
EXAMPLE 9
Cell Penetration of Cystine-Dense Peptides and Peptide Fragments
[0426] This example describes cell penetration of cystine-dense peptides and peptide fragments. The ability of various cell-penetrating cystine-dense peptides and cell-penetrating peptide fragments to enter cells, alone or with cargo cystine-dense peptides was measured as described in EXAMPLE 5 - EXAMPLE 8. Peptides were qualitatively ranked based on their ability to access a cell cytoplasm, nucleus, or both, as shown in TABLE 14. “+++” indicates strong cell uptake, and
Figure imgf000196_0001
denote decreasing cell uptake, as measured by SNAPP A, though results varied with different cell lines, cargo peptides, and other conditions.
TABLE 14 - Qualitative Relative Cell Penetration Capability of Cystine-Dense Peptides and Peptide Fragments
Figure imgf000196_0002
[0427] The cell-penetrating cystine-dense peptide, KR IpTxa (SEQ ID NO: 1, corresponding to SEQ ID NO: 59 with no N-terminal GS), showed consistently strong uptake into a variety of cells, both into the cytosol and the nucleus, and the short cell-penetrating peptide tags, C3A_MCa(l-9) (SEQ ID NO: 195, corresponding to SEQ ID NO: 210 with no N-terminal GS) and C5A_Had(l-l 1) (SEQ ID NO: 198, corresponding to SEQ ID NO: 213 with no N-terminal GS), were also very capable of delivering a payload into a variety of cell, both into the cytosol and the nucleus. The cell-penetrating cystine-dense peptides KR_HwTx-IV (SEQ ID NO: 8, corresponding to SEQ ID NO: 66 with no N-terminal GS), MCa_varl (SEQ ID NO: 18, corresponding to SEQ ID NO: 67 with no N-terminal GS), and MCa (SEQ ID NO: 212, corresponding to SEQ ID NO: 197 with no N-terminal GS) were also able to access the cytosolic and nuclear compartment of cells at significant levels. Some cystine-dense peptides exhibit lower levels of cellular uptake from the media, and some appeared have little to no access in certain cell lines.
EXAMPLE 10
Endocytotic Profiling via SNAP Penetration Assay
[0428] This example describes endocytotic profiling via SNAP penetration assay. pSNAPf cells were preincubated with a variety of endocytosis inhibitors to assess mechanisms responsible for uptake and cytosolic access of various BG-peptides. Macropinocytosis was inhibited using 50 pM ethylisopropyl amiloride (EIP A), which may block Na+/H+ exchangers and prevents formation of macropinosomes. Clathrin-mediated endocytosis was inhibited using either 20 pM nocodazole, which may inhibit microtubule polymerization, 3 pM cytochalasin D, which can interfere with actin polymerization, or 80 pM dynasore, which can inhibit dynamin GTPase. Endosomal acidification or lysosomal maturation can be inhibited using 50 nM bafilomycin A, which can inhibit the vacuolar ATPase complex, or 50 pM chloroquine, which can diffuse into endosomes as a weak base. pSNAPf cells were preincubated with the specified concentration of inhibitor in growth media for 1 hour prior to SNAPP A, which was conducted as described in EXAMPLE 5
[0429] To identify the dominant mechanisms of cellular uptake of these cell -penetrating peptides, cytosolic access of BG-peptides comprising KR IpTxa (SEQ ID NO: 59) in measured in pSNAPf cells preincubated with various chemicals inhibiting different mechanisms of endocytosis. Inhibition of macropinocytosis with 50 pM EIPA significantly decreased cellular uptake of BG-KR_IpTxa in all three cell lines, suggesting that micropinocytosis may be a primary driver for uptake of this cell-penetrating peptide, as shown in FIG. 14A. The data showed that the addition of 50 pM EIPA greatly reduced the level of cell penetration of KR IpTxa in all three cell lines, and that other inhibitors had some effect or no effect on uptake in different cell lines. Other more minor or unique effects were seen, such as a reduction of uptake in 293 cells by bafilomycin A, and potentially dynasore or chloroquine, and a potential reduction of uptake in 3T3 cells by nocodazole, indicating that additional mechanisms may be factors for certain cell lines.
[0430] To identify the dominant mechanisms of cellular uptake of these cell-penetrating peptides, cytosolic access of BG-peptides comprising MCa_varl (SEQ ID NO: 67) was measured in pSNAPf cells preincubated with various chemicals inhibiting different mechanisms of endocytosis. Similar to the BG-peptide comprising cell-penetrating peptide BG-KR_IpTxa, cellular uptake of the BG-peptide comprising cell-penetrating peptide BG-MCa_varl was significantly decreased upon inhibition of macropinocytosis with 50 pM EIPA in HeLa cells, though only moderately with 3T3 cells or 293 cells. Additionally, inhibition of lysosomal maturation with 50 nM bafilomycin A or 50 uM chloroquine also partially blocked uptake in all cell lines, implying that BG-KR_MCa_varl may be trafficked through endosomal/lysosomal maturation pathways. Uptake was also partially inhibited upon inhibition of clathrin-mediated endocytosis in some cell lines, in some cases with 20 pM nocodazole, 3 pM cytochalasin D, or 80 pM dynasore, as shown in FIG. 14B. The data showed that the addition of various inhibitors had some effect or no effect on uptake in different cell lines. These data indicate that various uptake and trafficking mechanisms may be important for cell-penetrating peptides of this disclose, which may vary to some extend between peptides and between cell lines.
EXAMPLE 11
Promoting Endosomal Escape of Cell-Penetrating Peptide Fusions
[0431] This example describes the potential for using additional endosomal escape peptides to further promote cell-penetrating peptides for promoting endosomal escape of cell-penetrating peptide fusions. Peptides S19 (PFVIGAGVLGALGTGIGGI; SEQ ID NO: 359), CM18 (KWKLFKKIGAVLKVLTTG; SEQ ID NO: 360), PAS (FFLIPKG; SEQ ID NO: 361), Aureinl.2 (GLFDIIKKIAESF; SEQ ID NO: 362), and B18 (LGLLLRHLRHHSNLLANI; SEQ ID NO: 363) purported to promote endosomal escape were conjugated by fusion to KR IpTxa (SEQ ID NO: 1, corresponding to SEQ ID NO: 59 without an N-terminal GS) in order to potentially further promote delivery of peptide to the cytosol, and then labeled with SNAP substrate as described in EXAMPLE 4. BG-KR_IpTxa-PAS (BG-SEQ ID NO: 322) and BG- KR_IpTxa-S19 (BG-SEQ ID NO: 323) were tested using SNAPPA in NIH3T3, HeLa and HEK293 cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously. The addition of the PAS and S19 sequences did not negatively affect cellular uptake and delivery to the cytosol, as shown in FIG. 15A, as all BG-peptide constructs showed high delivery including BG-peptides comprising KR IpTxa (SEQ ID NO: 59), KR_IpTxA-PAS (SEQ ID NO: 322), and KR_IpTxa-S19 (SEQ ID NO: 323) and as compared to the normalizing positive control BG- GLA-OH. The data showed that KR IpTxa without or with the addition of PAS or S19 had high levels of cell penetration in all cell lines tested.
[0432] The same SNAP substrate-peptides comprising BG-KR_IpTxa-PAS (BG-SEQ ID NO: 322) and BG-KR_IpTxa-S19 (BG-SEQ ID NO: 323) endosomal escape fusions were tested but in cells stably transfected with pSNAPf-H2B, limiting expression of the SNAP-tag to the nucleus. The same effect was observed, with the additional sequence not affecting cell penetration, and all SNAP substrate peptides comprising KR_IpTxa (SEQ ID NO: 59), KR_IpTxA-PAS (SEQ ID NO: 322), and KR_IpTxa-S19 (SEQ ID NO: 323) showing high levels of cell penetration and nuclear delivery as compared to the normalizing positive control BG-GLA-OH, as shown in FIG. 15B. The data showed that KR IpTxa (SEQ ID NO: 1) without or with the addition of PAS or S19 had high levels of nuclear penetration in all cell lines tested.
EXAMPLE 12
Peptide Cargos for Cellular Delivery
[0433] This example describes peptide cargos that are delivered to cells using cell-penetrating cystine-dense peptides or peptide fragments. FIG. 16A and FIG. 16B illustrates peptide cargos that can be conjugated, such as by chemical conjugation or recombinant fusion, to a cellpenetrating cystine-dense peptide or peptide fragment of the present disclosure. A peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 1 4, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is conjugated to, linked to, or fused to a peptide cargo and expressed recombinantly or chemically synthesized as a cell-penetrating peptide complex. The peptide cargo is conjugated to, linked to, or fused to the N-terminus or the C-terminus of the cell-penetrating cystine-dense peptide or peptide fragment. The peptide cargo and the cell-penetrating cystine-dense peptide or peptide fragment are connected by a linker of any one of SEQ ID NO: 225 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485, or any other linker, or no linker. The peptide cargo is a cystine-dense peptide, an affibody, a 13-hairpin, an avimer, an adnectin, a stapled peptide, a nannofittin, a kunitz domain, a fynomer, or a bicyclic peptide, as shown in FIG. 16A. Sometimes, the peptide is a nanobody, an antibody scFc fragment, or an antibody FAb fragment, as shown in FIG. 16B. One or more cell-penetrating peptides of this disclosure may be conjugated to any of these cargos. One or more of these cargos may be conjugates to a cell-penetrating peptide of this disclosure.
EXAMPLE 13
Peptide Radiolabeling
[0434] This example describes the radiolabeling of peptides. Peptides are radiolabeled by reductive methylation with 14C formaldehyde and sodium cyanoborohydride with standard techniques (such as those described in Jentoft et al. J Biol Chem. 254(11):4359-65. 1979). The sequences are engineered to have the amino acids, “G” and “S” at the N terminus. See Methods in Enzymology V9E 1983 p.570 and JBC 254(11): 1979 p. 4359. An excess of formaldehyde was used to drive complete methylation (dimethylation of every free amine). The labeled peptides are isolated via solid-phase extraction on Strata-X columns (Phenomenex 8B-S100-AAK), rinsed with water with 5% methanol, and recovered in methanol with 2% formic acid. Solvent is subsequently removed in a blowdown evaporator with gentle heat and a stream of nitrogen gas.
EXAMPLE 14
Peptide Detectable Agent Conjugates
[0435] This example describes the dye labeling of peptides. A peptide of the disclosure is expressed recombinantly or chemically synthesized, and then the N-terminus of the peptide is conjugated to, linked to, or fused to a detectable agent via an NHS ester using DCC or EDC to produce a peptide-detectable agent conjugate. The detectable agent is the fluorophore dye is a cyanine dye, such as Cy5.5 or an Alexa fluorophore, such as Alexa647.
EXAMPLE 15 Peptide Active Agent Conjugates
[0436] This example describes the conjugation of peptides to active agents. A cell-penetrating peptide of the disclosure is expressed recombinantly or chemically synthesized, and then the N- terminus of the peptide is conjugated to, linked to, or fused to an active agent via an NHS ester using DCC or EDC to produce a peptide-active agent conjugate Alternatively, the C-terminus of the peptide, or an internal residue of the peptide such as a Lys residue is used to conjugate to the active agent. Alternatively, a cell-penetrating peptide is conjugated to, linked to, or fused to a peptide active agent and expressed recombinantly or chemically synthesized as a peptide-active agent peptide complex. The active agent is a small molecule drug such as a chemotherapeutic agent or a biologic agent such as a peptide therapeutic agent. A linker may or may not be included between the cell-penetrating peptide of this disclosure and the active agent. The linker may be cleavable or stable.
[0437] The peptide active agent conjugates are administered to a subject. The subject can be a human or a non-human animal. After administration, the peptide active agent conjugates penetrate cells and localize to the cytoplasm, the nucleus, other subcellular organelles, or combinations thereof, thereby treating the subject. A therapeutic level of the active agent reaches the cytosol of the cell, thereby having a therapeutic effect on the subject’s disease. Optionally, the subject’s disease is cancer, neurodegeneration, or inflammatory /immune disease.
EXAMPLE 16
Extension of Peptide Serum Half-Life
[0438] This example describes a method of extending the serum half-life of a peptide as disclosed herein. A peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is modified in order to increase its serum half-life. Conjugation of the peptide to a near infrared dye, such as Cy5.5 is used to extend serum half-life of the peptide complex. Alternatively, conjugation of the peptide to an albumin, an albumin binder, such as Albu-tag, or a fatty acid, such as palmitic acid or S21, is used to extend serum half-life. Alternatively, conjugation or fusion of the peptide to an Fc domain or to PEG is used to extend the serum half-life.
Optionally, serum half-life is extended as a result of reduced immunogenicity by using minimal non-human protein sequences.
EXAMPLE 17
High-Throughput Method for Screening Cytosolic or Nuclear Access of Peptides [0439] This example describes a high-throughput method for screening cytosolic and nuclear access of peptides, including novel CDPs. Cells expressing a GFP -labeled SNAP -tag in either the cytoplasm or the nucleus are cultured in a microplate format. Candidate cell -penetrating peptides are tagged with SNAP substrate and purified using the method described in EXAMPLE 4. Each BG-candidate peptide is added to the cells expressing GFP-labeled SNAP- tag in a well of the microplate. Peptides capable of cell penetration cross the cell membrane and covalently bind to the GFP-labeled SNAP-tag expressed in the cytoplasm or the nucleus. Following incubation, excess BG-candidate peptides that did not enter the cells are washed off. Cells are pooled and lysed, and GFP-labeled SNAP-tag are purified by GFP immunoprecipitation. Candidate peptides that entered the cell are immunoprecipitated with the SNAP-tag. Cell-penetrating peptides are identified by mass spectrometry. EXAMPLE 18
Delivery of an Anti-Cancer Agent to Intercellular Nanolumen of Cancer Cells [0440] This example describes delivery of an anti-cancer agent to intercellular nanolumen. A cell-penetrating peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is conjugated to an anti-cancer active agent. The peptide-active agent conjugate is administered to a subject having cancer. The peptide-active agent conjugate penetrates a cellular layer (cell membrane, nuclear envelope, intercellular space, paracellular space, endosomal membrane, lysosomal membrane, other subcellular compartment membrane, blood brain barrier, or nanolumen) of cancer cells and enters the intercellular nanolumen, delivering the active agent to the nanolumen of the cancer cells. Upon delivery to the nanolumen, the anti -cancer agent may selectively kill a cancer cell, thereby treating the cancer.
