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WO2025076213A1 - Polypeptides de type élastine à phases séparables isothermiquement, compositions et leurs procédés d'utilisation - Google Patents

Polypeptides de type élastine à phases séparables isothermiquement, compositions et leurs procédés d'utilisation Download PDF

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Publication number
WO2025076213A1
WO2025076213A1 PCT/US2024/049779 US2024049779W WO2025076213A1 WO 2025076213 A1 WO2025076213 A1 WO 2025076213A1 US 2024049779 W US2024049779 W US 2024049779W WO 2025076213 A1 WO2025076213 A1 WO 2025076213A1
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elp
cleavage site
cell
domain
site
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Danielle J. MAI
Brendan M. WIRTZ
Xiaojing GAO
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • ELPs Elastin-like polypeptides
  • ELPs underlie advances in drug delivery, tissue engineering, and biomolecular actuators. ELPs have garnered attention for their thermoresponsive properties, such that they undergo phase separation into insoluble coacervates when raised above a transition temperature. Regulation of ELPs has also been demonstrated using pH and light, which influence ELP phase behavior by modulating hydrophilicity. pH-responsive ELPs contain acidic or basic guest residues with pH-dependent protonation states.
  • ELP hydrophilicity can also be tuned by integrating guest residues with nonstandard amino acids that undergo photochemical transformations. For example, photoisomerizable residues undergo reversible changes in polarity upon irradiation with different wavelengths of light. These changes in polarity impact ELP hydrophilicity and emergent ELP phase behavior. Temperature, however, is relatively constant in many biological contexts. Likewise, biological systems do not typically use pH or light to modulate their physical environments, limiting the effectiveness and applicability of existing ELPs. Thus, a need exists for new, isothermally phase-separable ELPs that can be modulated by more biologically relevant cues.
  • ELPs isothermally phase-separable elastin-like polypeptides
  • the ELPs comprising: i) a first ELP domain having a first lower critical solution temperature (LCST); and ii) a second ELP domain having a second LCST that is different from the first LCST, wherein the first and second ELP domains are separated by an enzymatic cleavage site.
  • an ELP may comprise a first LCST that is greater than the second LCST.
  • the first and second ELP domain of a subject may comprise one or more pentapeptide repeats.
  • each pentapeptide repeat of the first and second ELP domain may comprise an amino acid sequence VPGXG (SEQ ID NO:1), wherein X is any amino acid other than proline.
  • each pentapeptide repeat of the first and second ELP domain may comprise an amino acid sequence VPGXG (SEQ ID NO:2), wherein X is selected from valine, alanine, glycine, lysine, histidine, serine, and glutamate.
  • the first ELP domain of a subject ELP comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO: 5), VPGAG (SEQ ID NO:6), VPGGG (SEQ ID NO: 7), VPGKG (SEQ ID NO: 8), VPGSG (SEQ ID NO: 9), and VPGEG (SEQ ID NO: 10), and the second ELP domain comprises one or more pentapeptide repeats, each pentapeptide repeat of the second ELP domain comprising an amino acid sequence selected from: VPGVG (SEQ ID NO: 5) and VPGHG (SEQ ID NO: 11).
  • the first and second ELP domain each comprises from 1 to 250 pentapeptide repeats. In some cases, the first ELP domain comprises from 50 to 150 pentapeptide repeats, and the second ELP domain comprises 30 to 90 pentapeptide repeats. In some cases, the first ELP domain comprises from 75 to 125 pentapeptide repeats, and the second ELP domain comprises 45 to 75 pentapeptide repeats.
  • a subject ELP comprises a first and second ELP domain, wherein the first ELP domain comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:12, and the second ELP domain comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:13.
  • a subject ELP comprises a first and second ELP domain, wherein the first ELP domain comprises the amino acid sequence set forth in SEQ ID NO:12, and the second Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 ELP domain comprises the amino acid sequence set forth in SEQ ID NO:13.
  • the first and second ELP domains are separated by two or more enzymatic cleavage sites.
  • the enzymatic cleavage site of a subject ELP is a protease cleavage site.
  • the protease cleavage site is a cleavage site for a eukaryotic protease, a prokaryotic protease, or a viral protease.
  • the protease cleavage site may be, for example, a TEV protease cleavage site, an enterokinase cleavage site, a cleavage site for a protease of the clotting cascade (e.g., a thrombin cleavage site, a factor IXa cleavage site), a matrix metalloproteinase (MMP) cleavage site (e.g., a MMP-9, MMP-11, or MMP-26 cleavage site), a furin cleavage site, a caspase cleavage site (e.g., a Caspase 1 cleavage site) and the like.
  • a TEV protease cleavage site e.g., an enterokinase cleavage site, a cleavage site for a protease of the clotting cascade (e.g., a thrombin cleavage
  • a subject ELP further comprises i) a first linker between the first ELP domain and the enzymatic cleavage site; and/or ii) a second linker between the second ELP domain and the enzymatic cleavage site.
  • the first and second linkers may each comprise the amino acid sequence set forth in SEQ ID NO: 14, wherein n is an integer equal to or greater than 1.
  • the first and second linkers may each comprise the amino acid sequence set forth in SEQ ID NO: 15, wherein n is an integer equal to or greater than 1.
  • a subject ELP comprises, in order from N-terminus to C-terminus: a) a subject first ELP domain, b) a subject first linker, c) an enzymatic cleavage site, d) a subject second linker, and e) a subject second ELP domain.
  • a subject ELP comprises, in order from N-terminus to C-terminus: a) a subject second ELP domain, b) a subject second linker, c) an enzymatic cleavage site, d) a subject first linker, and e) a subject first ELP domain.
  • the present disclosure provides isothermally phase-separable ELPs, wherein the ELP further comprises a payload polypeptide.
  • the payload polypeptide comprises a single-chain antibody, a cytokine, a peptide hormone, or a therapeutic peptide.
  • the payload polypeptide is fused to the second ELP domain via a linker.
  • the payload polypeptide is fused N-terminally to the second ELP domain.
  • the payload polypeptide is fused C-terminally to the second ELP domain.
  • a subject ELP further comprises an N-terminal leader domain.
  • the N-terminal leader domain encodes a signal peptide.
  • a subject ELP is non-immunogenic.
  • the present disclosure also provides isothermally phase-separable ELPs, wherein the ELP is conjugated to a payload molecule.
  • the payload molecule may be, for example, a nucleic acid, a polypeptide, a carbohydrate, or a small molecule.
  • the payload molecule is conjugated to the second ELP domain.
  • the present Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 disclosure additionally provides ELP compositions, an ELP composition comprising a subject ELP as described herein, and a pharmaceutically acceptable excipient.
  • the present disclosure provides methods for assembling a cell-protective biopolymer on the surface of a target cell, the methods comprising: contacting the surface of a target cell with a subject ELP, as described herein, to assemble a cell-protective biopolymer, wherein the surface of the target cell comprises an enzyme that can cleave the enzymatic cleavage site of the ELP.
  • the present disclosure provides methods for assembling a cell-protective biopolymer on the surface of a target cell, the methods comprising: expressing a subject ELP, as described herein, in the target cell to assemble a cell-protective biopolymer on the surface of said cell, wherein the target cell comprises an enzyme that can cleave the enzymatic cleavage site of the ELP.
  • the target cell comprises a subject expression vector as described herein.
  • the enzyme is present on the surface of the target cell.
  • the enzyme is present inside of the target cell.
  • the target cell is a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell. In some cases, the mammalian cell is a human cell. In certain embodiments of the methods, the target cell is in vivo. In certain embodiments of the methods, the target cell expresses a transgene.
  • the present disclosure provides methods for reducing blood loss at a hemorrhage site in a subject, the method comprising: administering to a hemorrhage site in a subject an effective amount of a subject ELP composition, as described herein, to reduce blood loss at the hemorrhage site, wherein an enzyme that can cleave the enzymatic cleavage site of the ELP is present in or around the hemorrhage site.
  • the enzymatic cleavage site of the subject ELP is a protease cleavage site.
  • the protease cleavage site is a cleavage site for a protease of the clotting cascade (e.g., a thrombin cleavage site, a factor IXa cleavage site).
  • the hemorrhage Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 site is an external hemorrhage site.
  • the administering may comprise applying the ELP composition to the external hemorrhage site.
  • the hemorrhage site is an internal hemorrhage site.
  • the administering may comprise intravenously administering the ELP composition to the subject.
  • the present disclosure also provides methods for delivering a therapeutic agent to a target site in a subject, the method comprising: administering a subject ELP composition, as described herein, to a subject, wherein an enzyme that can cleave the enzymatic cleavage site of the ELP is present in or around the target site.
  • the administering comprises intravenously administering the ELP composition to the subject.
  • the administering comprises intramuscular injection, intradermal injection, or subcutaneous injection of the ELP composition in a subject.
  • the target site is a tumor.
  • the enzymatic cleavage site of the ELP is a protease cleavage site.
  • protease cleavage site is a matrix metalloproteinase cleavage site (e.g., a MMP-9, MMP-11, or MMP-26 cleavage site).