EXAMPLE 19
Delivery of CRISPR Components to a Target Nucleus
[0441] This example describes delivery of CRISPR components to a target nucleus using cellpenetrating peptides. A cell-penetrating peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195
- SEQ ID NO: 254 is conjugated to a guide nucleic acid (gRNA) directed to a target nucleic acid sequence. A second cell-penetrating peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195
- SEQ ID NO: 254 is conjugated to a Cas9 nuclease. The peptide-gRNA conjugate and the peptide-Cas9 nuclease conjugate are administered to a subject in need thereof. The peptide- gRNA conjugate and the peptide-Cas9 nuclease conjugate enter the nucleus of a target cell in the subject and edit a target nucleic acid sequence in the target cell.
EXAMPLE 20
Delivery of a Transcription Factor Inhibitor to a Target Cell
[0442] This example describes delivery of a transcription factor inhibitor or binder to a target cell using a cell-penetrating peptide. A cell-penetrating peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is conjugated to an inhibitor of a Forkhead family (FOXO) transcription factor. The peptide-transcription factor inhibitor conjugate is administered to a subject having cancer. The peptide-transcription factor inhibitor conjugate penetrates the cell membrane and the nuclear envelope and enters the cell nucleus. The FOXO transcription factor inhibitor, inhibits FOXO and activates genes promoting apoptosis, DNA repair, and cell cycle arrest, thereby treating the cancer.
EXAMPLE 21
Delivery of a Tau-binding Cystine-Dense Peptide Across the Blood Brain Barrier to Treat Alzheimer’s Disease
[0443] This example describes delivery of a Tau-binding cystine-dense peptide conjugated to a cell-penetrating peptide across the blood brain barrier (BBB) to treat Alzheimer’s disease. A cell-penetrating peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is conjugated to a cystine-dense peptide that binds to Tau (Tau-binding CDP). The peptide-Tau- binding CDP conjugate is administered to a subject showing signs of Alzheimer’s disease. The peptide-Tau-binding CDP conjugate penetrates the blood brain barrier and reaches the brain. The Tau-binding CDP binds to Tau and disrupts neurofibrillary tangles formed from aggregated Tau protein, thereby treating the Alzheimer’s disease.
EXAMPLE 22
Cell-Penetrating Peptide Variants
[0444] This example illustrates peptide variants that retain cell-penetrating properties, have increases cell-penetrating properties, are shorter or longer, or have increased stability or decreased immunogenicity.
[0445] Multiple peptides with cell-penetrating capabilities were used to identify variants that also have cell-penetrating capabilities. The peptides in TABLE 15 were identified as additional cell-penetrating peptides.
TABLE 15 - Exemplary Cell-Penetrating Peptide Variants
Figure imgf000203_0001
Figure imgf000204_0001
Figure imgf000205_0001
[0446] FIG. 17 illustrates a multiple sequence alignment of MCa_varl (SEQ ID NO: 67), KR Urocalcin (SEQ ID NO: 39), KR IpTxa (SEQ ID NO: 59), KR Hemicalcin (SEQ ID NO: 34), KR_Opicalcin-l (SEQ ID NO: 37), KR_Opicalcin-2 (SEQ ID NO: 38), KR Vejocalcin (SEQ ID NO: 35), and KR Intrepi calcin (SEQ ID NO: 36). The alignment identified permissive or preferred amino acids at a given location and provided a guide for discovery of novel peptide variants that could be generated and that could retain essential properties such as structure, function, peptide folding, biodistribution, or stability. Furthermore, based on the ability to substitute K residues with R residues and R residues with K residues, additional permissive amino acid variants were identified. The multiple sequence alignment identified peptides of the family of sequences of SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 22 - SEQ ID NO: 29, SEQ ID NO: 44 - SEQ ID NO: 58 as potential peptide variants that could be generated and that could retain essential properties such as structure, function, peptide folding, biodistribution, or stability. Other conserved regions within sequences of the present disclosure can be any one of SEQ ID NO: 4 - SEQ ID NO: 7, SEQ ID NO: 9 - SEQ ID NO: 17, SEQ ID NO: 21 - SEQ ID NO: 29, SEQ ID NO: 41 - SEQ ID NO: 58, SEQ ID NO: 62 - SEQ ID NO: 65, SEQ ID NO: 70, SEQ ID NO: 82 - SEQ ID NO: 84, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 214, or SEQ ID NO: 216. EXAMPLE 23
Peptide Immunogenicity and Design for Reduced Immunogenicity
[0447] This example illustrates the testing of the immunogenicity of a peptide and design of peptides for reduced immunogenic potential. NetMHC II version 2.3 prediction software was used to predict peptides more likely to be immunogenic based on a neural network alignment algorithm that predicts peptide binding to MHC Class II molecules.
[0448] The NetMHC II prediction software was utilized to determine the putative peptide binding capability to DR, DQ, and DP MHC II alleles and the strength of the interaction between peptide and MHC II molecules. TABLE 16 shows the resulting immunogenicity score of some select peptides. The numbers of strong versus weak peptides are tallied into each major MHC allele group (DR, DQ, and DP). Additionally, the numbers of ‘unique strong’ and ‘unique weak core’ peptides are also tallied. This data was used to predict which peptides are less likely to induce an immunogenic response in patients. For example, the stronger a peptide binds to an allele, the more likely it is to be presented in a MHC/peptide combination on an antigen presenting cell, thus triggering an immune response, and a peptide that is predicted to bind to fewer alleles is more likely to have weaker binding to given alleles and should be less immunogenic. The multiple sequence alignment technique from Example 22 was used to design peptide variants and the variants were also tested for immunogenic potential using NetMHC II prediction software, and peptide variants that were predicted to be less immunogenic were identified. Fusions and linkers between fused moieties can also be tested and designed using the same methods.
TABLE 16 - Exemplary Peptides and Immunogenicity Scores of Peptides
Figure imgf000206_0001
Figure imgf000207_0001
EXAMPLE 24
Immunogenicity Testing of Peptides
[0449] This example illustrates immunogenicity testing of peptides, including a peptide of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254
[0450] Dendritic cells and/or CD4+ T cells and/or peripheral blood mononuclear cells are isolated from healthy or diseased, such as affected by rheumatoid arthritis, autoimmune disease, or cancer, human donors. Multiple donors are tested. Any peptide or SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is applied in separate wells to the cells. The cells are assessed for proliferation and for secretion of cytokines such as IL-2 or IFN-gamma. Control peptides, such as those known to be immunogenic, non-immunogenic, or of known immunogenic potential are also incubated with the cells. Peptides that are less immunogenic by inducing less cell activation are identified.
[0451] Separately, one or more peptides of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 are injected into a mouse or rat or non-human primate one to four times, intravenously or subcutaneously. The serum from the animal is collected at various time points and tested for the presence of antibodies that bind or neutralize the peptide that was injected. Control proteins or peptides, such as those known to be immunogenic, non-immunogenic, or of known immunogenic potential are also separately tested in the animals. Peptides that are less immunogenic by inducing less antibody formation are identified.
[0452] By identifying which peptides are less immunogenic by the above methods, peptides with lower immunogenicity are selected for use in human therapeutic treatment, thereby reducing immunogenicity of the therapeutic treatment. Some of these peptides are listed in TABLE 16 of EXAMPLE 23
EXAMPLE 25
Targeted Protein Degradation Using a Cell-Penetrating Peptide
[0453] A cell-penetrating peptide of this disclosure, including any of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is fused or conjugated to a cystine-dense peptide. The complex is applied to a cell. The cystine-dense peptide causes the formation of a complex between an E3 ligase and a target protein. The target protein is a cytosolic or nuclear protein, such as an oncogenic transcription factor, whose activity, such as increased activity or novel activity brought on by gene amplification, missense mutation, nonsense mutation, aberrant splicing, or fusion protein brought on by chromosomal translocation, is associated with a disease or condition. Alternatively, a cell-penetrating peptide of this disclosure, including any of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 is fused or conjugated to a cystine-dense peptide dimer, where one cystine-dense peptide binds to an E3 ligand and the other cystine-dense peptide binds to a target protein. The complex is applied to a cell. The target protein is ubiquitinated and then degraded by the ubiquitin-proteasome system (UPS).
EXAMPLE 26
Identification of Cell-Penetrating Peptides by Mass Spectrometry
[0454] This example illustrates identification of cell-penetrating peptides by mass spectrometry. HeLa cells stably transfected and expressing the GFP-SNAP-tag ubiquitously were plated on a 96-well microtiter plate. 48 wells were incubated with 10 pM BG-KR_IpTxa (SEQ ID NO: 59), as described in EXAMPLE 5. The cells were then trypsinized, pooled, and lysed with Mammalian Protein Extraction Reagent (M-PER) from ThermoFisher, supplemented with protease and phosphatase inhibitors. GFP-SNAP-tag junctions with BG-KR IpTxa were immunoprecipitated using GFP-Trap magnetic beads from Chromotek and submitted for mass spectrometry analysis. Immunoprecipitated proteins were digested with trypsin and run on a ThermoFisher Orbitrap Fusion. GFP-SNAP-tag fragments were detected, with the blue bars indicated different unique tryptic fragments identified by the mass spectrometer. The tryptic fragment TALSGNPVPILIPCHR (SEQ ID NO: 613) contains the Cysl45 residue that is irreversibly modified by conjugation to BG-KR_IpTxa. The GFP-SNAP-tag including covalent complexes to BG-KR_IpTxa was detected and identified with a modification of 1268.61 Da, corresponding to the tryptic fragment GSDCLPHLR (SEQ ID NO: 615) from BG-KR_IpTxa (BG-SEQ ID NO: 59), as shown in FIG. 18. The MS2 analysis of that peptide provides the exact sequence and is depicted on the right of FIG. 18.
[0455] HeLa cells stably transfected and expressing the GFP-SNAP-tag ubiquitously were plated on a 96-well microtiter plate. 7 wells were incubated with 10 pM BG-KR_IpTxa (SEQ ID NO: 59). The remaining 41 wells were incubated with 2 pM hydrolyzed BG-GLA-NHS (BG- GLA-OH). Cells were pooled and prepared for mass spectrometry analysis as previously described. Detection of the modified GFP-SNAP-tag tryptic fragment TALSGNPVPILIPCHR (SEQ ID NO: 613) with a modification of 1268.61 Da was detected and identified by MS2, as shown in FIG. 19. HeLa GFP-SNAP cells were lysed with M-PER supplemented with protease and phosphatase inhibitors before incubating with 10 pM BG-KR_IpTxa for 2 hours at 4C. Samples were then prepared for mass spectrometry analysis as previously described. Detection of the modified GFP-SNAP-tag tryptic fragment TALSGNPVPILIPCHR (SEQ ID NO: 613) with a modification of 1268.61 Da was detected and identified by MS2, as shown in FIG. 20. Prototypic peptides from BG-KR_IpTxa (BG-SEQ ID NO: 59) were also detected under the previously described conditions with the tryptic fragment highlighted detected and identified by MS2, indicating the presence of intact BG-KR_IpTxa (BG-SEQ ID NO: 59), as shown in FIG. 21. HeLa GFP-SNAP cells were lysed with M-PER supplemented with protease and phosphatase inhibitors before incubating with 1 pM BG-KR_IpTxa for 2 hours at 4 °C. Samples were then prepared for mass spectrometry analysis as previously described. Detection of the modified GFP-SNAP-tag tryptic fragment TALSGNPVPILIPCHR (SEQ ID NO: 613) with a modification of 1268.61 Da was detected and identified by MS2, as shown in FIG. 22. When cells were incubated with 0.1 pM BG-KR IpTxa, the modified GFP-SNAP-tag trypic fragment TALSGNPVPILIPCHR (SEQ ID NO: 613) with a modification of 1268.61 Da was not detected. [0456] FIG. 23 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with PBS in the SNAPPA. Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP -Cell TMR-Star dye). FIG. 24 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with 2 pM hydrolyzed BG-GLA-NHS (BG-GLA-OH) in the SNAPPA. Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP-Cell TMR-Star dye). FIG. 25 shows representative images of HeLa cells stably transfected with pSNAPf and expressing the SNAP -tag ubiquitously after incubation with 10 pM BG-KR_IpTxa (BG-SEQ ID NO: 59) in the SNAPPA. Cells were fluorescently imaged with a Hoescht live-cell stain (left) and with Texas Red (right). Texas Red fluorescence was used to quantify the uptake of BG-fluorophore (SNAP-Cell TMR-Star dye).
EXAMPLE 27
High Throughput Production and Purification of Cystine-Dense Peptides
[0457] This example describes high throughput production and purification of cystine-dense peptides (CDPs). Suspension-adapted HEK-293ST cells were transfected in a 96-well format using a Hamilton Starlet liquid handler with lentiviral constructs encoding various CDPs. Each CDP was generated as a fusion protein linked to a His-tagged siderocalin chaperone protein (Sen) via a TEV protease-cleavable linker. Transfected HEK-293ST cells were fed once with valproic acid on day 2 before collecting lentivirus-containing media on day 4. Lentivirus- containing media was used to transduce HEK-293F cells which were monitored for transduction efficiency by expression of GFP. 7 days after lentiviral transduction, the fusion proteincontaining media was harvested and applied to a 96-well block containing Ni-beads for binding to the His-tagged Sen chaperone protein. Eluted fusion protein was then cleaved with TEV protease and CDP was purified away from the Sen chaperone using size-exclusion chromatography on an Agilent AdvanceBio SEC 130A 2.7um 7.8x300mm column.
EXAMPLE 28
Quantification of Cytosolic and Nuclear Levels of GFP-SNAP-Tag Protein
[0458] This example describes quantification of cytosolic and nuclear levels of GFP-SNAP-tag protein. In order to relate fluorescence intensities measured during SNAP assays, as utilized in EXAMPLE 5 - EXAMPLE 11, to intracellularly-delivered protein concentrations, levels of GFP-SNAP-tag protein expressed in various cell types was quantified. If a SNAP assay indicates 100% saturation of GFP-SNAP-tag protein, such as shown by complete inhibition of BG-fluorophore fluorescence generation, by a molecule such as a BG-peptide or a BG-peptide complex, that would indicate that the cytosolic or nuclear concentration of the BG-peptide or BG-peptide complex reached a level at least as high as the concentration of GFP-SNAP-tag protein in the cytosol or nucleus.
[0459] Cell lines expressing GFP-SNAP-tag protein in the cytosol or H2B-tagged GFP-SNAP- tag in the nucleus were treated with increasing concentrations of hydrolyzed SNAP-substrate (BG-GLA-OH). Following incubation, the cells were treated with SNAP substrate-tagged fluorophore (BG-fluorophore) and washed to remove unbound fluorophore. As small molecules, both the BG-GLA-OH and the BG-fluorophore freely diffuse across the cytoplasmic and nuclear membranes and bind to SNAP -tag protein. However, binding of the BG-GLA-OH to a SNAP- tag protein permanently occupies the binding site and prevents subsequent binding of BG- fluorophore. Therefore, fluorescence from the SNAP substrate-tagged fluorophore serves as a measure of available SNAP -tag protein binding sites. Presuming that the level of normalized fluorescence is zero when no BG-fluorophore is present, the concentration of BG-GLA-OH at which the fluorescence from the SNAP substrate-tagged fluorophore reaches background levels (normalized fluorescence reaches zero) was used to estimate the concentration of GFP-SNAP- tag protein in the cytosol or H2B-tagged GFP-SNAP-tag in the nucleus. Fluorescence from the BG-fluorophore was normalized to GFP fluorescence to account for potential differences in GFP-SNAP-tag protein expression levels.
[0460] Cytosolic concentrations of GFP-SNAP-tag protein (“pSNAPf ’) expressed in 3T3 cells (FIG. 26A), HeLa cells (FIG. 26B), and HEK-293 cells (FIG. 26C) was measured using increasing concentrations of hydrolyzed SNAP-substrate. FIG. 26A - FIG. 26C shows fluorescence from SNAP substrate-tagged fluorophore as a function of hydrolyzed SNAP- substrate concentration. Linear fits to the data were used to extrapolate the concentration of hydrolyzed SNAP-substrate at which the normalized fluorescence reached zero, and therefore the interpolated concentration of GFP-SNAP-tag protein in the cytosol. Based on this data, the interpolated concentration of GFP-SNAP-tag protein in the cytosol was determined to be 936 nM in 3T3 cells, 700 nM in HeLa cells, and 1378 nM in HEK-293 cells. Due to low levels of residual fluorescence, the actual concentration of GFP-SNAP-tag protein in the cytosol may be slightly lower than the interpolated concentration.
[0461] Nuclear concentrations of H2B-tagged GFP-SNAP-tag protein (“pSNAPf-H2B”) expressed in 3T3 cells (FIG. 27A), HeLa cells (FIG. 27B), and HEK-293 cells (FIG. 27C) was measured using increasing concentrations of hydrolyzed SNAP-substrate. FIG. 27A - FIG. 27C shows fluorescence from SNAP substrate-tagged fluorophore as a function of hydrolyzed SNAP-substrate concentration. Linear fits to the data were used to extrapolate the concentration of hydrolyzed SNAP-substrate at which the normalized fluorescence reached zero, and therefore the interpolated concentration of H2B-tagged GFP-SNAP-tag protein in the nucleus. Based on this data, the interpolated concentration of H2B-tagged GFP-SNAP-tag protein in the nucleus was determined to be 1632 nM in 3T3 cells, 471 nM in HeLa cells, and 1523 nM in HEK-293 cells.
[0462] This data indicates the level of GFP-SNAP-tag protein in the cytosol or nucleus of the cells. This data also indicates, when 100% saturation of fluorescence is achieved in the SNAP tag assay after applying a BG-peptide or BG-peptide complex, that the BG-peptide or BG- peptide complex has been delivered to the cytosol or nucleus of the cells to at least the concentration of the GFP-SNAP-tag protein.
EXAMPLE 29
Quantification of Uptake of a Cell-Penetrating Peptide
[0463] This example describes quantification of uptake of a cell-penetrating peptide. Using a standard response curve to BG-GLA-OH, such as generated in EXAMPLE 28, to determine the cytosolic and nuclear concentrations of GFP-SNAP-tag protein or H2B-tagged GFP-SNAP-tag protein in 3T3, HeLa, and HEK-293 cells, the concentration and percent uptake of a KR IpTxa cell-penetrating peptide (SEQ ID NO: 59, corresponding to SEQ ID NO: 1 with an N-terminal GS) into various cells was quantified. Increasing concentrations of SNAP substrate-tagged KR IpTxa (BG- KR IpTxa, “Extracellular concentration of BG-KR_IpTxa”) was applied extracellularly to 3T3, HeLa, and HEK-293 cells expressing either GFP-SNAP-tag protein in the cytosol or H2B-tagged GFP-SNAP-tag protein in the nucleus, followed by incubation with BG- fluorophore, and compared to the standard concentration curves of BG-GLA-OH measured in EXAMPLE 28. The concentration of BG-KR_IpTxa delivered into the cells was determined using the BG-GLA-OH standard curve, and the % of extracellularly applied BG-KR_IpTxa that was delivered was calculated by dividing the cytosolic or nuclear concentration of BG- KR IpTxa by the applied extracellular concentration of BG-KR_IpTxa. The delivered cytosolic and nuclear concentrations (values provided in nM) of BG- KR IpTxa in 3T3, HeLa, and HEK- 293 cells, along with the percent delivered (parenthetical percentages), are provided in TABLE 171 TABLE 17 -Concentrations and Percent Delivered of a Cell-Penetrating Peptide Delivered to the Cytosol or Nucleus
Figure imgf000213_0001
[0464] Based on these results, the cell -penetrating peptide complex of SEQ ID NO: 59 reached cytosolic concentrations of up to 600.8 nM in 3T3 cells and nuclear concentrations of up to 960.8 nM in HEK-293 cells. Up to 27% of the cell -penetrating peptide of SEQ ID NO: 59 reached the cytosol in HeLa cells and HEK-293 cells, and up to 38% of the cell-penetrating peptide of SEQ ID NO: 59 reached the nucleus in HEK-293 cells.
EXAMPLE 30
Characterization of Uptake Mechanisms for Cell-Penetrating Peptides Fused to Cargo Cystine-Dense Peptides
[0465] This example describes characterization of uptake mechanisms for two different cellpenetrating peptides fused to cargo cystine-dense peptides (CDPs). SNAP penetration assays were performed to measure cytosolic uptake of SEQ ID NO: 320 (comprising a cell -penetrating peptide of SEQ ID NO: 210 fused to a cargo peptide of SEQ ID NO: 295) and SEQ ID NO: 321 (comprising a cell-penetrating peptide of SEQ ID NO: 213 fused to a cargo peptide of SEQ ID NO: 295) in the presence of various chemical inhibitors of different mechanisms of endocytosis. Cells were preincubated for 1 hour with 50 pM EIP A which inhibits macropinocytosis, 3 pM cytochalasin D which inhibits clathrin-mediated endocytosis, 20 pM nocodazole which inhibits endosomal transport by disrupting microtubule polymerization, 100 pM dynasore which inhibits dynamin-dependent endocytosis, 50 nM bafilomycin A which inhibits lysosomal maturation, or 100 pM of chloroquine which also inhibits lysosomal maturation. Subsequently, cells were incubated with 10 pM of SNAP substrate-tagged SEQ ID NO: 320 (FIG. 28A) or 10 pM of SNAP substrate-tagged SEQ ID NO: 321 (FIG. 28B) in the continued presence of chemical inhibitors. Assays were conducted in 3T3, HeLa, and HEK-293 cell lines expressing GFP- SNAP-tag protein in the cytosol.
[0466] As shown in FIG. 28A, cytosolic uptake of SEQ ID NO: 320 was inhibited by bafilomycin A and chloroquine, suggesting that SEQ ID NO: 320 is trafficked through endosomal pathways. Additionally, cytosolic uptake of SEQ ID NO: 320 was inhibited by cytochalasin D, particularly in 3T3 and HeLa cells, indicating that clathrin-mediated endocytosis is involved in uptake of SEQ ID NO: 320 in these cell lines. Furthermore, EIPA inhibited cytosolic uptake, particularly in HEK-293 cells, suggesting that SEQ ID NO: 320 may utilize macropinocytosis in 3T3 and HEK-293 cells. Nocodazole and dynasore induced less or no inhibition of cytosolic uptake of SEQ ID NO: 320 in these cell types. Together, this data demonstrates that SEQ ID NO: 320 can be trafficked through endosomal pathways but that there is some variation in predominant endocytotic mechanisms between cell lines. As shown in FIG. 28B, cytosolic uptake of SEQ ID NO: 321 showed less overall inhibition by the various endocytosis inhibitors. The trends, with smaller magnitudes, were similar to those found with SEQ ID NO: 320 (FIG. 28A), where bafilomycin, chloroquine, EIPA, and cytochalasin D showed some inhibition in some cell lines, whereas nocodazole and dynasore induced negligible inhibition. The smaller magnitudes of inhibition in FIG. 28B suggests that SEQ ID NO: 321 may exhibit more trafficking through non-endocytotic mechanisms.
EXAMPLE 31
Identification of Cell-Penetrating Peptides that are Cystine Dense Peptides
[0467] This example describes identification of cell-penetrating peptides that are cystine dense peptides. Cystine-dense peptides (CDPs) that function as cell-penetrating peptides were identified using an in silica algorithm designed to predict cell-penetrating CDPs. Exemplary cell-penetrating CDPs identified using this algorithm are provided as SEQ ID NO: 408 - SEQ ID NO: 457. The peptide of SEQ ID NO: 1 (corresponding to SEQ ID NO: 59 with no GS), experimentally validated as cell-penetrating in EXAMPLE 6 - EXAMPLE 9, was also identified as cell-penetrating using the same algorithm.
EXAMPLE 32
Targeted Degradation Complexes for Degradation of a Target Protein [0468] This example describes targeted degradation complexes for degradation of a target protein. A targeted degradation complex includes a cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) is fused or conjugated to a target-binding peptide (e.g., any one of SEQ ID NO: 295, SEQ ID NO: 298, or SEQ ID NO: 364 - SEQ ID NO: 407) that binds a target protein The target protein is a cytosolic or nuclear protein, such as an oncogenic transcription factor, whose activity, such as increased activity or novel activity brought on by gene amplification, missense mutation, nonsense mutation, aberrant splicing, or fusion protein brought on by chromosomal translocation, is associated with a disease or condition. The cell-penetrating peptide/target-binding peptide complex is further linked to a molecule that binds an E3 ubiquitin ligase via a linker to form a targeted degradation complex. The molecule capable of binding the E3 ubiquitin ligase is an immunomodulatory imide drug (IMiD) such as thalidomide, pomalidomide, or lenalidomide (which bind cereblon (CRBN)), methyl bestatin or bestatin (which bind cellular inhibitor of apoptosis protein 1 (cIAPl)), nutlin- 3 (which binds MDM2), or VHL ligand 1 (which binds von Hippel-Lindau protein (VHL)). Examples of molecules capable of binding various E3 ubiquitin ligases that may be incorporated into a complex for targeted degradation are shown in FIG. 30. The linker is a peptide linker (e g., any one of SEQ ID NO: 255 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485) or a small molecule linker (e.g., a linker provided in TABLE 11) of sufficient length to permit ternary complex formation between the target protein, the E3 ubiquitin ligase, and the targeted degradation complex.
[0469] Upon administration to a cell expressing the target protein and the E3 ubiquitin ligase, the targeted degradation complex localizes to the cytosol or the nuclease and binds the target protein and the E3 ubiquitin ligase. Recruitment of the E3 ubiquitin ligase to the target protein facilitates ubiquitination and subsequent degradation of the target protein.