  • the present disclosure further provides methods for producing a tissue scaffold at a site of injury in a subject, the method comprising: administering a subject ELP composition, as described herein, to a subject, wherein an enzyme that can cleave the enzymatic cleavage site of the ELP is present in or around the site of injury.
  • the administering comprises applying the ELP composition to the site of injury.
  • the administering comprises intramuscular injection, intradermal injection, or subcutaneous injection of the ELP composition in a subject.
  • kits comprising a) a subject ELP, as described herein, and b) a pharmaceutical excipient.
  • the ELP is lyophilized.
  • the kit further comprises a substrate material.
  • the substrate material comprises a gauze.
  • the kit further comprises a syringe.
  • FIGs.1A-1C Molecular design of protease-cleavable elastin-like polypeptides.
  • A Phase behavior that is sensitive to temperature and sequence.
  • B A protease derived from Tobacco Etch Virus (TEV) will generate insoluble ELP components that assemble into protective Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 coatings (SEQ ID NO: 111).
  • FIGs.2A-2C (A) Protease-responsive ELP generates a self-assembling fragment upon protease exposure.
  • C Turbidity of ELP solutions without and with TEV protease over time.
  • FIGs.3A-3C (A) Polyacrylamide gel of TEV protease-responsive ELP without and with TEV protease under different incubation times.
  • B Expected molecular weights (MW) of the TEV protease-responsive ELP, ELP fragments, and the TEV protease.
  • FIG.4 MALDI-TOF mass spectrometry analysis of TEV protease-responsive ELP.
  • FIGs. 5A-5B Isothermal phase separation of ELPs proceeds through proteolytic cleavage.
  • A Protease-cleavable ELPs are diblock ELPs with hydrophilic and hydrophobic blocks separated by a protease cleavage site. These ELPs are soluble before protease cleavage and produce an insoluble ELP post-cleavage.
  • B Primary sequences for protease-cleavable ELP variants.
  • FIGs.6A-6B Incubation of TEVP-cleavable ELP with TEVP produces a hydrophobic ELP cleavage product that undergoes phase separation under isothermal conditions.
  • A Time-resolved turbidimetry at 37 °C shows dynamic phase separation of TEVP-cleavable ELP co- incubated with TEVP (purple).
  • Cleavable-ELP (blue) and TEVP-only (red) controls show no changes in turbidity.
  • FIG.7 At all concentrations, transition temperatures of TEVP-cleavable ELP (blue) are higher than cleavage products (purple) and recombinant ELP[V]60 (black). The concentration of cleavage products is reported as the initial concentration of TEVP-cleavable ELP before incubation with TEVP. Unless shown, horizontal and vertical error bars are smaller than data points. In region I (light red), cleavage reactions result in protease-driven phase separation. In Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 region II (light blue), cleavage reactions do not result in ELP phase separation.
  • FIG.8 Four cleavable ELP variants undergo isothermal phase separation in response to their respective proteases: TEVP, thrombin, factor Xa, or enterokinase. Final OD 350 values are reported from time-resolved turbidimetry of 30 ⁇ M cleavable ELP solutions at 37 °C for 6 hours. Time traces of OD 350 and replicates are included in FIG.19.
  • FIG.9 Cloning of cleavable ELP variants proceeded with two rounds of Golden Gate assembly and two rounds of directional ligation. Custom gene fragments (Twist Bioscience) flanked by BsmBI recognition sites were purchased to assemble genes for the hydrophilic and hydrophobic ELP blocks. Golden Gate assembly overhangs (5’ ⁇ 3’) are indicated above the BsmBI recognition sites.
  • two Golden Gate assembly reactions with BsmBI-v2 proceeded in parallel to assemble genes for the hydrophilic and hydrophobic ELP blocks in the pGGAselect vector (step 1).
  • Genes encoding the ELP blocks were directionally ligated using SacI-HF and HindIII-HF to encode for the full-length diblock ELP (step 2).
  • This diblock ELP gene was moved into a modified pET-22b(+) expression vector (SI Section 4) using XhoI and NdeI (step 3).
  • ssDNA oligos encoding protease recognition sequences (SI Section 5) were annealed and inserted into the diblock ELP gene using a second round of Golden Gate Assembly with BsaI-HF v2 (step 4).
  • Golden Gate assembly overhangs (5’ ⁇ 3’) are indicated above the BsaI recognition sequences.
  • FIG.10 The hydrophilic ELP cleavage product between the 37 kDa and 50 kDa standards (lanes 2 and 5, lower gel; same image as FIG.1B) cannot be visualized with Coomassie Brilliant Blue dye (lanes 2 and 5, upper gel).
  • Upper and lower gels show 12% SDS-PAGE analysis of sample compositions at the end of a 3-hour turbidimetry experiment with TEVP-cleavable ELP and TEVP. Protein bands were visualized using Coomassie Brilliant Blue staining (upper gel) or Stain-Free imaging (lower gel).
  • FIG.11 Replicates of time-resolved turbidimetry at 37 °C. Purple: TEVP-cleavable ELP and TEVP; Blue: TEVP-cleavable ELP; Red: TEVP. Attorney Docket No.: STAN-2151WO Stanford No.: S23-364
  • FIGs.12A-12E MALDI-TOF MS of ELPs.
  • A MALDI-TOF MS of TEVP-cleavable ELP (expected 64.0 kDa). Raw data (gray curve) was smoothed (blue curve) with a moving average over 21 data points.
  • B MALDI-TOF MS of Thr-cleavable ELP (expected 63.7 kDa). Raw data (gray curve) was smoothed (blue curve) with a moving average over 21 data points.
  • C MALDI- TOF MS of FXa-cleavable ELP (expected 63.6 kDa). Raw data (gray curve) was smoothed (blue curve) with a moving average over 21 data points.
  • FIGs.13A-13C Transition temperature determination from turbidity of A) TEVP-cleavable ELP, B) cleavage products, and c) ELP[V] 60 . Temperature-dependent OD 350 at each concentration were min-max normalized.
  • FIGs.14A-14B Incubation of TEVP-cleavable ELP with TEVP did not result in ELP phase separation at 32 °C.
  • Each plot is a replicate of the time-resolved turbidimetry experiment at 32 °C. The bottom right plot does not contain the TEVP-only control.
  • TEVP was added to a 30 ⁇ M ELP[V]60 solution at a volume ratio of 1:25, respectively, to match the ratio used in time-resolved turbidimetry. Turbidimetry was subsequently performed on 30 ⁇ M ELP[V]60 solutions with (green) and without (red) TEVP. Transition temperatures (temperature at which dOD350,norm/dT was maximized) remained unchanged upon addition of TEVP. Replicates are offset vertically.
  • FIGs. 16A-16B The presence of 30 ⁇ M hydrophilic ELP[V1A8G7]6 cleavage product in solution does not influence the transition temperature of 30 ⁇ M hydrophobic ELP[V]60 cleavage product.
  • 30 ⁇ M TEVP-cleavable ELP cleavage products were obtained by incubating 100 ⁇ M TEVP-cleavable ELP with TEVP (10 units/ ⁇ L) at a 60:1 volume ratio at 34 °C for 3 hours. This sample was subsequently diluted to 30 ⁇ M with PBS. The hydrophobic cleavage product was isolated from a sample of 100 ⁇ M cleavage products via centrifugation at 17000 ⁇ g at 37 °C for 15 mins. The insoluble pellet was resuspended in PBS and diluted to a concentration of 30 ⁇ M, which was confirmed by UV absorbance at 280 nm.
  • FIG.17 Time-resolved turbidimetry of unique cleavable ELP–protease pairings. Each row corresponds to a different cleavable ELP variant (TEVP-, Thr-, FXa-, EK-cleavable ELP from top to bottom). Each column is an experimental replicate.
  • FIG.18 SDS-PAGE analysis of unique cleavable ELP–protease pairings at the endpoint of time-resolved turbidimetry. Each column is a replicate with samples taken from an independent time-resolved turbidimetry experiment. Each row corresponds to a different cleavable-ELP variant (TEVP-, Thr-, FXa-, EK-cleavable ELP from top to bottom).
  • Lane 1 Cleavable ELP
  • Lane 2 TEVP and cleavable ELP
  • Lane 3 Thr and cleavable ELP
  • Lane 4 FXa and cleavable ELP
  • Lane 5 EK and cleavable ELP
  • Lane 6 Protease (varies by row, corresponds to the intended compatible protease for the cleavable ELP). Due to low protease concentration, bands for thrombin, factor Xa, and enterokinase are not visible.
  • FIGs.19A-19F Cleavage kinetics of cleavable ELP influences time-resolved profile of OD350.
  • Turbidity profiles were similar despite the slight dilution of FXa and FXa-cleavable ELP in the 160 ⁇ L reaction volume condition.
  • the turbidity profile with 3 ⁇ g of FXa in panel C) is anomalous and is expected to more closely match the FXa and FXa-cleavable ELP profiles reported in FIG. 19 (153 ⁇ L reaction volume).
  • F) SDS-PAGE analysis of samples reported in panel e) reveals near complete conversion of FXa-cleavable ELP (lane 1) with 3 ⁇ g of FXa in a reaction volume of 160 ⁇ L (lane 2) and 153 ⁇ L (lane 3).