EXAMPLE 33 Targeted Degradation of TEAD
[0470] This example describes targeted degradation of transcriptional enhancer factor TEF-1 (TEAD) using a targeted degradation complex comprising cell-penetrating peptide. A targeted degradation complex is constructed as described in EXAMPLE 32. Components for targeted degradation of TEAD are illustrated in FIG. 29A. A cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) is fused or conjugated to a cystine-dense peptide (CDP) capable of binding TEAD (e.g., a CDP of any one of SEQ ID NO: 295, SEQ ID NO: 298, or SEQ ID NO: 364 - SEQ ID NO: 407) and further linked to an immunomodulatory imide drug (IMiD) capable of binding a cereblon (CRBN) E3 ubiquitin ligase via a linker, as shown in FIG. 29B. Optionally, the cell-penetrating peptide/TEAD-binding peptide complex comprises a sequence of SEQ ID NO: 307 or SEQ ID NO: 308. Optionally, the IMiD is thalidomide, pomalidomide, or lenalidomide. The linker is a peptide linker (e g., any one of SEQ ID NO: 255 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485) or a small molecule linker (e.g., a linker provided in TABLE 11) of sufficient length to permit ternary complex formation between TEAD, CRBN, and the resulting cell -penetrating peptide/TEAD-binding CDP/IMiD (“CDP + Linker + IMiD”) complex.
[0471] The complex is administered to a cell expressing TEAD and CRBN. The cell-penetrating peptide of the complex facilitates uptake of the complex to the nucleus of the cell. The complex binds to TEAD via the TEAD-binding CDP, as shown in FIG. 290C, and to CRBN via the IMiD, as shown in FIG. 29D. Upon formation of the ternary complex, CRBN ubiquitinates TEAD, thereby targeting TEAD for degradation. The ubiquitinated TEAD is subsequently degraded.
EXAMPLE 34
Targeted Degradation of a Cold-Inducible RNA-Binding Protein
[0472] This example describes targeted degradation of a cold-inducible RNA-binding protein, (CIRP) using a targeted degradation complex comprising cell-penetrating peptide. A targeted degradation complex is constructed as described in EXAMPLE 32. A cell-penetrating peptide (e g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) is fused or conjugated to a cystine-dense peptide (CDP) capable of binding CIRP and further linked to a molecule capable of binding an E3 ubiquitin via a linker. The linker is a peptide linker (e.g., any one of SEQ ID NO: 255 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485) or a small molecule linker (e.g., a linker provided in TABLE 11) of sufficient length to permit ternary complex formation between CIRP, the E3 ubiquitin ligase, and the resulting cell -penetrating peptide/CIRP -binding CDP/E3 ubiquitin ligase-binding molecule complex. The complex is administered to a cell expressing CIRP and the E3 ubiquitin ligase. The cell -penetrating peptide of the complex facilitates uptake of the complex to the cytosol of the cell, the nucleus of the cell, or both. The complex binds to CIRP via the CIRP -binding CDP and to the E3 ubiquitin ligase via the the E3 ubiquitin ligase-binding molecule. Upon formation of the ternary complex, the E3 ubiquitin ligase ubiquitinates CIRP, thereby targeting CIRP for degradation. The ubiquitinated CIRP is subsequently degraded. EXAMPLE 35
Targeted Degradation of an Oncogenic Protein
[0473] This example describes targeted degradation of an oncogenic protein using a targeted degradation complex comprising cell-penetrating peptide. A targeted degradation complex is constructed as described in EXAMPLE 32. A cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) is fused or conjugated to a cystine-dense peptide (CDP) capable of binding a tumor-specific growth and/or homeostasis factor or transcription factor, including STAT3, STAT5, Runx, NF-KB, P-catenin, Notch, GLI, c-JUN, TEAD, Myc, Fos, AR, ER, Bcl-2, Ras, Src, Abll, Aik, EGFR, HER2, ERBB3, or Kit. Transmembrane proteins, such as EGFR, HER2, or ERBB3, can thus be targeted for degradation intracellularly via the cytosolic end of the proteins. The complex binds to a tumor-specific growth and/or homeostasis factor, transcription factor, or other oncogenic protein via the tumor-specific growth and/or homeostasis factor or transcription factor-binding CDP and to the E3 ubiquitin ligase via the E3 ubiquitin ligase-binding molecule. Upon formation of the ternary complex, the E3 ubiquitin ligase facilitates ubiquitination of a tumor-specific growth and/or homeostasis factor or transcription factor, thereby targeting a tumor-specific growth and/or homeostasis factor, transcription factor, or other oncogenic protein for degradation. The ubiquitinated tumor-specific growth and/or homeostasis factor, transcription factor, or other oncogenic protein is subsequently degraded.
EXAMPLE 36
Inhibition of a Cold-Inducible RNA-Binding Protein
[0474] This example describes inhibition of a cold-inducible RNA-binding protein, (CIRP) using a cell-penetrating CIRP -binding cystine dense peptide (CDP). A CIRP -binding CDP is linked or fused to a cell -penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254). Optionally, the CIRP -binding CDP is fused to the cell -penetrating peptide via a peptide linker (e.g., any one of SEQ ID NO: 255 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485). The resulting peptide complex is administered to a cell expressing CIRP, and the cell-penetrating peptide facilitates uptake of the complex into the nucleus of the cell. Upon entry into the nucleus, the CIRP -binding CDP binds to and inhibits CIRP. EXAMPLE 37
Cytoplasmic Delivery of a Cystine-Dense Peptide Using a Cell-Penetrating Peptide [0475] This example describes cytoplasmic delivery of a cystine-dense peptide using a cellpenetrating peptide. Delivery of BG-peptides to the cytosol of HeLa cells was measured using a gel shift assay. A western blot of showing a molecular weight shift upon binding of BG-peptides to GFP-SNAP-tag protein expressed in the cytosol is shown in FIG. 31. Intact HeLa cells expressing GFP-SNAP-tag protein in the cytosol were incubated with 10 pM BG-SEQ ID NO: 320 or 10 pM BG-SEQ ID NO: 321 for 15, 30 or 60 minutes. Cells were then washed with PBS and treated with trypsin to detach the cells from the plate and remove surface-bound peptides. The detached cells were pelleted and washed with PBS before lysis. The cell lysates were then analyzed by western blot with staining via an anti-GFP antibody. A shift in the GFP-SNAP-tag protein band size (from slightly below 55 kDa to between the 55 kDa and the 73 kDa markers) is observed at 15 and 30 minutes indicating the presence of full-length peptide in the cytosol, contacting and reacting with the GFP-SNAP protein. The fainter band between 95 and 130 kDa may represent a dimer of two GFP-SNAP proteins linked and occupied by a BG-peptide that contains at least two BG moieties (Lys residues and the N-terminus may react with BG-GLA- NHS). This data indicates that the peptides of SEQ ID NO: 320 and SEQ ID NO: 321 penetrated the cells and entered the cytosol. The loss of the shifted GFP-SNAP band over time suggests some intracellular degradation of exposed BG-peptide, where the cleavage could be on the N- terminal GS, the cell penetrating MCa or Had sequences, the GGS linker, the TEAD binding peptide, or residues of the SNAP protein proximal to the reactive site, may be occurring, resulting in the continued occupation of GFP-SNAP-tag protein, and preventing subsequent BG- fluorophore binding. Together these results show that the cell-penetrating peptide was able to deliver the cystine-dense peptide to the cytosol.
EXAMPLE 38 Treatment of a Cancer with a Cell-Penetrating Peptide Complex
[0476] This example describes treatment of a cancer with a cell-penetrating peptide complex. A cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) is fused or conjugated to a cargo molecule to form a cell-penetrating peptide complex. The cargo molecule is a molecule that binds to a cytosolic or nuclear target. Optionally, the cargo molecule comprises a CDP. Optionally the cargo molecule binds to modulators of any oncogene implicated in tumor-specific growth and homeostasis. Optionally the molecule binds a transcription factors such as STAT3, STAT5, Runx, NF-KB, P-catenin, Notch, GLI, c-JUN, TEAD, Myc, Fos, AR, ER, or MYB. Alternatively, the molecule binds a protein such as Bcl-2, Ras, K-Ras, H-Ras, N-Ras, Src, Abll, Aik, EGFR, HER2, ERBB3, Kit, RHO, RAN, or RAB When applied to a cell, the cell-penetrating complex may penetrate the cell and bind to the target and block protein-protein interactions. Alternatively, when applied to a cell, the cell -penetrating peptide complex may penetrate the cell and bind to the target and promote degradation of the target, such as by the UPS.
[0477] The cell-penetrating peptide complex is administered to a subject with cancer. The cellpenetrating peptide complex is administered intravenously (e.g., by infusion or bolus), subcutaneously, or directly into the affected organ or tumor. The cell -penetrating peptide complex reaches cancer cells, penetrates the cells, and the cargo molecule interacts with the cytosolic and/or nuclear target. Optionally, the target is inhibited, prevented from binding other proteins, or degraded. The cancer is thus effectively treated in the patient, such as by causing tumor regression, stabilizing disease, preventing metastasis, or improving subject quality of life.
EXAMPLE 39
Treatment of Inflammation with a Cell-Penetrating Peptide Complex
[0478] This example describes treatment of an inflammatory disorder with a cell -penetrating peptide complex. A cell penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) is fused or conjugated to a cargo molecule to form a cell-penetrating peptide complex. The cargo molecule is a molecule that binds to a cytosolic or nuclear target.
Optionally, the cargo molecule comprises a CDP. The cargo molecule binds to or otherwise affects cytosolic or nuclear targets that are involved in an inflammatory disease. Optionally, the cell-penetrating peptide complex delivers an agent that targets the inflammosome, and thus reduces NLRP3 activation or otherwise reduces activity of the inflammosome.
[0479] The cell-penetrating peptide complex is administered to a subject The subject has an inflammatory disease such as rheumatoid arthritis, type 1 or type 2 diabetes, psoriasis, fatty liver disease, NASH, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, lupus, ankylosing spondylitis, psoriasis, multiple sclerosis, or autoimmune disease. The cellpenetrating peptide complex is administered intravenously (e.g., by infusion or bolus), subcutaneously, intramuscularly, or orally. The cell -penetrating peptide complex reaches cells involved in inflammation, penetrates the cells, and the molecule interacts with the cytosolic or nuclear target. Optionally, the target is inhibited, prevented from binding other proteins, or degraded. The inflammation is thus reduced in the patient, resulting in reduced pain, reduced inflammation, increased mobility, increased function, improved organ function, or improved quality of life.
EXAMPLE 40 Treatment of a Cancer with a Cell-Penetrating Peptide-RIG-I Complex [0480] This example describes treatment of a cancer with a cell-penetrating peptide-RIG-I ligand complex. A cell penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) is conjugated or fused to RIG-I ligand to form a cell-penetrating peptide- Rig-I ligand complex. The RIG-I ligand and the cell penetrating peptide are joined by a linker, which may be stable or cleavable. The RIG-I ligand may be a dsRNA or hairpin RNA comprising 5-60 base pairs and containing at least one 5’ triphosphate or 5 ’ diphosphate group. Optionally, an additional component is added to the complex that directs the cell-penetrating Rig-I ligand complex to tumor cells.
[0481] The cell-penetrating peptide-RIG-I ligand complex is administered to a subject that has cancer. The cell-penetrating peptide-RIG-I ligand complex is administered intravenously (e.g., by infusion or bolus), subcutaneously, or directly into the affected organ or tumor. The cellpenetrating peptide-RIG-I ligand complex reaches cells in the tumor and penetrates the cells. The RIG-I ligand binds to RIG-I in the cytosol of the cells. The RIG-I pathway is activated. A type I interferon response, inflammosome activity, apoptosis, or other responses to the RIG-I ligand lead to an anti-tumor response. The cancer is thus effectively treated in the subject, such as by causing tumor regression, stable disease, preventing metastasis, or improving subject quality of life.
EXAMPLE 41
Treatment of a Disease with a Cell-Penetrating Peptide-Oligonucleotide Complex [0482] This example describes treatment of a disease with a cell-penetrating peptideoligonucleotide complex. A cell penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) is conjugated to an oligonucleotide, forming a cell-penetrating peptide-oligonucleotide complex. The oligonucleotide may comprise a nucleotide antisense RNA, a complementary RNA, an inhibitory RNA, an interfering RNA, a nuclear RNA, an antisense oligonucleotide, a microRNA, a sequence complementary to a natural antisense transcript, a small interfering RNA, a small nuclear RNA, an aptamer, a gapmer, an anti-miR sequence, a splice blocker antisense oligonucleotide, or a U1 adapter. The oligonucleotide interacts with a target molecule. When applied to the cell, the cell-penetrating peptideoligonucleotide complex enters the cytoplasm or the nucleus of the cell. The oligonucleotide component of the cell-penetrating peptide-oligonucleotide complex may modulate alternative splicing of a targeted sequence, dictate the location of the polyadenylation (poly A) site of a targeted sequence, recruit RNaseHl to induce cleavage of a targeted sequence, directly bind to microRNA (miRNA) or messenger (mRNA) sequences. siRNAs, which are targeted to a specific sequence for its regulation, may alternatively be used to bind and regulate a targeted sequence in the cytosol, engaging an RNA-induced silencing complex (RISC) which is a multiprotein complex that incorporates one strand of a small interfering RNA (siRNA) or micro RNA (miRNA), using the siRNA or miRNA as a template for recognizing complementary mRNA of the targeted sequenc (when it finds a complementary strand, the RISC complex cleaves the targeted sequence), or bind and regulate a target molecule. The cell-penetrating peptide and the oligonucleotide may be conjugated by a stable or cleavable linker. The oligonucleotide may comprise modified bases that have increased stability or half-life in vivo. [0483] The cell-penetrating peptide-oligonucleotide complex is given to a subject. The subject has a disease, such as cancer, an inflammatory or immune-mediated disease, or a disease of the CNS. The cell-penetrating peptide-oligonucleotide complex is formulated and administered to the subject intravenously (e.g., by infusion or bolus), subcutaneously, intrathecally, or directly into the affected organ or tumor. The cell-penetrating peptide-oligonucleotide complex reaches cells and penetrates the cells, thereby delivering the oligonucleotide to the cytosol or the nucleus. The oligonucleotide modulates the activity of the target molecule, such as by increasing the expression of the target molecule, reducing translation of the target molecule, degrading the target molecule, reducing a level of the target molecule, modifying the processing of the target molecule, modifying the splicing of the target molecule, inhibiting processing of the target molecule, reducing a level of a protein encoded by the target molecule, or blocking an interaction with the target molecule. The disease in the subject is thus treated, and a condition of the subject improved by this modulation.