  • phase transition and “phase separation”, as used herein, refer to physical processes whereby a material, substance, composition, or the like undergoes a change from one physical state to another (e.g., from a liquid to a solid, from a state of solubility to a state of Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 insolubility) and/or is separated or de-mixed from another material, substance, or composition by undergoing a change from one physical state to another (e.g., in the case of a liquid-liquid phase separation).
  • a material, substance, composition, or the like undergoes a change from one physical state to another (e.g., from a liquid to a solid, from a state of solubility to a state of Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 insolubility) and/or is separated or de-mixed from another material, substance, or composition by undergoing a change from one physical state to another
  • LCST lower critical solution temperature
  • polypeptide refers to a polymeric form of amino acids of any length, which can include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • amino acid includes, but is not limited to, naturally-occurring ⁇ -amino acids and their stereoisomers.
  • Stepoisomers of amino acids refer to mirror image isomers of the amino acids, such as L-amino acids or D-amino acids.
  • a stereoisomer of a naturally- occurring amino acid refers to the mirror image isomer of the naturally-occurring amino acid (i.e., the D-amino acid).
  • Naturally-occurring ⁇ -amino acids are those encoded by the genetic code as well as those amino acids that are later modified (e.g., hydroxyproline, ⁇ -carboxyglutamate, and O- phosphoserine).
  • Naturally-occurring ⁇ -amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof.
  • Stereoisomers of a naturally-occurring ⁇ -amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D- Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature.
  • an L-amino acid may be represented herein by its commonly known three letter symbol (e.g., Arg for L-arginine) or by an upper-case one-letter amino acid symbol (e.g., R for L-arginine).
  • a D-amino acid may be represented herein by its commonly known three letter symbol (e.g., D-Arg for D-arginine) or by a lower-case one-letter amino acid symbol (e.g., r for D-arginine).
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
  • terms “polynucleotide” and “nucleic acid” encompass single-stranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • small molecule is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 900 amu or less, 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In some instances, the small molecule is not made of repeating molecular units such as are present in a polymer.
  • a “restriction site” refers to a nucleotide sequence recognized and cleaved by a given restriction endonuclease.
  • the restriction site present in the circular nucleic acid template is for a restriction endonuclease that generates cohesive (or “sticky”) ends, including but not limited to, AscI, Aval, BamHI, BclI, BglII, BstEI, Bst, BI, BstYI, EcoRI, MluI, NarI, NheI, NotI, PstI, PvuI, SacI, SalI, SpeI, StyI, XbaI, XhoI and XmaI.
  • AscI AscI, Aval, BamHI, BclI, BglII, BstEI, Bst, BI, BstYI, EcoRI, MluI, NarI, NheI, NotI, PstI, PvuI, SacI, SalI, SpeI, StyI, XbaI, XhoI and XmaI.
  • the restriction site present in the circular nucleic acid template is for a restriction endonuclease that generates blunt ends, including but not limited to, EcoRV, FspI, NaeI, NruI, PvuII, SmaI, SnaBI, and StuI.
  • antibody may include an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to the cell surface molecule of the target cell, including, but not limited to single chain Fv (scFv), Fab, (Fab’)2, (scFv’)2, and diabodies; Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments
  • the antibody is selected from an IgG, Fv, single chain antibody, scFv, Fab, F(ab')2, or Fab'.
  • the antibody is a nanobody (an antibody fragment consisting of a single monomeric variable antibody domain – also known as a single-domain antibody (sdAb)), a monobody (a synthetic binding protein constructed using a fibronectin type III domain (FN3) as a molecular scaffold), or a Bi-specific T- cell engager (BiTE).
  • the present disclosure provides isothermally phase-separable elastin-like polypeptides (ELPs), the ELPs comprising: i) a first ELP domain having a first lower critical solution temperature (LCST); and ii) a second ELP domain having a second LCST that is different from the first LCST, wherein the first and second ELP domains are separated by an enzymatic cleavage site.
  • ELPs phase-separable elastin-like polypeptides
  • conjugates of the subject ELPs conjugates of the subject ELPs, nucleic acids encoding the subject ELPs, cells comprising nucleic acids encoding the subject ELPs, methods of use for the subject ELPs, and related kits.
  • the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.
  • Elastin-like Polypeptides Elastin-like polypeptide domains
  • the present disclosure provides isothermally phase-separable elastin-like polypeptides (ELPs) that can be phase-separated from surrounding solute in an inducible manner under isothermal conditions.
  • Elastin-like polypeptides are a class of synthetic polypeptides derived, in part, from the human tropoelastin protein.
  • ELPs comprise ELP domains that are generally composed of repeated units of a pentapeptide sequence.
  • the pentapeptide repeat of an ELP domain is of the sequence VPGXG, where X is any amino acid other than proline.
  • ELPs undergo a phase separation when raised above a transition temperature (i.e., when raised above the lower critical solution temperature of the ELP) to form insoluble coacervates.
  • a transition temperature i.e., when raised above the lower critical solution temperature of the ELP
  • ELPs in solution can transition from a soluble liquid phase to an insoluble disperse phase composed of ELP particles.
  • the ELPs of the present disclosure can undergo a phase transition in isothermal conditions, that is without a change in temperature.
  • the ELPs of the present disclosure comprise: i) a first ELP domain having a first lower critical solution temperature (LCST); and ii) a second ELP domain having a second LCST that is different from the first LCST, wherein the first and second ELP domains are separated by an enzymatic cleavage site.
  • LCST lower critical solution temperature
  • a second ELP domain having a second LCST that is different from the first LCST wherein the first and second ELP domains are separated by an enzymatic cleavage site.
  • the first LCST i.e., the LCST of the first ELP domain
  • the second LCST i.e., the LCST of the second ELP domain
  • the first LCST is greater than the second LCST, such that the whole ELP, comprising the first and second ELP domains has a phase transition temperature that is from 2 °C to 10 °C higher than the second LCST of the second ELP domain alone, e.g., the whole ELP has a phase transition temperature that is from 2 °C to 9 °C, from 2 °C to 8 °C, from 2 °C to 8 °C, from 3 °C to 7 °C, from 3 °C to 6 °C, from 4 °C to 5 °C higher than the second LCST Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 of the second ELP domain alone.
  • the phase transition temperature of the whole ELP can be modulated by the choice of the first and second LCSTs of the first and second ELPs, respectively.
  • the first LCST of the first ELP domain is 39 °C or higher, e.g., in some cases the first LCST is 40 °C or higher, 41 °C or higher, 42 °C or higher, 43 °C or higher, 44 °C or higher, 45 °C or higher, 46 °C or higher, 47 °C or higher, 48 °C or higher, 49 °C or higher, 50 °C or higher, 60 °C or higher, or 70 °C or higher.
  • the second LCST of the second ELP domain ranges from 30 °C to 38°C, e.g., the LCST of the second ELP domain ranges from 31°C to 37 °C, from 32 °C to 36 °C, or from 33 °C to 35 °C.
  • the whole ELP has a phase transition temperature that is from 4 °C to 5 °C higher than the second LCST of the second ELP domain, wherein the first ELP domain has a first LCST that is 45 °C or higher, and the second ELP domain has a second LCST from 33 °C to 35 °C.
  • the first and/or second ELP domain comprises one or more pentapeptide repeats.
  • a pentapeptide repeat is a contiguous, five residue long, amino acid sequence of the form VPGXG, where X is any amino acid other than proline.
  • the one or more pentapeptide repeats of an ELP domain may all be identical in sequence to one another or they may differ in sequence.
  • the lower critical solution temperature (LCST) of an ELP domain (i.e., the transition temperature at, or above, which the ELP domain undergoes a phase transition) can be influenced by both the choice of residue for X in each pentapeptide repeat and the total number of pentapeptide repeats in the ELP.
  • LCST lower critical solution temperature
  • increasing the hydrophilicity of the X residues and/or decreasing the number of pentapeptide repeats will increase the LCST of the ELP domain.
  • increasing the hydrophobicity of the X residues and/or increasing the number of pentapeptide repeats will decrease the LCST of the ELP domain.
  • the sequence and length of an ELP domain can be varied to achieve a desired LCST for the ELP domain.
  • a pentapeptide repeat comprises the amino acid sequence VPGXG (SEQ ID NO:1), wherein X is any amino acid other than proline.
  • each pentapeptide repeat of the first and/or second ELP domain comprises the amino acid sequence VPGXG (SEQ ID NO:2), wherein X is selected from valine, alanine, glycine, lysine, histidine, serine, and glutamate.
  • Elastin-like polypeptides comprising pentapeptide repeats as described herein exhibit little to no immunological response.
  • a subject ELP is non-immunogenic.
  • the LCST of an ELP domain can be increased (e.g., the ELP can be made more hydrophilic), as an example, by the inclusion of pentapeptide repeats having the amino acid sequence VPGXG (SEQ ID NO:3), wherein X is selected from alanine, glycine, lysine, serine, and glutamate.
  • the LCST of an ELP domain can be decreased (e.g., the ELP can be made more hydrophobic), as an example, by the inclusion of pentapeptide repeats having the amino acid sequence VPGXG (SEQ ID NO:4), wherein X is selected from valine or histidine.
  • the first ELP domain of a subject ELP comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5), VPGAG (SEQ ID NO:6), VPGGG (SEQ ID NO:7), VPGKG (SEQ ID NO:8), VPGSG (SEQ ID NO:9), and VPGEG (SEQ ID NO:10).
  • the second ELP domain of a subject ELP comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5) and VPGHG (SEQ ID NO:11).
  • the first ELP domain comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5), VPGAG (SEQ ID NO:6), VPGGG (SEQ ID NO:7), VPGKG (SEQ ID NO:8), VPGSG (SEQ ID NO:9), and VPGEG (SEQ ID NO:10); and ii) the second ELP domain comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5) and VPGHG (SEQ ID NO:11).
  • the first ELP domain of a subject ELP comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGAG (SEQ ID NO:6), VPGSG (SEQ ID NO:9), and VPGEG (SEQ ID NO:10).
  • the second ELP domain of a subject ELP comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5) and VPGHG (SEQ ID NO:11).
  • the Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 first ELP domain comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGAG (SEQ ID NO:6), VPGSG (SEQ ID NO:9), and VPGEG (SEQ ID NO:10); and ii) the second ELP domain comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5) and VPGHG (SEQ ID NO:11).
  • the first ELP domain of a subject ELP comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5), VPGAG (SEQ ID NO:6), VPGGG (SEQ ID NO:7).
  • the second ELP domain of a subject ELP comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5) and VPGHG (SEQ ID NO:11).
  • the first ELP domain comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5), VPGAG (SEQ ID NO:6), VPGGG (SEQ ID NO:7); and ii) the second ELP domain comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5) and VPGHG (SEQ ID NO:11).
  • the first and/or second ELP domains of a subject ELP each comprises from 1 to 250 pentapeptide repeats.
  • the first and/or second ELP domains of a subject ELP each comprises from 5 to 140 pentapeptide repeats, from 10 to 130 pentapeptide repeats, from 20 to 120 pentapeptide repeats, from 30 to 110 pentapeptide repeats, from 40 to 100 pentapeptide repeats, from 50 to 90 pentapeptide repeats, from 60 to 80 pentapeptide repeats, or from 65 to 75 pentapeptide repeats.
  • first and/or second ELP domains of a subject ELP can each comprise from 40 to 160 pentapeptide repeats, e.g., from 50 to 150 pentapeptide repeats, from 75 to 125 pentapeptide repeats, and from 85 to 95 pentapeptide repeats.
  • first and/or second ELP domains of a subject ELP can each comprise from 20 to 100 pentapeptide repeats, e.g., from 30 to 90 pentapeptide repeats, from 40 to 80 pentapeptide repeats, from 50 to 70 pentapeptide repeats, and from 55 to 65 pentapeptide repeats.
  • the first ELP domain comprises from 50 to 150 pentapeptide repeats; and ii) the second ELP domain comprises 30 to 90 pentapeptide repeats. In some embodiments of a subject ELP: i) the first ELP domain comprises from 75 to 125 pentapeptide repeats; and ii) the second ELP domain comprises 45 to 75 pentapeptide repeats.
  • the first ELP domain comprises from 85 to 95 pentapeptide repeats; and ii) the second ELP domain comprises 55 to 65 pentapeptide repeats.
  • the first ELP domain comprises an amino acid sequence having 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:12, below: VPGVGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGGGVPGGGVPGGGVPGGGVPGGGVPGVGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGGGVPGGGVPGGGVPGGGVPGGGVPGVPGGGVPGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGGG VPGGGVPGGGVPGGGVPGGGVPGVGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG VPGAGVPGGGVPGGGVPGGGVPGGGVPGGGVPGGGVPGGGVPGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAG V
  • the second ELP domain comprises an amino acid sequence having 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:13, below: VPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVGVPGVGVPGVGVGVPGVGVPGVGVGVPGVGVPGVGVGVPGVGVPGVGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG
  • the first ELP domain comprises an amino acid sequence having 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:12; and ii) the second ELP domain comprises an amino acid sequence having 80% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:13.
  • Enzymatic Cleavage Sites As noted above, a subject ELP of the present disclosure comprises a first and second ELP domain, wherein the first and second ELP domain are separated by an enzymatic cleavage site.
  • An enzymatic cleavage site refers to an amino acid sequence in a polypeptide that renders the polypeptide susceptible to enzymatic cleavage (e.g., through hydrolysis of a peptide bond).
  • the Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 susceptibility to enzymatic cleavage can be generated due to the amino acid sequence of the enzymatic cleavage site preferentially interacting with the enzyme at or near the active site of the enzyme.
  • the enzymatic cleavage of the polypeptide can occur within the amino acid sequence of the enzymatic cleavage site or adjacent to the enzymatic cleavage site.
  • the first and second ELP domains are separated by two or more enzymatic cleavages, e.g., in some cases, the first and second ELP domains are separated by two enzymatic cleavage sites, are separated by 3 enzymatic cleavage sites, are separated by four enzymatic cleavage sites, or are separated by 5 or more enzymatic cleavage sites. In certain embodiments, the two or more enzymatic cleavage sites are different enzymatic cleavage sites. In such cases, phase-separation of the subject ELP may be induced by a plurality of different enzyme stimuli.
  • any suitable enzyme cleavage site may be chosen, e.g., any suitable enzyme for the desired application.
  • the enzymatic cleavage is a protease cleavage site.
  • the protease cleavage site is a cleavage site for a eukaryotic protease, a prokaryotic protease, or a viral protease.
  • the suitable eukaryotic protease cleavage site is a mammalian protease cleavage site.
  • a suitable mammalian protease cleavage site is a human protease cleavage site.
  • Suitable human protease cleavage sites include, without limitation, an enterokinase cleavage site, a clotting cascade protease cleavage site, a furin cleavage site, a matrix metalloproteinase (MMP) cleavage site, a caspase cleavage site, a paracaspase cleavage site, a granzyme B cleavage site, and a proline-endopeptidase cleavage site.
  • MMP matrix metalloproteinase
  • a suitable enterokinase cleavage site comprises the amino acid sequence: X1X2X3K, wherein X1 is selected from D or E, X2 is selected from D or E, and X3 is selected from D or E.
  • a suitable furin cleavage site comprises the amino acid sequence RXXR, wherein X is any amino acid.
  • a suitable granzyme B cleavage site comprises the amino acid sequence IEXDG (SEQ ID NO: 20), wherein X is any amino acid.
  • a suitable proline-endopeptidase cleavage site comprises the amino acid sequence X1PX2, wherein X1 is selected from H, K, or R; and X2 is any amino acid except P.
  • a suitable clotting cascade protease cleavage site includes, without limitation, a thrombin cleavage site, a factor Xa cleavage site, a factor VIIa cleavage site, a factor IXa cleavage site, a factor XIa cleavage site, a factor XIIa cleavage site, a kallikrein Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 cleavage site, or a plasmin cleavage site.
  • a suitable thrombin cleavage site comprises the amino acid sequence X 1 X 2 PRX 3 X 4 , wherein X 1 is selected from A, F, G, I, L, T, V or M, X2 is selected from A, F, G, I, L, T, V, W or A, X 3 is any amino acid except D or E, and X4 is any amino acid except D or E.
  • a suitable FXa cleavage site comprises the amino acid sequence X 1 X 2 GR, wherein X 1 is selected from A, F, G, I, L, T, V or M, and X 2 is any amino acid except D or E.
  • a MMP cleavage site includes, without limitation, a MMP-1 cleavage site, a MMP-13 cleavage site, a MMP-11 cleavage site, a MMP-12 cleavage site, a MMP-7 cleavage site, a MMP-26 cleavage site, a MMP-2 cleavage site, a MMP-9 cleavage site, a MMP-14 cleavage site, a MMP-16 cleavage site, a MMP-17 cleavage site, a MMP-24 cleavage site, and a MMP-25 cleavage site.
  • a suitable MMP cleavage site comprises the amino acid sequence X 1 AX 2 X 3 X 4 X 5 , wherein X 1 is selected from P, A, or V, X 2 is selected from N, A, or G, X 3 is selected from L, I, C, M, V, or G, X 4 is selected from V, I, K, or H, and X 5 is selected from A,G, or V.
  • X 1 is selected from P, A, or V
  • X 2 is selected from N, A, or G
  • X 3 is selected from L, I, C, M, V, or G
  • X 4 is selected from V, I, K, or H
  • X 5 is selected from A,G, or V.
  • the protein substrate specificities of MMPs are known in the art and described, for example, in Eckhard, Ulrich, et al.
  • a caspase cleavage site includes, without limitation, a Caspase 1 cleavage site, a Caspase 1 cleavage site, a Caspase 2 cleavage site, a Caspase 3 cleavage site, a Caspase 4 cleavage site, a Caspase 5 cleavage site, a Caspase 6 cleavage site, a Caspase 7 cleavage site, a Caspase 8 cleavage site, a Caspase 9 cleavage site, and a Caspase 10 cleavage site.