EXAMPLE 42
Blocking Protein-Protein Interactions using a Cell-Penetrating Peptide Complex [0484] This example describes blocking protein-protein interactions using a cell-penetrating peptide complex. A cell-penetrating peptide (e.g., any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) is fused or conjugated to a cargo molecule, such as a peptide (e g., any one of SEQ ID NO: 295, SEQ ID NO: 298, or SEQ ID NO: 364 - SEQ ID NO: 407), that blocks a protein-protein interaction (PPI). The cell-penetrating peptide and the PPI-inhibiting molecule are optionally linked by a stable or cleavable linker. The PPI-inhibiting molecule is optionally a CDP. The resulting cell -penetrating peptide-PPI inhibiting complex is applied to a cell and the complex enters the cytosol or the nucleus of the cell. The PPI-inhibiting molecule inhibits, blocks, or reduces the formation of a PPI inside the cell. The PPI may be the binding of TEAD to YAP.
[0485] Optionally, the cell-penetrating peptide-PPI inhibiting complex is administered to a subject. The subject has a disease such as cancer. The cell-penetrating peptide-PPI inhibiting complex is administered intravenously (e.g., by bolus or infusion), subcutaneously, or by any other route. The cell-penetrating peptide-PPI inhibiting complex reaches effected cells, such as cancer cells and penetrates the cells to reach the cytoplasm or nucleus. The cell-penetrating peptide-PPI inhibiting complex, or a cleavage product thereof, inhibits a PPI, such as the binding of TEAD to YAP. The PPI inhibition modulates signaling in a biological pathway of the cell. The subject’s disease is thereby treated, such as by causing tumor regression, stable disease, reduced pain or inflammation, or improve quality of life.
EXAMPLE 43
Synthesis of a Peptide Oligonucleotide Complex for Antisense Therapy
[0486] A gene targeted for silencing in order to address a disease is identified and the desired single-stranded antisense oligonucleotide sequence is designed and synthesized based on the target coding or complementary sequence. The antisense oligonucleotide is conjugated to any cell-penetrating peptide disclosed herein, including peptides of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 by any of the methods disclosed herein, for example, in accordance with EXAMPLE 50 - EXAMPLE 53, such as with a cleavable or stable linker Optionally, a nucleotide (including the backbone) is modified, such as to increase in vivo stability, to increase resistance to enzymes such as nucleases, increase protein binding including to serum proteins, increase in vivo half-life, to modify the tissue biodistribution, or to reduce immune system activation.
[0487] Any peptide oligonucleotide complexes of the present disclosure (e.g., including an oligonucleotide of any one of SEQ ID NO: 488 - SEQ ID NO: 573, linked or conjugated to SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) are described. Any peptide oligonucleotide complexes of the present disclosure can have oligonucleotides that bind to any target in TABLE 12, or to any of SEQ ID NO: 574 - SEQ ID NO: 611, or to any genomic or ORF sequence provided in TABLE 6.
EXAMPLE 44
Synthesis of a peptide-oligonucleotide conjugate for RNAi therapy
[0488] A gene targeted for silencing in order to address a disease is identified and the desired double-stranded RNAi sequence is designed and synthesized based on the target coding or complementary sequence. The sense or the antisense oligonucleotide of the RNAi is conjugated to any cell-penetrating peptide disclosed herein, including a peptide of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 by any of the methods disclosed herein, for example, in accordance with EXAMPLE 50 - EXAMPLE 53, such as with a cleavable or stable linker. Optionally the peptide is conjugate to the sense (passenger) strand of the oligonucleotide. Optionally, a nucleotide (including the backbone) is modified, such as to increase in vivo stability, to increase resistance to enzymes such as nucleases, increase protein binding including to serum proteins, increase in vivo half-life, to modify the tissue biodistribution, or to reduce immune system activation. The sense and antisense strands are hybridized together, either before or after the conjugation.
[0489] Any peptide oligonucleotide complexes of the present disclosure (e.g., including an oligonucleotide of any one of SEQ ID NO: 488 - SEQ ID NO: 573, linked or conjugated to SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) are described. Any peptide oligonucleotide complexes of the present disclosure can have oligonucleotides that bind to any target in TABLE 12, or to any of SEQ ID NO: 574 - SEQ ID NO: 611, or to any genomic or ORF sequence provided in TABLE 6.
EXAMPLE 45
Synthesis of a peptide-oligonucleotide conjugate for U1 adaptor therapy
[0490] A gene targeted for silencing in order to address a disease is identified and the desired oligonucleotide sequence for U1 adaptor therapy is designed and synthesized based on the target coding or complementary sequence. The oligonucleotide is conjugated to any to any cellpenetrating peptide disclosed herein, including peptide of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 by any of the methods disclosed herein, for example, in accordance with EXAMPLE 50 - EXAMPLE 53, such as with a cleavable or stable linker. Optionally, a nucleotide (including the backbone) is modified, such as to increase in vivo stability, to increase resistance to enzymes such as nucleases, increase protein binding including to serum proteins, increase in vivo half-life, to modify the tissue biodistribution, or to reduce immune system activation.
[0491] Any peptide oligonucleotide complexes of the present disclosure (e.g., including an oligonucleotide of any one of SEQ ID NO: 488 - SEQ ID NO: 573, linked or conjugated to SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) are described. Any peptide oligonucleotide complexes of the present disclosure can have oligonucleotides that bind to any target in TABLE 12, or to any of SEQ ID NO: 574 - SEQ ID NO: 611, or to any genomic or ORF sequence provided in TABLE 6.
EXAMPLE 46
Synthesis of a peptide-oligonucleotide conjugate for aptamer therapy
[0492] An aptamer sequence that interacts with a target molecule is selected to address a disease is identified against the target and synthesized. The aptamer oligonucleotide is conjugated to any cell-penetrating peptide disclosed herein, including any peptide of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254 by any of the methods disclosed herein, for example, in accordance with EXAMPLE 50 - EXAMPLE 53, such as with a cleavable or stable linker. Optionally, a nucleotide (including the backbone) is modified, such as to increase in vivo stability, to increase resistance to enzymes such as nucleases, increase protein binding including to serum proteins, increase in vivo half-life, to modify the tissue biodistribution, or to reduce immune system activation.
[0493] Any peptide oligonucleotide complexes of the present disclosure (e g , including an oligonucleotide of any one of SEQ ID NO: 488 - SEQ ID NO: 573, linked or conjugated to SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) are described. Any peptide oligonucleotide complexes of the present disclosure can have oligonucleotides that bind to any target in TABLE 12, or to any of SEQ ID NO: 574 - SEQ ID NO: 611, or to any genomic or ORF sequence provided in TABLE 6. EXAMPLE 47
Conjugation of an Oligonucleotide and a Peptide using Click Chemistry
[0494] An alkyne or azide group is installed in an oligonucleotide, such as by adding a hexynyl group to the 5’ end or the 3’ end of the oligonucleotide, installation of a 5 -Octadiynyl dU, installation of a DIBO at the 5’ end using, which is optionally installed using a DIBO phosphoramidite, or installation of an azide group by use of an NHS ester reaction linking an azide group to a dT base. An azide or an alkyne group is installed on a peptide, such as by incorporating an N-terminal 6-azidohexanoic acid, an azidohomoalanine residue, or homopropargyl glycine residue. Optionally, the alkyne group comprises a strained ring such as strained cyclooctyne ring, such as DIBO. The oligonucleotide is conjugated to any cellpenetrating peptide disclosed herein, including any peptide of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254. The oligonucleotide and the peptide are conjugated together by combining an azide group on one with the alkyne group on the other using a copper-catalyzed azide-alkyne cycloaddition or a strain-promoted azide-alkyne cycloaddition to form a triazole bond.
[0495] Any peptide oligonucleotide complexes of the present disclosure may be so modified with an alkyne or azide group and are described. Any peptide oligonucleotide complexes of the present disclosure (e.g., including an oligonucleotide of any one of SEQ ID NO: 488 - SEQ ID NO: 573, linked or conjugated to SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 SEQ ID NO: 457, or SEQ ID NO: 195 SEQ ID NO: 254) are described. Any peptide oligonucleotide complexes of the present disclosure can have oligonucleotides that bind to any target in TABLE 12, or to any of SEQ ID NO: 574 - SEQ ID NO: 611, or to any genomic or ORF sequence provided in TABLE 6.
EXAMPLE 48
Conjugation of an RNAi Sequence and a Peptide using Click Chemistry
[0496] An alkyne group within a strained cyclooctyne is installed on an oligonucleotide, optionally linked to either the 5 ’ or the 3 ’ end of a sense or antisense strand. Optionally the strained cyclooctyne is DIBO, which is optionally installed on the 5’ end using a DIBO phosphoramidite. An azide group is installed on a peptide. Optionally, a 6-azidohexanoyl group is added to the N-terminus of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254, optionally with a linker in between the 6-azidohexanoyl group and the peptide. Optionally, the peptide is prepared as a TFA salt form. The alkyne-containing oligonucleotide and the azide- containing peptide are contacted together, such as in a buffer, solution, or solvent. The azide and the alkyne react to form a triazole bond that links the oligonucleotide and the peptide. The sense and antisense strands of the RNAi are hybridized together, either before or after the conjugation reaction.
[0497] Any peptide oligonucleotide complexes of the present disclosure may be modified to include an alkyne group within a strained cyclooctyne and are described. Any peptide oligonucleotide complexes of the present disclosure (e.g., including an oligonucleotide of any one of SEQ ID NO: 488 - SEQ ID NO: 573, linked or conjugated to SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) are described. Any peptide oligonucleotide complexes of the present disclosure can have oligonucleotides that bind to any target in TABLE 12, or to any of SEQ ID NO: 574 - SEQ ID NO: 611, or to any genomic or ORF sequence provided in TABLE 6
EXAMPLE 49
Conjugation of a U1 Adapter Sequence and a Peptide using Click Chemistry [0498] An alkyne group within a strained cyclooctyne is installed on an oligonucleotide, optionally linked to either the 5 ’ or the 3 ’ end of a sequence, designed for U 1 adapter therapy. Optionally the strained cyclooctyne is DIBO, which is optionally installed on the 5’ end using a DIBO phosphoramidite. An azide group is installed on a peptide. Optionally, the peptide is prepared as a TFA salt form. The alkyne-containing oligonucleotide and the azide-containing peptide are contacted together, such as in a buffer, solution, or solvent. The azide and the alkyne react to form a triazole bond that links the oligonucleotide and the peptide.
[0499] Any peptide oligonucleotide complexes of the present disclosure may be modified with an alkyne group within a strained cyclooctyne are described. Any peptide oligonucleotide complexes of the present disclosure (e.g., including an oligonucleotide of any one of SEQ ID NO: 488 - SEQ ID NO: 573, linked or conjugated to SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) are described. Any peptide oligonucleotide complexes of the present disclosure can have oligonucleotides that bind to any target in TABLE 12, or to any of SEQ ID NO: 574 - SEQ ID NO: 611, or to any genomic or ORF sequence provided in TABLE 6. EXAMPLE 50
Installation of a Thiol Group, an Amine Group, or an Aldehyde Group in an Oligonucleotide
[0500] This example describes incorporation of a thiol group, an amine group, or an aldehyde group in RNA or DNA or any oligonucleotide. FIG. 33A - FIG. 33E illustrates incorporation or addition of these groups on RNA or DNA. A thiol group is added on an oligonucleotide, using EDC and imidazole to activate the 5’ phosphate group to a phosphorylimidazolide, and by subsequently reacting the resulting product with cystamine. This is followed by reduction with dithiothreitol (DTT) to form a phosphoramidite linkage to a free thiol group. A thiol group is, alternatively, added on an oligonucleotide by incorporating a phosphoramidite that contains a thiol during solid-phase phosphoramidite oligonucleotide synthesis, at either the 5’- end or the 3 ’-end of the oligonucleotide as shown in FIG. 33A - FIG. 33E. The phosphoramidite used during synthesis can have a protecting group on the thiol during synthesis that is removed during cleavage, purification, and workup. FIG. 33A illustrates structures of oligonucleotides containing a 5 ’-thiol (thiohexyl; C6) modification (left), and a 3 ’-thiol (C3) modification (right), as shown at https://www.atdbio.com/content/50/Thiol-modified-oligonucleotides.
[0501] An amine group is added on RNA or DNA by incorporating a phosphoramidite during synthesis that contains a protected amino group that is later deprotected. FIG. 33B illustrates an MMT-hexylaminolinker phosphoramidite. FIG. 33C illustrates a TFA-pentylaminolinker phosphoramidite, as shown at https: //www. sigmaaldrich. com/ catalog/product/ sigma/mO 1023 hh?lang=en&region=U S .
[0502] Alternatively, thiol or amine containing oligonucleotide residues are included within the sequence at any chosen location in RNA or DNA, such as described by Jin et al. (J Org Chem. 2005 May 27;70(l l):4284-99). FIG. 33D illustrates RNA residues incorporating amine or thiol residues, as presented in Jin et al. (J Org Chem. 2005 May 27;70(l l):4284-99). Also, an oligonucleotide residue that contains a phosphorothioate group within the phosphodiester backbone (where a sulfur atom replaces a non-bridging oxygen in the phosphate backbone of the oligonucleotide) provides a reactive group that is similarly used for conjugation to a thiol group. Use of the phosphorothioate containing residues can also make the RNA more resistant to nuclease degradation.