  • a suitable Caspase 1 cleavage site comprises the amino acid sequence X1X2X3DX4, wherein X1 is selected from F, W, Y, or L, X2 is any amino acid, X3 is selected from H, A, or T, and X4 is any amino acid except for P, E, D, Q, K, or R.
  • a suitable Caspase 2 cleavage site comprises the amino acid sequence DVADX (SEQ ID NO:21), wherein X is any amino acid other than P, E, D, Q, K or R.
  • a suitable Caspase 3 cleavage site comprises the amino acid sequence DMQDX (SEQ ID NO:22), wherein X is any amino acid other than P, E, D, Q, K or R.
  • Caspase 4 cleavage site comprises the amino acid sequence LEVDX (SEQ ID NO:23), wherein X is any amino acid other than P, E, D, Q, K or R.
  • a suitable Caspase 5 cleavage site comprises the amino acid sequence XEHD, wherein X is L or W.
  • a suitable Caspase 6 cleavage site comprises the amino acid sequence VEX 1 DX 2 , wherein X 1 is I or L, and X 2 any amino acid other than P, E, D, Q, K or R.
  • a suitable Caspase 7 cleavage site comprises the amino acid sequence DEVDX (SEQ ID NO:24), wherein X is any amino acid other than P, E, D, Q, K or R.
  • a suitable Caspase 8 cleavage site comprises the amino acid sequence X 1 ETDX 2 , wherein X 1 is I or L, and X 2 is any amino acid other than P, E, D, Q, K or R.
  • a suitable Caspase 9 cleavage site comprises the amino acid sequence LEHD (SEQ ID NO:25).
  • a suitable Caspase 10 cleavage site comprises the amino acid sequence IEAD (SEQ ID NO:26).
  • the suitable eukaryotic protease cleavage site is a plant protease cleavage site. Suitable plant protease cleavage sites include, without limitation, a plant metacaspase cleavage site, a phytaspase cleavage site, a plant subtilase cleavage site, and a plant papain-like protease.
  • Plant proteases and their cleavage substrates are known in the art and described, for example, in Minina, Maria A., et al. "Plant metacaspase activation and activity.” Caspases, paracaspases, and metacaspases: Methods and protocols (2014): 237-253, Tsiatsiani, Liana, et al. "Metacaspases.” Cell Death & Differentiation 18.8 (2011): 1279-1288, . Chichkova, Nina V., et al. "Plant phytaspases and animal caspases: structurally unrelated death proteases with a common role and specificity.” Physiologia Plantarum 145.1 (2012): 77-84, Vartapetian, A.
  • the protease cleavage site is a bacterial protease cleavage site.
  • Suitable bacterial protease cleavage sites include, without limitation, a ftsH protease and the like, a rseP protease and the like, a IClip protease and the like, a GxGD-aspartyl protease, and a lepB protease and the like.
  • Bacterial proteases and their cleavage substrates are known in the art and described, for example, in De Castro, Rosana E., and Ansgar Poetsch. "Proteases of the prokaryotic cell envelope.” Frontiers in Microbiology 13 (2022): 988067 and Culp, Elizabeth, and Gerard D. Wright.
  • the protease cleavage site is a viral protease cleavage site.
  • Suitable viral protease cleavage sites include, without limitation, a TEV protease (TEVP) cleavage site, a HIV-1 protease cleavage site, a HCV NS3/4A protease cleavage site, a HTLV-1 protease cleavage site, a flavivirus protease cleavage site, an enterovirus protease cleavage site, and a coronavirus protease cleavage site.
  • Viral proteases and their cleavage substrates are known in the art and described, for example, in Zephyr, Jacqueto, Nese Kurt Yilmaz, and Celia A. Schiffer.
  • a suitable protease cleavage site for use in a subject ELP includes, without limitation, any one of the protease cleavage sites set forth in Table 1.
  • a subject ELP further comprises: i) a first linker between the first ELP domain and the enzymatic cleavage site; and/or ii) a second linker between the second ELP domain and the enzymatic cleavage site.
  • a subject ELP comprises: i) a first linker between the first ELP domain and the enzymatic cleavage site; and ii) a second linker between the second ELP domain and the enzymatic cleavage site.
  • the first and/or second linker may be a flexible linker.
  • a Gly-Ser (GS) linker is employed, non-limiting examples of which are those comprising one or more GGGGS (SEQ ID NO: 18) units, i.e., (GGGGS) n (SEQ ID NO: 19) where n is an integer of 1 or greater, e.g., 1 to 10.
  • a suitable linker may comprise one or more GGGS (SEQ ID NO: 16) units, i.e., (GGGS) n (SEQ ID NO: 14), where n is an integer of 1 or greater, e.g., 1 to 10.
  • a suitable linker may comprise one or more SGGG (SEQ ID NO: 17) units, i.e., (SGGG) n (SEQ ID NO: 15), where n is an integer of 1 or greater, e.g., 1 to 10.
  • a flexible linker comprising Gly and Ser, and one or more threonine (Thr) residues is employed.
  • a flexible linker comprising Gly or Ser residues, and one or more Thr residues.
  • a subject ELP comprises: i) a first linker between the first ELP domain and the enzymatic cleavage site, wherein the first linker comprises the amino acid sequence (GGGS)n (SEQ ID NO: 14); and ii) a second linker between the second ELP domain and the enzymatic cleavage site, wherein the second linker comprises the amino acid sequence (SGGG)n (SEQ ID NO: 15), and wherein n is an integer of 1 or greater, e.g., 1 to 10.
  • a subject ELP comprises: i) a first linker between the first ELP domain and the enzymatic cleavage site, wherein the first linker comprises the amino acid sequence (SGGG)n (SEQ ID NO: 15); and ii) a second linker between the second ELP domain and the enzymatic Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 cleavage site, wherein the second linker comprises the amino acid sequence (GGGS) n (SEQ ID NO: 14), and wherein n is an integer of 1 or greater, e.g., 1 to 10.
  • a subject ELP of the present disclosure comprises, in order from N-terminus to C-terminus: a) the first ELP domain; b) a first linker; c) the enzymatic cleavage site; d) a second linker; and e) the second ELP domain.
  • a subject ELP of the present disclosure comprises, in order from N-terminus to C-terminus: a) the first ELP domain; b) a first linker; c) two or more enzymatic cleavage sites; d) a second linker; and e) the second ELP domain.
  • Payload Polypeptides In some embodiments, a subject ELP further comprises a payload polypeptide.
  • a “payload polypeptide”, as used herein, is an amino acid sequence or polypeptide domain that can exert a desired pharmacological, biological, or physical effect.
  • the payload polypeptide can be fused to a subject ELP so that the ELP may function as an isothermally phas-separable delivery vehicle for the payload polypeptide.
  • Suitable payload polypeptides include, without limitation, single-chain antibodies, cytokines, growth factors, peptide hormones, or therapeutic peptides.
  • the payload polypeptide is fused to the second ELP domain via a linker (e.g., a flexible linker as disclosed herein).
  • the payload polypeptide is fused N-terminally to the second ELP domain. In some embodiments of a subject ELP, the payload polypeptide is fused C-terminally to the second ELP domain. In cases where the payload polypeptide is fused to the second ELP domain, the LCST of the second ELP domain may be less than the LCST of the first ELP domain, such that, upon cleavage of the subject ELP, the second ELP domain that is fused to the payload polypeptide undergoes a phase transition.
  • Suitable single-chain antibodies for use as a payload polypeptide in a subject ELP include, without limitation, a single-chain antibody comprising variable regions derived from Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, Dusigitumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab, Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 Lucatumumab, Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab, Panitumum
  • Suitable cytokines for use as a payload polypeptide in a subject ELP include, without limitation, Interleukin-1, Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-7, Interleukin-9, Interleukin-11, Interleukin-12, Interleukin-13, Interleukin-15 , Interleukin-18, Interleukin-21 , Interleukin-23, Interleukin-27, TGF- ⁇ , IFN- ⁇ , TNF- ⁇ , PD-L1, 4- 1BBL, OX40L, ICOSLG, and variants thereof.
  • Suitable peptide hormones and/or growth factors for use as a payload polypeptide in a subject ELP include, without limitation, Human growth hormone (HGH), angiotensin, insulin, glucagon, glucagon-like peptide-1, vasopressin, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and variants thereof.
  • HGH Human growth hormone
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • Therapeutic peptides suitable for use as a payload polypeptide are known in the art and described, for example, in Wang, Lei, et al.
  • a subject ELP of the present disclosure is conjugated to a payload molecule.
  • conjugated it is meant that the payload molecule is covalently linked to the ELP via any suitable chemistry.
  • the payload molecule can be any molecule that can exert a desired pharmacological, biological, or physical effect.
  • the payload molecule can be conjugated to the ELP so that the ELP may function as an isothermally phas-separable delivery vehicle for the conjugated payload molecule.
  • the payload molecule may be a nucleic acid, a polypeptide, a carbohydrate, or a small molecule.
  • the payload molecule may be conjugated to a lysine residue present in the subject ELP.
  • the linker moiety and/or payload molecule may be covalently attached via the primary amine of the lysine residue. In such cases, a linker moiety and/or the payload molecule may be reacted with the lysine via amide coupling using an activated carboxylic ester in the linker moiety.
  • the primary amine of the lysine can be reacted with N- hydroxysuccinimide (NHS) esters introduced into the linker moiety, forming a stable amide bond.
  • N- hydroxysuccinimide (NHS) esters introduced into the linker moiety, forming a stable amide bond.
  • exemplary linker types that may be conjugated to a lysine residue include, without limitation, N- succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) linkers, sulfo-SPDB linkers, maleimidomethyl cyclohexane-1-carboxylate (MCC), 4-(4-acetylphenoxy)butanoic acid (AcBut) linkers, and derivatives thereof.
  • SPDB N- succinimidyl-4-(2-pyridyldithio)butanoate
  • MCC maleimidomethyl cyclohexane-1-carboxylate
  • the payload molecule may be conjugated to a cysteine residue present in the subject ELP.
  • the linker moiety and/or payload molecule may be covalently attached via the thiol group of the cysteine residue.
  • free thiol groups of any cysteines in the subject ELP may serve as reactive attachment sites to conjugate the linker moiety and/or payload molecule via a variety of chemical reactions.
  • the subject ELP may be introduced to reducing conditions to ensure any disulfide bonds are reduced.
  • the linker moiety may be reacted with the free thiol group by Michael addition, a-halo carbonyl alkylation, or disulfide formation.
  • linker types that may be conjugated to a cysteine residue include, without limitation, maleimidocaproyl (MC) linkers, maleimidomethyl cyclohexane-1-carboxylate (MCC) linkers, and derivatives thereof.
  • MC maleimidocaproyl
  • MCC maleimidomethyl cyclohexane-1-carboxylate
  • Available conjugation chemistries for attaching linker moieties to lysine and cysteine residues of a polypeptide are known in the art and reviewed, Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 for example, in Lu et al. 2016 (Lu, Jun, et al.
  • Linkers having a crucial role in antibody–drug conjugates International journal of molecular sciences 17.4 (2016): 561), McDonagh et al.2006 (McDonagh, Charlotte F., et al. "Engineered antibody–drug conjugates with defined sites and stoichiometries of drug attachment.” Protein Engineering, Design and Selection 19.7 (2006): 299- 307), and Sun et al. 2005 (Sun, Michael MC, et al. "Reduction ⁇ alkylation strategies for the modification of specific monoclonal antibody disulfides.” Bioconjugate chemistry 16.5 (2005): 1282-1290).
  • methods for conjugating linkers to cysteine residues of a polypeptide are described, for example, in WO 2014/197612.
  • linker moieties for conjugating a payload molecule to a subject ELP include, without limitation, maleimidocaproyl (MC) linkers, maleimidomethyl cyclohexane-1-carboxylate (MCC) linkers, N-succinimidyl-4-(maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linkers, and derivatives thereof.
  • the linker moiety may be a cleavable linker moiety.
  • the cleavable linker may be a reducible or glutathione sensitive linker or comprises a reducible or glutathione sensitive moiety.
  • a payload molecule from a reducible linker can be facilitated by a reducing environment and/or high glutathione concentration.
  • glutathione is released during cell replication and thus proliferating cancer cells exhibit high concentrations of glutathione, offering an additional dimension of target-selectivity for delivery of the payload molecule.
  • a suitable glutathione sensitive moiety for incorporation into a reducible linker is a disulfide moiety.
  • the linker moiety may be a pH sensitive (i.e., acid labile) linker moieties.
  • pH sensitive linker moieties can offer an additional dimension of target-selectivity for delivery of the payload molecule to a tumors.
  • Suitable pH sensitive moieties for incorporation into an acid-labile linker include hydrazone moieties, which are hydrolyzed under acidic conditions. Additional acid-labile moieties that may be incorporated into a pH sensitive include carbonate and cis-aconityl moieties.
  • Exemplary cleavable linker types that incorporate pH sensitive moieties include, without limitation, AcBut-like linkers incorporating a hydrazone moiety, MC-like linkers incorporating a hydrazone moiety, and MCC-like linkers incorporating a hydrazone moiety.
  • the payload molecule is conjugated to the second ELP domain.
  • the LCST of the second ELP domain may be less than the LCST of the first ELP Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 domain, such that, upon cleavage of the subject ELP, the second ELP domain that is conjugated to the payload molecule undergoes a phase transition.
  • the payload molecule may be a nucleic acid, e.g., an RNA or DNA oligonucleotide.
  • a feature of nucleic acid payloads is the capability to offer highly selective modulation of, in theory, any gene of interest by designing to the nucleic acid payload to bind to a target nucleic acid through relatively simple Watson-Crick base pairing mechanisms.
  • Nucleic acid payloads can be used to, for example, modulate the expression of a target gene through a variety of mechanisms that involve base-pairing to the target, e.g., base-pairing to a complementary sequence of a transcript of the target gene.
  • the nucleic acid payload molecule may be an anti-sense oligonucleotide (ASO).
  • ASOs are designed to be complementary (i.e., anti-sense) to a target nucleic acid RNA transcript (e.g., an mRNA).
  • a target nucleic acid RNA transcript e.g., an mRNA
  • an ASO can modulate expression of a desired gene (e.g., a disease associated gene) by triggering degradation of the target gene transcript.
  • a desired gene e.g., a disease associated gene
  • an DNA, or DNA-like, ASO can be designed to be complementary to a target RNA transcript and hybridize to form a DNA/RNA complex with the transcript.
  • the ASO/transcript complex is then susceptible to the activity of RNAse H, which selectively degrades the RNA strands of DNA/RNA complexes, thereby degrading the hybridized target transcript.
  • the ASO can be designed to alter the expression of a specific isoform of a gene through modulation of RNA splicing.
  • an ASO can be designed to bind the intron-exon junction of the pre-mRNA of a target gene and sterically interfere with the spliceosome machinery and thereby alter splicing events.
  • the nucleic acid payload molecule may be a short-interfering RNA (siRNA).
  • siRNA is a double-stranded RNA molecule, a strand of which can hybridize to a complementary target RNA, that can induce degradation of the target RNA through recruitment of and incorporation into an RNA-induced silencing complex (RISC) in a target cell.
  • RISC RNA-induced silencing complex
  • the payload molecule may be a polypeptide (i.e., a polypeptide that is covalently linked to the subject ELP, other than through a peptide bond as part of the ELP polypeptide chain.
  • Suitable polypeptides for use as a payload molecule for a subject ELP include, without limitation, single-chain antibodies, cytokines, growth factors, peptide hormones, or therapeutic peptides as described herein.
  • the nucleic acid payload molecule of a subject ELP of the present disclosure comprises one or more modifications (e.g., a nucleobase modification, a sugar modification, a backbone modification) to improve, for example, affinity or the stability of the Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 nucleic acid payload in circulating plasma.
  • Suitable nucleic acid modifications include, by way of example, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, 2’-O-Methyl modified nucleotides, 2’ fluoro modified nucleotides, and phosphorothioate linkages, among others.
  • Nucleic acid modifications useful for nucleic acids with therapeutic applications are known in the art and described, for example, in WO 2007/047913, Ochoa et al.2020 (Ochoa, Steven, and Valeria T. Milam. "Modified nucleic acids: Expanding the capabilities of functional oligonucleotides.” Molecules 25.20 (2020): 4659), Kulkarni et al. 2021 (Kulkarni, Jayesh A., et al. "The current landscape of nucleic acid therapeutics.” Nature nanotechnology 16.6 (2021): 630-643) and Moumné et al.
  • the payload molecule may be a carbohydrate. Suitable carbohydrates for use a payload molecule include hyaluronic acids, glycosaminoglycans, chitosans, and the like. In some embodiments, the payload molecule may be a small molecule.
  • Candidate small molecules comprise functional groups for structural interaction with proteins, particularly hydrogen bonding, and may include an amine, carbonyl, hydroxyl or carboxyl group, and in some instances two or more of the functional chemical groups.
  • the candidate agents may include cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • the small molecule may be a modulator of a G-protein Couple Receptor (GPCR).
  • GPCRs encompass the largest protein family encoded in the human genome and are involved in diverse physiological processes and have been implicated in many diseases ranging from type 2 diabetes to schizophrenia.
  • GPCRs transduce extracellular signals in the form of varying ligands which induce a conformational change in the GPCR upon binding, subsequently leading to activation of G proteins and other downstream intracellular molecular pathways.
  • GPCRs can be grouped into four classes based on their amino acid sequences: class A (rhodopsin-type), class B (secretin/adhesion-type), class C (glutamate-type), and class F (frizzled- type) GPCR subfamilies.
  • the class A type GPCRs can be further subdivided into aminergic, peptide, lipid, protein, nucleotide, and steroid receptor GPCRs.
  • the small molecule may be a microtubule inhibitor.
  • Microtubule inhibitors are commonly used, e.g., to treat tumors. Microtubule assembly plays a critical role in cell division and cellular transport, among other key cellular functions, and inhibition of microtubule assembly leads to arrest of cell division and/or cell death.
  • the microtubule inhibitor may be an auristatin or analogues and derivatives thereof.