[0503] FIG. 33E illustrates oligonucleotides with aminohexyl modifications at the 5’ (left) and 3’ ends (right).
[0504] Aldehyde functional groups can be incorporated at the 3’ end of RNA by using periodate oxidation to convert the diol into two aldehyde groups. [0505] Other methods of incorporating or modifying functional groups are carried out using techniques set forth in Bioconjugate Techniques, by Greg Hermanson, 3rd edition.
[0506] Any peptide oligonucleotide complexes of the present disclosure may be modified with a thiol group, an amine group, or an aldehyde group and are described. Any peptide oligonucleotide complexes of the present disclosure (e.g., including an oligonucleotide of any one of SEQ ID NO: 488 - SEQ ID NO: 573, linked or conjugated to SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) are described. Any peptide oligonucleotide complexes of the present disclosure can have oligonucleotides that bind to any target in TABLE 12, or to any of SEQ ID NO: 574 - SEQ ID NO: 611, or to any genomic or ORF sequence provided in TABLE 6
EXAMPLE 51
Generation of Cleavable Linkers Between an oligonucleotide with a Peptide [0507] This example describes generation of cleavable linkers between an oligonucleotide with any one of peptides of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254. A disulfide linker is generated by combining a thiol-containing oligonucleotide with a peptide comprising a free thiol group. The thiol is incorporated on the peptide using Traut’s reagent, SATA, SPDP or other appropriate reagents on a reactive amine (such as a heterobifunctional SPDP and NHS ester linker with the N-terminus or a lysine residue), or by incorporating a free cysteine residue in the peptide, as shown in FIG. 34, wherein “CPP” indicates a cell-penetrating peptide of this disclosure. The disulfide linker is cleaved in the reducing environment of the cytoplasm or in the endosomal/lysosomal pathway.
[0508] An ester linkage is generated by combining a free hydroxyl group (such as on the 3 ’ end of an oligonucleotide) with a carboxylic acid group on the peptide (such as from the C-terminus, an aspartic acid, glutamic acid residue, or introduced via a linker to a lysine residue or the N- terminus) such as via Fisher esterification or via use of an acyl chloride. The ester linker is cleaved by hydrolysis, which is accelerated by the lower pH of endosomes and lysosomes, or by enzymatic esterase cleavage.
[0509] An oxime or hydrazone linkage is generated by combining an aldehyde group on the oligonucleotide with a peptide that has been functionalized with an aminooxy group (to form an oxime linkage) or a hydrazide group (to form a hydrazone linkage). The stability or lability of an oxime or hydrazone linkage is tailored by neighboring groups (Kalia et al., Angew Chem Int Ed Engl. 2008;47(39):7523-6.), and hydrolytic cleavage is accelerated in acidic compartments such as the endosome/lysosome.
[0510] A hydrazide group is incorporated on a peptide by reacting adipic acid dihydrazide or carbohydrazide with carboxylic acid groups in the C-terminus or in aspartic or glutamic acid residues. An aminooxy group is incorporated on a peptide by reacting the N-terminus or a lysine residue with a heterobifunctional molecule containing an NHS ester on one end and a phthalimidoxy group on the other end, followed by cleavage with hydrazine. The reaction is, optionally, catalyzed by addition of aniline.
[0511] The cleavage rate of any linker is tuned, for example, by modifying the electron density in the vicinity of the cleavable link or by sterically affecting access to the cleavage site (e.g., by adding bulky groups, such as methyl groups, ethyl groups, or cyclic groups).
[0512] Cleavable linkers are, alternatively, generated using methods set forth in Bioconjugate Techniques, by Greg Hermanson, 3rd edition.
[0513] Installation of a thiol, amine, or aldehyde groups in RNA or DNA as a functional handle is carried out as described above in EXAMPLE 50.
[0514] Any peptide oligonucleotide complexes of the present may contain a cleavable linker and are described. Any peptide oligonucleotide complexes of the present disclosure (e.g., including an oligonucleotide of any one of SEQ ID NO: 488 - SEQ ID NO: 573, linked or conjugated to SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) are described. Any peptide oligonucleotide complexes of the present disclosure can have oligonucleotides that bind to any target in TABLE 12, or to any of SEQ ID NO: 574 - SEQ ID NO: 611, or to any genomic or ORF sequence provided in TABLE 6.
EXAMPLE 52
Generation of Stable Linkers Between an oligonucleotide and a Peptide
[0515] This example describes generation of a stable linkers between RNA, DNA, or any oligonucleotide, with any one of peptides of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254. A stable linker through a secondary amine is generated by reductive amination, achieved by combining an aldehyde-containing oligonucleotide with the amine at the N-terminus of a peptide or in a lysine residue, followed by reduction with sodium cyanoborohydride. [0516] A stable amide linkage is generated by combining an amine group on an oligonucleotide with the carboxylate at the C-terminus of a peptide or in an aspartic acid or glutamic acid residues.
[0517] A stable carbamate linkage is generated by activating a hydroxyl group in an oligonucleotide with carbonyldiimidazole (CDI) or N,N’-disuccinimidyl carbonate (DSC) and subsequently reacted with a peptide’s N-terminus or lysine residue.
[0518] A maleimide linker is created by combining a thiol-containing oligonucleotide with a maleimide functionalized peptide. The peptide is functionalized using an NHS-X-maleimide heterobifunctional agent on a reactive amine in the peptide, wherein X is any linker. A maleimide linker is used as a stable linker or as a slowly cleavable linker, which is cleaved by exchange with other thiol-containing molecules in biological fluids. The maleimide linker is also stabilized by hydrolyzing the succinimide moiety of the linker using various substituents, including those described in Fontaine et al., Bioconjugate Chem., 2015, 26 (1), pp 145-152.
[0519] Other methods of incorporating, adding, or modifying functional groups in polynucleotides, for example, are carried out using techniques set forth in Bioconjugate Techniques, by Greg Hermanson, 3rd edition.
[0520] Installation of a thiol, amine, or aldehyde groups in an oligonucleotide, as a functional handle, is carried out as described above in EXAMPLE 50.
[0521] Any peptide oligonucleotide complexes of the present disclosure may contain a stable linker and are described. Any peptide oligonucleotide complexes of the present disclosure (e.g., including an oligonucleotide of any one of SEQ ID NO: 488 - SEQ ID NO: 573, linked or conjugated to SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) are described. Any peptide oligonucleotide complexes of the present disclosure can have oligonucleotides that bind to any target in TABLE 12, or to any of SEQ ID NO: 574 - SEQ ID NO: 611, or to any genomic or ORF sequence provided in TABLE 6.
EXAMPLE 53
Generation of an Enzyme Cleavable Linkage between an oligonucleotide and a Peptide [0522] This example describes generation of an enzyme cleavable linkage between RNA, DNA, or any oligonucleotide, and any one of peptides of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254. An enzymatically cleavable linkage is generated between an oligonucleotide and a peptide. The conjugate with a cleavable linkage is administered in vitro or in vivo and is cleaved by enzymes in the cells or body, releasing the oligonucleotide. The enzyme is present in the endosome/lysosome, the cytosol, the cell surface, or is upregulated in the tumor microenvironment or the tissue microenvironment. These enzymes include, but are not limited to, cathepsins (such as all those listed in Kramer et al., Trends Pharmacol Sci. 2017 Oct;38(10):873-898) such as cathepsin B, glucoronidases including beta-glucuronidase, hyaluronidase and matrix metalloproteases, such as MMP-1, 2, 7, 9, 13, or 14 (Kessenbrock et al., Cell. 2010 Apr 2; 141(1): 52-67). Cathepsin or MMPs cleave amino acid sequences of any one of SEQ ID NO: 458 - SEQ ID NO: 489, shown below in TABLE 18 (see also Nagase, Hideaki. "Substrate specificity of MMPs." Matrix Metalloproteinase Inhibitors in Cancer Therapy. Humana Press, 2001. 39-66; Dal Corso et al., Bioconjugate Chem., 2017, 28 (7), pp 1826-1833; Dal Corso et al., Chemistry-A European Journal 21.18 (2015): 6921-6929;
Doronina et al., Bioconjug Chem. 2008 Oct;19(10):1960-3.). Glucuronidase linkers include any one of those described in Jeffrey et al., Bioconjugate Chem., 2006, 17 (3), pp 831-840.
TABLE 18 - Enzymatically Cleavable Linkers
Figure imgf000231_0001
Figure imgf000232_0001
[0523] A Val-Cit-PABC enzymatically cleavable linker, such as described in Jain et al., Pharm Res. 2015 Nov;32(l 1 ) :3526-40., is created by conjugating the PABC end to an amine group on the oligonucleotide. The valine end is further linked to the peptide, for example, by generating an amide bond to the C-terminus of the peptide. A spacer on either side of the molecule is optionally incorporated in order to facilitate steric access of the enzyme to the Val-Cit linkage (SEQ ID NO: 461). The linkage to the peptide is, alternatively, generated by activating the N- terminus of the peptide with SATA and creating a thiol group, which is subsequently reacted to a maleimidocaproyl group attached to the N-terminus of the Val-Cit pair (SEQ ID NO: 461). Upon cleavage by cathepsin B, the self-immolative PABC group spontaneously eliminates, releasing the amine-containing oligonucleotide with no further chemical modifications. Other amino acid pairs include Glu-Glu, Glu-Gly, and Gly-Phe-Leu-Gly (SEQ ID NO: 612).
[0524] Installation of a thiol, amine, or aldehyde group in RNA or DNA, as a functional handle, is carried out as described above in EXAMPLE 50.
[0525] Any peptide oligonucleotide complexes of the present disclosure may contain an enzyme cleavable linker and are described. Any peptide oligonucleotide complexes of the present disclosure (e g., including an oligonucleotide of any one of SEQ ID NO: 488 - SEQ ID NO: 573, linked or conjugated to SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254) are described. Any peptide oligonucleotide complexes of the present disclosure can have oligonucleotides that bind to any target in TABLE 12, or to any of SEQ ID NO: 574 - SEQ ID NO: 611, or to any genomic or ORF sequence provided in TABLE 6.
[0526] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A peptide complex comprising a cell-penetrating peptide comprising a sequence that has:
(a) at least 80% sequence identity with any one of SEQ ID NO: 4, SEQ ID NO:
7, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20 - SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 44 - SEQ ID NO:
54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO:
69, SEQ ID NO: 70, SEQ ID NO: 75, SEQ ID NO: 77, or SEQ ID NO: 80, or a fragment thereof;
(b) at least 85% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40 - SEQ ID NO: 42, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 67, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 81 - SEQ ID NO: 83, or a fragment thereof;
(c) at least 90% sequence identity with any one of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 43, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 72, or SEQ ID NO: 84, or a fragment thereof; or
(d) at least 95% sequence identity with any one of SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 61, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 73, or SEQ ID NO: 74, or a fragment thereof.
2. The peptide complex of claim 1, comprising a sequence that has at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, or a fragment thereof.
3. The peptide complex of claim 1 or claim 2, comprising a sequence of any one of SEQ ID NO 1 - SEQ ID NO: 84
4. The peptide complex of any one of claims 1-3, comprising a sequence of:
(a) SEQ ID NO: 1;
(b) SEQ ID NO: 2;
(c) SEQ ID NO: 3;
(d) SEQ ID NO: 5;
(e) SEQ ID NO: 8; (f) SEQ ID NO: 18;
(g) SEQ ID NO: 19;
(h) SEQ ID NO: 20;
(i) SEQ ID NO: 21;
(j) SEQ ID NO: 40;
(k) SEQ ID NO: 41; or
(l) SEQ ID NO: 42.
5. The peptide complex of any one of claims 1-4, wherein the cell -penetrating peptide is fused or linked to a cargo molecule.
6. A peptide complex comprising a cell-penetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof.
7. A peptide complex comprising a cell-penetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254, or a fragment thereof, and wherein the cargo molecule is a cystine-dense peptide, a DNA-binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or combinations thereof.
8. The peptide complex of claim 7, wherein the cell-penetrating peptide comprises a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, SEQ ID NO: 408 - SEQ ID NO: 457, or SEQ ID NO: 195 - SEQ ID NO: 254.
9. The peptide complex of claim 7 or claim 8, comprising a sequence of:
(a) SEQ ID NO: 195;
(b) SEQ ID NO: 197; or
(c) SEQ ID NO: 198.
10. The peptide complex of any one of claims 1-8, wherein the cell -penetrating peptide comprises a sequence of:
(a) SEQ ID NO: 1;
(b) SEQ ID NO: 2;
(c) SEQ ID NO: 3;
(d) SEQ ID NO: 5;
(e) SEQ ID NO: 8;
(f) SEQ ID NO: 18;
(g) SEQ ID NO: 19;
(h) SEQ ID NO: 20;
(i) SEQ ID NO: 21;
(j) SEQ ID NO: 40;
(k) SEQ ID NO: 41; or
(l) SEQ ID NO: 42.
11. A peptide complex comprising a cell-penetrating peptide and a cargo molecule fused or linked to the cell-penetrating peptide, wherein the cell-penetrating peptide comprises a sequence of any one of SEQ ID NO: 325 - SEQ ID NO: 342 or SEQ ID NO: 343 - SEQ ID NO: 351, and wherein the cargo molecule wherein the cargo molecule is a cystine-dense peptide, a DNA- binding protein, an RNA-binding protein, a transcriptional activator, a transcriptional inhibitor, a promotor of protein degradation, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or combinations thereof.
12. The peptide complex of any one of claims 1-11, wherein the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, at least 3.5, at least 3.7, or at least 4.0 arginine amino acid residues per ten amino acid residues.
13. The peptide complex of any one of claims 1-12, wherein the cell-penetrating peptide comprises no more than 0.5, no more than 0.8, no more than 0.9, no more than 1.0, no more than 1.1, no more than 1.2, no more than 1.3, no more than 1.4, no more than 1.5, no more than 1.6, no more than 1.7, no more than 1.8, no more than 1.9, no more than 2.0, no more than 2.1, no more than 2.2, no more than 2.3, no more than 2.4, no more than 2.5, no more than 3.0, no more than 3.3, no more than 3.5, no more than 3.7, or no more than 4.0 arginine amino acid residues per ten amino acid residues.
14. The peptide complex of any one of claims 1-13, wherein the cell-penetrating peptide comprises no arginine amino acid residues.
15. The peptide complex of any one of claims 1-14, wherein the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, at least 3.5, at least 3.7, or at least 4.0 lysine amino acid residues per ten amino acid residues.
16. The peptide complex of any one of claims 1-15, wherein the cell-penetrating peptide comprises no more than 0.5, no more than 0.8, no more than 0.9, no more than 1.0, no more than 1.1, no more than 1.2, no more than 1.3, no more than 1.4, no more than 1.5, no more than 1.6, no more than 1.7, no more than 1.8, no more than 1.9, no more than 2.0, no more than 2.1, no more than 2.2, no more than 2.3, no more than 2.4, no more than 2.5, no more than 3.0, no more than 3.3, no more than 3.5, no more than 3.7, or no more than 4.0 lysine amino acid residues per ten amino acid residues.
17. The peptide complex of any one of claims 1-14, wherein the cell-penetrating peptide comprises no lysine amino acid residues.
18. The peptide complex of any one of claims 1-17, wherein the cell-penetrating peptide comprises at least 0.5, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, at least 3.5, at least 3.7, or at least 4.0 positively charged amino acid residue per 10 amino acid residues at a pH of about 7.4.
19. The peptide complex of any one of claims 1-18, wherein the cell-penetrating peptide comprises no more than 4.0, no more than 3.9, no more than 3.8, no more than 3.7, no more than 3.6, no more than 3.5, no more than 3.4, no more than 3.3, no more than 3.2, no more than 3.1, no more than 3.0, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, no more than 2.0, no more than 1.5, no more than 1.2, or no more than 1.0 positively charged amino acid residue per 10 amino acid residues at a pH of about 7.4.
20. The peptide complex of any one of claims 1-19, wherein the cell-penetrating peptide comprises at least 0.03, at least 0.05, at least 0.1, at least 0.2, at least 0.5, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, negatively charged amino acid residues per ten amino acid residues at a pH of about 7.4.
21. The peptide complex of any one of claims 1-20, wherein the cell-penetrating peptide comprises a ratio of positively charged amino acid residues to negatively charged amino acid residues of at least 1.0, at least 1.5, at least 2, at least 2.5, at least 2.75, at least 3, at least 3.5, at least 4, at least 5, at least 6, or at least 7.
22. The peptide complex of any one of claims 1-21, wherein the cell-penetrating peptide comprises a ratio of negatively charged amino acid residues to positively charged amino acid residues of no more than 1.0, no more than 1.5, no more than 2, no more than 2.5, no more than 2.75, no more than 3, no more than 3.5, or no more than 4.
23. The peptide complex of any one of claims 1-22, wherein the cell-penetrating peptide comprises at least 1, at least 2, at least 3, or at least 4 negatively charged amino acids.
24. The peptide complex of any one of claims 1-23, wherein the cell-penetrating comprises at least 1, at least 2, at least 3, or at least 4 histidine amino acid residues.
25. The peptide complex of any one of claims 1-24, wherein the cell-penetrating comprises at least 1, at least 2, at least 3, or at least 4 proline amino acid residues.
26. The peptide complex of any one of claims 1-25, wherein the cell-penetrating peptide comprises an amphipathic a-helix.
27. The peptide complex of any one of claims 1-26, wherein the cell-penetrating peptide comprises at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues.
28. The peptide complex of any one of claims 1-27, wherein the cell-penetrating peptide comprises no cysteine amino acid residues.
29. A peptide complex comprising a cell-penetrating peptide comprising:
(a) at least 1.0, at least 1.8, at least 1.9, at least 2.0, at least 2.2, at least 2.5, at least 3.0, at least 3.3, or at least 3 5 arginine amino acid residues per 10 amino acid residues; and
(b) at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues; wherein the cell-penetrating peptide is fused or linked to a cargo molecule.
30. The peptide complex of claim 29, wherein the cell-penetrating peptide comprises at least four cysteine amino acid residues.
31. The peptide complex of claim 29 or claim 30, wherein the cell-penetrating peptide comprises no more than 3.3, no more than 3.2, no more than 3.1, no more than 3.0, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, or no more than 2.0 positively charged amino acid residue per 10 amino acid residues at a pH of 7.4.
32. A peptide complex comprising a cell-penetrating peptide comprising:
(a) at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, or at least 2.7 arginine amino acid residues per 10 amino acid residues; and
(b) no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, or no more than 2.0 positively charged amino acid residue per 10 amino acid residues at a pH of 7.4; wherein the cell-penetrating peptide is fused or linked to a cargo molecule.
33. A peptide complex comprising a cell-penetrating peptide comprising:
(a) at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 3.0, at least 3.3, or at least 3.5 arginine amino acid residues per 10 amino acid residues; and
(b) no more than 3.3, no more than 3.2, no more than 3.1, no more than 3.0, no more than 2.9, no more than 2.8, no more than 2.7, no more than 2.6, no more than 2.5, no more than 2.4, no more than 2.3, no more than 2.2, no more than 2.1, or no more than 2.0 positively charged amino acid residue per 10 amino acid residues at a pH of 7.4; wherein the cell-penetrating peptide is fused or linked to a cargo molecule.
34. The peptide complex of claim 32 or claim 33, wherein the positively charged residues are arginine, lysine, or any combination thereof.
35. The peptide complex of any one of claims 28-32, wherein the cell -penetrating peptide comprises no cysteine amino acid residues.
36. The peptide complex of any one of claims 32-34, wherein the cell-penetrating peptide comprises at least two, at least three, at least four, at least five, or at least six cysteine amino acid residues.
37. The peptide complex of any one of claims 1-34, wherein the cell-penetrating peptide comprises a disulfide through disulfide knot.
38. The peptide complex of any one of claims 1-34, wherein the cell-penetrating peptide comprises a plurality of disulfide bridges formed between cysteine residues.
39. The peptide complex of any one of claims 1-34, wherein the cell-penetrating peptide comprises three or more disulfide bridges formed between cysteine residues, wherein one of the disulfide bridges passes through a loop formed by two other disulfide bridges.
40. The peptide complex of any one of claims 1-39, wherein the cell-penetrating peptide comprises at least 1.1 arginine amino acid residues per 10 amino acid residues.
41. The peptide complex of any one of claims 1-40, wherein the cell-penetrating peptide comprises no more than 2.7 positively charged amino acid residue per 10 amino acid residues.
42. The peptide complex of any one of claims 1-41, wherein the cell-penetrating peptide is derived from maurocalcin, imperatoxin, hadrucalcin, hemicalcin, opicalcin-1, opicalcin-2, midkine, MCoTI-II, chlorotoxin, huwentoxin, vejocalcin, intrepicalcin, or urocalcin.
43. The peptide complex of any one of claims 1-42, wherein the cell-penetrating peptide or the fragment thereof comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least, 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 40, at least 50, at least 60, at least 70, or at least 80 residues.
44. The peptide complex of any one of claims 1-43, wherein the cell-penetrating peptide or the fragment thereof comprises no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 21, no more than 22, no more than 23, no more than 24, no more than 25, no more than 26, no more than 27, no more than 28, no more than 29, no more than 30, no more than 31, no more than 32, no more than 33, no more than 34, no more than 35, no more than 40, no more than 50, no more than 60, no more than 70, or no more than 80 residues.
45. The peptide complex of any one of claims 1-44, wherein the cell-penetrating peptide comprises an isoelectric point within a range from about 6.0 to about 12.0, from about 6.0 to about 10.0, from about 6.5 to about 7.5, from about 7.0 to about 10.0, or from about 8.0 to about 10.0.
46. The peptide complex of any one of claims 1-45, wherein the cell-penetrating peptide is stable at pH of from 6.5 to 7.5.
47. The peptide complex of any one of claims 1-46, wherein the cell-penetrating peptide is stable at pH values within a range from pH 5.0 to pH 7.0.
48. The peptide complex of any one of claims 5-47, wherein the cargo molecule comprises a cargo peptide comprising four or more cysteine amino acid residues and at least two disulfide bonds.
49. The peptide complex of any one of claims 5-48, wherein the cargo molecule is fused or linked to the cell-penetrating peptide at an N-terminus or a C-terminus of the cell-penetrating peptide.
50. The peptide complex of any one of claims 5-49, wherein the cargo molecule comprises at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors.
51. The peptide complex of any one of claims 5-50, wherein the cargo molecule comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors.
52. The peptide complex of any one of claims 5-51, wherein the cargo molecule comprises a molecular weight of at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, or at least 1000 Da.
53. The peptide complex of any one of claims 5-52, wherein the cargo molecule comprises a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0.
54. The peptide complex of any one of claims 5-53, wherein the cargo molecule comprises an antibody, an antibody fragment, an Fc domain, a single chain Fv, an intrabody, or a nanobody.
55. The peptide complex of any one of claims 5-54, wherein the cargo molecule comprises a cystine-dense peptide, an affibody, a 13-hairpin, an avimer, an adnectin, a stapled peptide, a nanofittin, a kunitz domain, a fynomer, or a bicyclic peptide.
56. The peptide complex of any one of claims 5-55, wherein the cargo molecule comprises an immunomodulatory imide drug, a Boc3Arg tag, an adamantyl group, or a carborane.
57. The peptide complex of any one of claims 5-56, where the cargo molecule comprises a target-binding molecule.
58. The peptide complex of claim 57, wherein the target-binding molecule comprises a target-binding peptide.
59. The peptide complex of claim 58, wherein the target-binding peptide comprises a cystine-dense peptide.
60. The peptide complex of claim 58 or claim 59, wherein the target-binding peptide is capable of binding a transcription factor or a tyrosine kinase.
61. The peptide complex of any one of claims 58-60, wherein the target-binding peptide is capable of binding TEAD, cold-inducible RNA-binding protein, androgen receptor, ikaros, aiolos, a nuclear receptor, CKl-a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-u, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-8, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, tau, AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, p-catenin, IKK, TEAD, NF-KB, PKC, AP- 1, Jun, Fos, or REL.
62. The peptide complex of any one of claims 58-61, wherein the target-binding peptide comprises a sequence with at least 90% or at least 95% sequence identity to any one of SEQ ID NO: 295, SEQ ID NO: 298, or SEQ ID NO: 364 - SEQ ID NO: 407.
63. The peptide complex of any one of claims 58-62, wherein the target-binding peptide comprises a sequence of any one of SEQ ID NO: 295, SEQ ID NO: 298, or SEQ ID NO: 364 - SEQ ID NO: 407.
64. The peptide complex of any one of claims 58-61, wherein the target-binding peptide comprises a sequence of SEQ ID NO: 295.
65. The peptide complex of any one of claims 57-64, wherein the peptide complex comprises a sequence of SEQ ID NO: 307 or SEQ ID NO: 308.
66. The peptide complex of any one of claims 57-65, wherein the target-binding molecule binds a ubiquitin ligase.
67. The peptide complex of claim 66, wherein the ubiquitin ligase comprises cereblon, cellular inhibitor of apoptosis protein 1, MDM2, DCAF15, DCAF16, cullin-4A, a Cul2-Rbxl- EloN/C-VHL E3 ubiquitin ligase, APC/C activator protein CDH1, or von Hippel-Lindau protein.
68. The peptide complex of claim 66 or claim 67, wherein the target-binding molecule comprises an immunomodulatory imide drug.
69. The peptide complex of any one of claims 66-68, wherein the target-binding molecule comprises a thalidomide, a pomalidomide, a lenalidomide, a methyl bestatin, a bestatin, a nutlin- 3, or a VHL ligand 1.
70. The peptide complex of any one of claims 5-69, wherein the cargo molecule comprises an anti-cancer agent, a transcription factor binding agent, an inhibitor of protein-protein interactions, a promoter of protein-protein interactions, a transcription factor, an RNA, a CRISPR component, a molecular glue, a proteolysis targeting chimera, an oligonucleotide, a protein-protein interaction inhibitor, or an immunomodulating agent.
-240-
71. The peptide complex of claim 70, wherein the oligonucleotide comprises a DNA, an RNA, an antisense oligonucleotide, an aptamer, an miRNA, an alternative splicing modulator, an mRNA-binding sequence, an miRNA-binding sequence, an siRNA-binding sequence, an RNaseHl -binding oligonucleotide, a RISC-binding oligonucleotide, a polyadenylation modulator, a gapmer, a RIG-I ligand, an mRNA, an antisense RNA, a small interfering RNA, a guide RNA, a U1 adaptor, a micro RNA, or a combination thereof.
72. The peptide complex of claim 70 or claim 71, wherein the oligonucleotide comprises:
(a) a sequence of any one of SEQ ID NO: 488 - SEQ ID NO: 573;
(b) a sequence that binds to any one of SEQ ID NO: 574 - SEQ ID NO: 611;
(c) a sequence of any one of SEQ ID NO: 574 - SEQ ID NO: 611, or a fragment thereof; or
(d) a sequence targeting or encoding a gene target provided in TABLE 12.