  • Auristatins are synthetic analogues of the natural molecule Dolostatin 10, isolated from Dolabella Auricularia. Suitable auristatins and auristatin derivatives for use as a payload include, without limitation, monomethyl auristatin E (MMAE; CAS Registry No.
  • the microtubule inhibitor may be a maytansinoid or derivatives thereof.
  • Maytansinoids are derived from the natural molecule maytansine, isolated from Maytenus ovatus. Suitable maytansinoids and maytansinoid derivatives for use as a payload include, without limitation, maytansine (CAS Registry No. 35846-53-8) and mertansine (DM1; CAS Registry No.139504-50-0).
  • microtubule inhibitors include, by way of example and without limitation, colchicine (CAS Registry No. 64868), halichondrin B (CAS Registry No. 103614-76-2), rhizoxin (CAS Registry No.90996-54-6), paclitaxel (CAS Registry No.33069-62- 4), and vinca alkaloids such as vindesine (CAS Registry No. 53643-48-4).
  • Microtubule inhibitors/disrupting agents are known in the art and described, for example, in Wang et al.2023 (Wang, Xingyu, et al.
  • the small molecule may be a DNA-damaging agent.
  • DNA- damaging agents are commonly used, e.g., to treat tumors.
  • DNA-damaging agents act through a variety of mechanisms and include, for example, alkylating agents, DNA intercalators, and topoisomerase inhibitors.
  • the DNA-damaging agent may be a calicheamicin or derivatives thereof.
  • Calicheamicins are anticancer antiobiotics isolated from the actinomycete Micromonospora Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 echinospora spp. Calichensis. Calicheamicins bind the minor groove of DNA in a site-specific manner and reductive cleavage by thiols present in a cell generates reactive diradical species that lead to strand scission of the DNA and, subsequently, cell death. Suitable calicheamicins and calicheamicin derivatives for use as a payload include, without limitation, calicheamicin ⁇ 1 (CAS Registry No.
  • the DNA-damaging agent may be a pyrrolobenzodiazepine (PBD) or derivatives thereof.
  • PBDs can alkylate and cross-link opposite DNA strands, preventing strand separation during genomic replication and inducing cell death.
  • Suitable PBDs for use as a payload include, without limitation, tesirine (SG3249; CAS Registry No.1595275-62-9) and SJG-136 (CAS Registry No.232931-57-6).
  • PBDs and derivatives thereof are known in the art and are described, for example, in: Mantaj et al. 2017 (Mantaj, Julia, et al.
  • the DNA-damaging agent may be a topoisomerase inhibitor, e.g., a topoisomerase I or topoisomerase II inhibitor. Topoisomerases are necessary for regulating DNA topology and torsional stresses during DNA replication, repair and transcription.
  • the topoisomerase inhibitor may be a topoisomerase I inhibitor, e.g., a camptothecin or a derivative thereof.
  • Camptothecin and its derivates inhibit topoisomerase I, inhibiting replication and causing cleavage of the DNA.
  • Suitable camptothecins and camptothecin derivatives for use as a payload include, without limitation, camptothecin (CAS Registry No.7689- 03-4), topotecan (CAS Registry No. 123948-87-8), irinotecan (CAS Registry No. 97682-44-5), belotecan (CAS Registry No. 256411-32-2), exatecan (CAS Registry No. 171335-80-1), deruxtecan (CAS Registry No. 1599440-13-7), and SN-38 (CAS Registry No. 86639-52-3).
  • the topoisomerase inhibitor may be a topoisomerase II inhibitor, e.g., an anthracycline, anthracenedione, acridine, epipodophyllotoxin, or derivatives thereof.
  • Anthracyclines intercalate DNA and poison DNA-topoisomerase II complexes, inhibiting DNA replication and causing cell death. Additionally, anthracyclines generate free-radicals in an iron- dependent manner, further enhancing their cytotoxic effects.
  • Suitable anthracycline and Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 anthracycline derivatives for use as a payload include, without limitation, doxorubicin (CAS Registry No.
  • Anthracenediones, acridines, epipodophyllotoxins, and derivatives thereof intercalate DNA and poison DNA-topoisomerase II complexes, inhibiting DNA replication and causing cell death.
  • Suitable anthracenediones and anthracenedione derivatives for use as a payload include, without limitation, mitoxantrone (CAS Registry No.
  • Suitable epipodophyllotoxins and epipodophyllotoxin derivatives include, without limitation, etoposide (CAS Registry No.33419-42-0) and teniposide (CAS Registry No.29767-20-2).
  • etoposide CAS Registry No.33419-42-0
  • teniposide CAS Registry No.29767-20-2
  • topoisomerase inhibitors are known in the art and described, for example, in Yakkala et al.2023 (Yakkala PA, Penumallu NR, Shafi S, Kamal A. “Prospects of Topoisomerase Inhibitors as Promising Anti-Cancer Agents”.
  • the DNA-damaging agent may be an alkylating agent.
  • Alkylating agents interact with DNA and form covalent adducts to cross-link DNA or cross-link DNA with protein, leading to DNA cleavage, inhibition of replication, and cell death.
  • Suitable alkylating agents for use as a payload include, without limitation, adozelesin (CAS Registry No.
  • a subject ELP of the present disclosure comprises an N-terminal leader domain.
  • the N-terminal leader domain is located at the N-terminus of the ELP and can generally range from, without limitation, 5 to 50 amino acids in length.
  • the N-terminal leader domain may serve a variety of desired functions.
  • an N-terminal leader domain may comprise tryptophan residues that facilitate visualization of the subject ELP using stain-free PAGE gels.
  • an N-terminal leader domain may encode an epitope to facilitate immuno-detection of the ELP or to facilitate affinity purification of the ELP.
  • the N-terminal leader domain encodes a signal peptide.
  • a signal peptide may enable secretion of the subject ELP when the ELP is expressed in a cell (e.g., when Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 expressed in a eukaryotic cell).
  • suitable signal peptide amino acid sequences for use in a subject N-terminal leader domain include, without limitation, MKWVTFISLLFSSAYS (SEQ ID NO: 105), METDTLLLWVLLLWVPGSTG (SEQ ID NO: 106), and MKCLLYLAFLFIGVNC (SEQ ID NO: 107.
  • Signal peptide sequences, in addition to methods for identifying them, are known in the art and described, for example, in Teufel, Felix, et al.
  • Nucleic Acids The present disclosure also provides nucleic acids encoding a subject ELP as described herein.
  • the subject nucleic acids may be DNA or RNA (e.g., double-stranded DNA or single- stranded RNA) comprising an open reading frame that encodes a subject ELP.
  • the nucleic acids of the present disclosure are of any sufficient length to comprise an open reading frame that encodes a subject ELP.
  • nucleotide sequences of the nucleic acids of the present disclosure may be codon- optimized. “Codon-optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest.
  • nucleic acid of the present disclosure encoding a subject ELP may be codon- optimized for optimal production from the host organism selected for expression, e.g., human cells.
  • a subject expression vector may further comprise a nucleic acid sequence encoding a transgene.
  • transgene it is meant any gene that is heterologous or exogenous to the host cell harboring the expression vector. Any suitable transgene may be chosen by the person of skill in the art, depending on the desired goal or application.
  • expression vector includes a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the subject ELP in a particular host organism.
  • Expression vectors can provide for extrachromosomal maintenance in a host cell or can provide for integration into the host cell genome.
  • the expression vector provides transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression, where the coding region (e.g., encoding the subject ELP or encoding a transgene) is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region.
  • the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • Suitable promoters for use in an expression vector described herein include any eukaryotic promoter (i.e., a promoter functional in a eukaryotic cell, e.g., a mammalian cell or a human cell).
  • the promoter can be a constitutively active promoter (i.e., a promoter always in an active or “ON” state), and inducible promoter (i.e., a promoter whose activity is controlled by an external stimulus, e.g., a specific temperature, small molecule, or protein).
  • Suitable eukaryotic promoters are well known in the art and include, without limitation, cytomegalovirus (CMV) immediate early promoters, herpes simplex virus (HSV) thymidine kinase promoters, SV40 early and late promoters, Rous-Sarcoma virus (RSV) promoters, ⁇ -actin promoters, tubulin promoters, and EF1 ⁇ promoters.
  • CMV cytomegalovirus
  • HSV herpes simplex virus
  • RSV40 early and late promoters SV40 early and late promoters
  • Rous-Sarcoma virus (RSV) promoters Rous-Sarcoma virus (RSV) promoters
  • ⁇ -actin promoters ⁇ -actin promoters
  • tubulin promoters tubulin promoters
  • EF1 ⁇ promoters EF1 ⁇ promoters.
  • the eukaryotic promoter may be a cell or tissue-specific promote
  • the nucleic acid encoding the ELP or the transgene may further comprise a ribosome binding site for initiation of translation (e.g., an internal ribosome entry site; IRES) and/or a transcription terminator.
  • IRES internal ribosome entry site
  • Expression constructs generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest.
  • a selectable marker operative in the expression host may be present to facilitate selection of cells containing the vector.
  • the expression construct may include additional elements.
  • the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example in mammalian cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selectable genes are well known in the art and will vary with the host cell used.