73. The peptide complex of claim 70, wherein the CRISPR component is a guide RNA, a tracrRNA, a crRNA, or a Cas nuclease.
74. The peptide complex of any one of claims 5-73, wherein the cargo molecule comprises a detectable agent or a therapeutic agent.
75. The peptide complex of claim 74, wherein the detectable agent is a fluorophore, a nearinfrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a radioisotope, or a radionuclide chelator.
76. The peptide complex of any one of claims 1-75, further comprising one or more chemical modifications.
77. The peptide complex of 76, wherein the chemical modification extends the half-life or modifies a pharmacokinetics of the peptide complex.
78. The peptide complex of claim 76 or claim 77, wherein the chemical modification blocks an N-terminus of the peptide complex.
79. The peptide complex of any one of claims 76-78, wherein the chemical modification comprises methylation, acetylation, or acylation.
80. The peptide complex of any one of claims 76-79, wherein the chemical modification comprises:
-241- (a) methylation of one or more lysine residues or analogue thereof;
(b) methylation of the N-terminus; or
(c) methylation of one or more lysine residue or analogue thereof and methylation of the N-terminus.
81. The peptide complex of any one of claims 1-80, further comprising a half-life modifying agent, a nuclear localization signal, or an endosomal escape motif.
82. The peptide complex of claim 81, wherein the half-life modifying agent comprises a polymer, a polyethylene glycol (PEG), a hydroxy ethyl starch, polyvinyl alcohol, a water soluble polymer, a zwitterionic water soluble polymer, a water soluble poly(amino acid), a water soluble polymer of proline, alanine and serine, a water soluble polymer containing glycine, glutamic acid, and serine, an Fc region, a fatty acid, palmitic acid, an albumin, or a molecule that binds to albumin.
83. The peptide complex of any one of claims 1-82, further comprising an additional active agent.
84. The peptide complex of claim 83, wherein the additional active agent is fused to the cellpenetrating peptide or the cargo molecule at an N-terminus, at the epsilon amine of an internal lysine residue, at the carboxylic acid of an aspartic acid or glutamic acid residue, or a C- terminus of the cell-penetrating peptide or the cargo molecule by a linker.
85. The peptide complex of claim 84, wherein the linker comprises an amide bond, an ester bond, a carbamate bond, a carbonate bond, a hydrazone bond, an oxime bond, a disulfide bond, a thioester bond, a thioether bond, or a carbon-nitrogen bond.
86. The peptide complex of claim 84 or claim 85, wherein the linker comprises a peptide linker.
87. The peptide complex of claim 86, wherein the peptide linker comprises a sequence of any one of SEQ ID NO: 255 - SEQ ID NO: 292 or SEQ ID NO: 458 - SEQ ID NO: 485.
88. The peptide complex of any one of claims 84-87, wherein the linker is a cleavable linker or a pH sensitive linker.
89. The peptide complex of claim 88, wherein the cleavable linker comprises a cleavage site for matrix metalloproteinases, thrombin, cathepsins, or beta-glucuronidase.
-242-
90. The peptide complex of any one of claims 84-89, wherein the linker is a hydrolytically labile linker.
91. The peptide complex of any one of claims 84-89, wherein the linker is a stable linker.
92. The peptide complex of any one of claims 83-91, wherein the additional active agent comprises at least 5, at least 6, at least 7, at least 8, 9, at least 10, at least 15, or at least 20 hydrogen bond donors.
93. The peptide complex of any one of claims 83-92, wherein the additional active agent comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 hydrogen bond acceptors.
94. The peptide complex of any one of claims 83-93, wherein the additional active agent comprises a molecular weight of at least 500 Da, at least 600 Da, at least 700 Da, at least 800 Da, at least 900 Da, or at least 1000 Da.
95. The peptide complex of any one of claims 83-94, wherein the additional active agent comprises a log of a partition coefficient (logP) of at least 5.0, at least 5.2, at least 5.4, at least 5.5, at least 5.6, at least 5.8, or at least 6.0.
96. The peptide complex of any one of claims 83-95, wherein the additional active agent is a detectable agent or a therapeutic agent.
97. The peptide complex of claim 96, wherein the detectable agent is a fluorophore, a nearinfrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a radioisotope, or a radionuclide chelator.
98. The peptide complex of any one of claims 1-97, wherein the cell-penetrating peptide is a membrane-penetrating peptide.
99. The peptide complex of any one of claims 1-98, wherein the cell-penetrating peptide is a nuclear envelope-penetrating peptide.
100. The peptide complex of any one of claims 1-99, wherein the cell-penetrating peptide is a blood brain barrier-penetrating peptide.
101. The peptide complex of any one of claims 1-100, wherein the cell-penetrating peptide is arranged in a multimeric structure with at least one other peptide.
-243-
102. The peptide complex of any one of claims 1-101, wherein the cell-penetrating peptide lacks an immunogenic sequence.
103. The peptide complex of any one of claims 1-102, wherein the cell-penetrating peptide is modified to increase homology to a human protein sequence.
104. The peptide complex of any one of claims 1-103, wherein the cell-penetrating peptide is modified to increase resistance to degradation.
105. The peptide complex of any one of claims 1-104, wherein the cell-penetrating peptide is modified to reduce an affinity of the peptide for a human leukocyte antigen complex, a major histocompatibility complex, or both.
106. The peptide complex of any one of claims 1-105, wherein the cell-penetrating peptide is an active agent.
107. A pharmaceutical composition comprising the peptide complex of any one of claims 1- 106, or a salt thereof, and a pharmaceutically acceptable carrier.
108. The pharmaceutical composition of claim 107, wherein the pharmaceutical composition is formulated for administration to a subject.
109. The pharmaceutical composition of claim 107 or claim 108, wherein the pharmaceutical composition is formulated for oral administration, intravenous administration, subcutaneous administration, intramuscular administration, or a combination thereof.
110. A method of delivering a cargo molecule across a cellular layer of a cell, the method comprising:
(a) contacting the cell with a peptide complex comprising a cell-penetrating peptide fused or linked to cargo molecule, wherein the cell -penetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof;
(b) penetrating the cellular layer with the cell -penetrating peptide; and
(c) delivering the cargo molecule across the cellular layer.
111. A method of delivering a cargo molecule across a cellular layer of a cell, the method comprising:
-244- (a) contacting the cell with the peptide complex of any one of claims 5-106 comprising a cell-penetrating peptide fused or linked to cargo molecule;
(b) penetrating the cellular layer with the cell-penetrating peptide; and
(c) delivering the cargo molecule across the cellular layer, thereby treating the condition.
112. A method of treating a condition in a subject in need thereof, the method comprising:
(a) administering to the subject a composition comprising a peptide complex comprising a cell-penetrating peptide fused or linked to cargo molecule, wherein the cellpenetrating peptide comprises a sequence that has at least 80%, at least 90%, or at least 95% sequence identity with any one of SEQ ID NO: 1 - SEQ ID NO: 84, SEQ ID NO: 85 - SEQ ID NO: 194, or SEQ ID NO: 408 - SEQ ID NO: 457, or a fragment thereof;
(b) penetrating a cellular layer of a cell of the subject with the cell -penetrating peptide; and
(c) delivering the cargo molecule across the cellular layer, thereby treating the condition.
113. A method of treating a condition in a subject in need thereof, the method comprising:
(a) administering to the subject a composition comprising the peptide complex of any one of claims 5-106 comprising a cell-penetrating peptide fused or linked to cargo molecule;
(b) penetrating a cellular layer of a cell of the subject with the cell -penetrating peptide; and
(c) delivering the cargo molecule across the cellular layer.
114. The method of claim 112 or claim 113, wherein the disease or condition is cancer, a neurological disorder, an inflammatory disorder, an immune disorder, a neurodegenerative disorder, or a genetic disorder.
115. The method of claim 114, wherein the cancer is liver cancer, breast cancer, colon cancer, lung cancer, prostate cancer, brain cancer, skin cancer, pancreatic cancer, leukemia, or lymphoma.
116. The method of any one of claims 110-115, wherein the composition is administered by inhalation, intranasally, orally, topically, intravenously, subcutaneously, intramuscularly
-245- administration, intraperitoneally, intratumorally, intrathecally, intravitreally, or a combination thereof.
117. The method of any one of claims 110-116, wherein the composition is administered intravenously as a bolus, injection, infusion, or prolonged infusion.
118. The method of any one of claims 110-117, wherein the cellular layer is a cell membrane, a nuclear envelope, an endosomal membrane, a lysosomal membrane, or a blood brain barrier.
119. The method of any one of claims 110-118, comprising penetrating a cell membrane of the cell.
120. The method of any one of claims 110-119, comprising penetrating a nuclear envelope of the cell.
121. The method of any one of claims 110-120, comprising delivering the cargo molecule to a cytosol of the cell.
122. The method of claim 121, comprising producing a cargo molecule concentration of at least 10 nM, at least 50 nM, at least 100 nM, at least 300 nM, at least 500 nM, at least 700 nM, at least 1000 nM, at least 1200 nM, at least 1400 nM, or at least 1600 nM in the cytosol of the cell.
123. The method of any one of claims 110-122, comprising delivering the cargo molecule to a nucleus of the cell.
124. The method of claim 123, comprising producing a cargo molecule concentration of at least 10 nM, at least 50 nM, at least 100 nM, at least 300 nM, at least 500 nM, at least 700 nM, at least 1000 nM, at least 1200 nM, at least 1400 nM, or at least 1600 nM in the nucleus of the cell.
125. The method of any one of claims 110-124, wherein the cargo molecule comprises an oligonucleotide that binds a target sequence, and wherein the method further comprises modulating alternative splicing of the target sequence, dictating the location of a polyadenylation site of the target sequence, inhibiting translation of the target sequence, inhibiting binding of the target sequence to a secondary target sequence, recruiting RISC to the target sequence, recruiting RNaseHl to the target sequence, inducing cleavage of the target
-246- sequence, or regulating the target sequence upon binding of the oligonucleotide to the target sequence.
126. The method of any one of claims 110-125, comprising delivering the cargo molecule into an intracellular space or a paracellular space.
127. The method of claim 126, wherein the intracellular space is a nanolumen.
128. The method of any one of claims 110-127, wherein the cell has uncontrolled or dysregulated cell growth.
129. The method of any one of claims 110-128, wherein the cell is a cancerous cell or a tumor cell.
130. The method of any one of claims 110-129, wherein the cell is a pancreatic cell, liver cell, colon cell, smooth muscle cell, ovarian cell, breast cell, lung cell, brain cell, skin cell, ocular cell, blood cell, lymph cell, immune system cell, reproductive cell, reproductive organ cell, prostate cell, fibroblast, kidney cell, adenocarcinoma cell, glioma stem cell, tumor cell, or any combination thereof.
131. The method of any one of claims 110-130, further comprising binding the cargo molecule to a target molecule.
132. The method of claim 131, wherein the target molecule comprises a transcription factor or a tyrosine kinase.
133. The method of claim 131 or claim 132, wherein the target molecule comprises TEAT), cold-inducible RNA-binding protein, androgen receptor, ikaros, aiolos, a nuclear receptor, CK1- a, GSPT1, RBM39, BTK, BCR-Abl, FKBP12, aryl hydrocarbon receptor, estrogen receptor, BET, c-ABL, RIPK2, ERR-a, SMAD3, FRS2-a, PI3K, BRD9, CDK6, BRD4, zinc finger proteins, CDK4, p38-a, p38-5, IRAK4, CRABP-I, CRABP-II, retinoic acid receptor, TACC3, BRD2, BRD3, GST-al, eDHFR, CSNK1A1, GSPT1, ZFP91, ZNF629, ZNF91, ZNF276, ZNF653, ZNF827, WIZ, ZNF692, GZF1, ZNF98, BCL6, cMYC, tau, AKT, mTOR, STAT3, RAS, RAF, MEK, ERK, p-catenin, IKK, TEAD, NF-KB, PKC, AP-1, Jun, Fos, or REL.
134. The method of any one of claims 131-133, further comprising inhibiting the target molecule.
-247-
135. The method of any one of claims 110-134, further comprising binding the cargo molecule to a ubiquitin ligase.
136. The method of claim 135, wherein the ubiquitin ligase comprises cereblon, cellular inhibitor of apoptosis protein 1, MDM2, DCAF15, DCAF16, cullin-4A, a Cul2-Rbxl-EloN/C- VHL E3 ubiquitin ligase, APC/C activator protein CDH1, or von Hippel -Lindau protein.
137. The method of claim 135 or claim 136, wherein the cargo molecule comprises an immunomodulatory imide drug, a thalidomide, a pomalidomide, a lenalidomide, a methyl bestatin, a bestatin, a nutlin-3, or a VHL ligand 1.
138. The method of any one of claims 135-137, further comprising ubiquitinating the target molecule upon binding of the cargo molecule to the target molecule and the ubiquitin ligase.
139. The method of any one of claims 110-138, wherein the cargo molecule comprises a detectable agent.
140. The method of claim 139, further comprising imaging the cell.
141. The method of claim 139 or claim 140, further comprising detecting a presence, absence, location, or a combination thereof of the detectable agent in the cell.
142. The method of any one of claims 110-141, comprising delivering at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% of the peptide complex across the cellular layer.
-248-
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CN116444678A (en) * 2022-10-18 2023-07-18 百葵锐(深圳)生物科技有限公司 Novel polypeptide with fungus antibacterial and bacteriostatic properties
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WO2024211847A1 (en) * 2023-04-07 2024-10-10 Vanderbilt University Albumin binding recombinant cas9 proteins and uses thereof
WO2025017349A1 (en) * 2023-07-18 2025-01-23 Hosseinkhani Saman Peptide-based dna vaccine platform
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