  • the expression vector may be an episome (i.e., a plasmid). The episome may include one or more elements in addition to the promoter region and the region that encodes the ELP (or transgene).
  • the episome may include an origin of replication, one or more regions that encode a protein that confers antibiotic resistance to the host cell (e.g., ampicillin resistance (AmpR), hygromycin resistance, and/or the like), one or more poly(A) signals, a pause site, an SV40 late poly(A) signal, an SV40 enhancer, an SV40 early promoter, etc., and any desired combination of such elements.
  • the expression vector may be a viral vector. Viral vectors are known in the art and described, for example, in Warnock, James N., Claire Daigre, and Mohamed Al-Rubeai.
  • Suitable viral vectors for use in the present methods and compositions include, without limitation, adenovirus vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655), adeno-associated virus (AAV) vectors (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:69166921, 1997), lentiviral and retroviral vectors (e.g., lentivirus, Rous- Sarcoma virus, Murine Leukemia virus, and human immunodeficiency virus related vectors).
  • adenovirus vectors see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/
  • the expression vector may be a non-viral vector.
  • Non-viral vectors are known in the art and described, for example, in Yin, Hao, et al. "Non-viral vectors for gene-based therapy.” Nature Reviews Genetics 15.8 (2014): 541-555, the entirety of which is incorporated Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 herein by reference.
  • Suitable non-viral vectors for use in the present methods and compositions include, without limitation, lipid-based vectors (such as a liposome or lipid nanoparticle, see, e.g., Liposomes: Methods and Protocols, Volume 1: Pharmaceutical Nanocarriers: Methods and Protocols. (ed. Weissig).
  • polymeric vectors e.g., polymeric poly(L-lysine), poly(lactic acid), and poly(D,L-lactide-co-glycolide) vectors, see, e.g., Functional Polymer Colloids and Microparticles volume 4 (Microspheres, microcapsules & liposomes). (eds. Arshady & Guyot). Citus Books, 2002 and Polymers in Drug Delivery. (eds. Uchegbu & Schatzlein).
  • Host Cells The present disclosure provides cell comprising an expression vector as described herein.
  • the cell comprising a subject expression vector is also referred to as a “host cell” herein.
  • the host cell is a eukaryotic cell.
  • Suitable eukaryotic host cells include, without limitation, a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C.
  • a plant e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco
  • seaweeds e.g. kelp
  • a fungal cell e.g., a yeast cell, a cell from a mushroom
  • an animal cell e.g., a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.)
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like.
  • the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).
  • a host cell may also be an in vitro cell (e.g., a cell culture line).
  • a host cell may be an ex vivo cell, (e.g., derived and cultures from an individual).
  • a host cell may also be an in vivo cell (e.g., a cell in an individual).
  • a cell can be an isolated cell.
  • a cell can be a cell inside of an organism.
  • a cell can be an organism.
  • a cell can be a cell in a cell culture (e.g., in vitro cell culture).
  • a cell can be one of a collection of cells.
  • a cell can be a eukaryotic cell or derived from a eukaryotic cell.
  • a cell can be a plant cell or derived from a plant cell.
  • a cell can be an animal cell or derived from an Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 animal cell.
  • a cell can be an invertebrate cell or derived from an invertebrate cell.
  • a cell can be a vertebrate cell or derived from a vertebrate cell.
  • a cell can be a mammalian cell or derived from a mammalian cell.
  • a cell can be a rodent cell or derived from a rodent cell.
  • a cell can be a human cell or derived from a human cell.
  • ELP Compositions comprising: a) a subject ELP (or ELP conjugate) as described herein; and b) a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient may comprise a pharmaceutically acceptable salt. Suitable salts for use in a present composition include, without limitation, sodium chloride, sodium citrate, sodium benzoate, sodium phosphates (e.g., phosphate buffered saline), potassium phosphate, potassium citrate, calcium carbonate, and calcium phosphate, or combinations thereof.
  • the pharmaceutically acceptable excipient may comprise a pharmaceutically acceptable sugar or polyol.
  • Suitable sugars or polyols for use in a present composition include, without limitation, sucrose, trehalose, mannose, mannitol, raffinose, lactitol, sorbitol and lactobionic acid, glucose, maltulose, iso-maltulose, lactulose, maltose, lactose, iso-maltose, maltitol, palatinit, stachyose, melezitose, dextran, or combinations thereof.
  • the pharmaceutically acceptable excipient may comprise an amino acid(s).
  • Suitable amino acids for use in a present composition include, without limitation, glycine, arginine, lysine, and glutamine.
  • the pharmaceutically acceptable excipient may comprise water in combination with any other of the compounds described above.
  • Methods Cell-Protective Biopolymers The present disclosure provides methods for assembling a cell-protective biopolymer on the surface of a target cell.
  • the method for assembling the cell-protective biopolymer comprises contacting the surface of a target cell with a subject ELP, as disclosed herein, to assemble a cell-protective biopolymer, wherein the surface of the target cell comprises an enzyme that can cleave the enzymatic cleavage site of the ELP.
  • the ELP may be part of an ELP composition as described herein.
  • the enzyme may (e.g., a protease, a membrane-bound protease) be present on the surface of the cell.
  • the LCST of the second ELP domain may be less than the LCST of the first ELP domain, such that, Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 upon contact of the ELP with the enzyme on the surface of the target cell, the ELP is cleaved and thus enables the second ELP domain to undergo a phase transition and form a cell-protective biopolymer on the surface of the target cell.
  • the ELP may further comprise a payload polypeptide as disclosed herein (e.g., a payload polypeptide fused to the second ELP domain via linker).
  • the ELP may be conjugated to a payload molecule as disclosed herein (e.g., a payload molecule conjugated to the second ELP domain).
  • the method for assembling the cell-protective biopolymer comprises expressing a subject ELP, as disclosed herein, in the target cell to assemble a cell- protective biopolymer on the surface of said cell, wherein the ELP is secreted by the target cell, and wherein the target cell comprises an enzyme that can cleave the enzymatic cleavage site of the ELP.
  • the target cell may comprise an expression vector encoding the subject ELP, as disclosed herein.
  • the enzyme e.g., a protease, a membrane- bound protease
  • the enzyme is present on the surface of the target cell.
  • the LCST of the second ELP domain may be less than the LCST of the first ELP domain, such that, upon secretion of the ELP, the ELP contacts the enzyme on the surface of the target cell. Following contact with the enzyme, the ELP is cleaved and thus enables the second ELP domain to undergo a phase transition and form a cell-protective biopolymer on the surface of the target cell.
  • the enzyme is present inside the target cell.
  • the enzyme e.g., a protease
  • the endosomal network e.g., the endoplasmic reticulum, the golgi, etc.
  • the LCST of the second ELP domain may be less than the LCST of the first ELP domain, such that, during secretion of the ELP, the ELP contacts the enzyme inside of the target cell. Following contact with the enzyme inside the cell (e.g., inside the endosomal network) the ELP is cleaved and the second ELP domain is liberated.
  • the LCST of the second ELP domain may vary depending on pH, such that the second ELP domain is soluble inside the acidic endosomal system but undergoes a phase transition upon secretion from the target cell, thus forming a cell-protective biopolymer on the surface of the target cell.
  • the ELP may further comprise a payload polypeptide as disclosed herein (e.g., a payload polypeptide fused to the second ELP domain via linker).
  • the ELP may further comprise an N-terminal leader domain, wherein the N-terminal leader domain encodes a signal peptide as disclosed herein.
  • the target cell comprises an expression vector encoding the subject ELP, as disclosed herein. In some particular Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 embodiments, said expression vector further comprises a nucleic acid sequence encoding a transgene, as described herein.
  • the target cell may be a eukaryotic cell.
  • Suitable eukaryotic cells include, without limitation, a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C.
  • a plant e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco,
  • seaweeds e.g. kelp
  • a fungal cell e.g., a yeast cell, a cell from a mushroom
  • an animal cell e.g., a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.)
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like.
  • the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).
  • the target cell may also be an in vitro cell (e.g., a cell culture line).
  • a target cell may be an ex vivo cell, (e.g., derived and cultures from an individual).
  • a target cell may also be an in vivo cell (e.g., a cell in an individual).
  • a cell can be an isolated cell.
  • a cell can be a cell inside of an organism.
  • a cell can be an organism.
  • a cell can be a cell in a cell culture (e.g., in vitro cell culture).
  • a cell can be one of a collection of cells.
  • a cell can be a eukaryotic cell or derived from a eukaryotic cell.
  • a cell can be a plant cell or derived from a plant cell.
  • a cell can be an animal cell or derived from an animal cell.
  • a cell can be an invertebrate cell or derived from an invertebrate cell.
  • a cell can be a vertebrate cell or derived from a vertebrate cell.
  • a cell can be a mammalian cell or derived from a mammalian cell.
  • a cell can be a rodent cell or derived from a rodent cell.
  • a cell can be a human cell or derived from a human cell.
  • the ELP may be non-immunogenic.
  • the cell-protective biopolymer formed by the subject ELP will likewise be non-immunogenic.
  • Such cell-protective biopolymers find use in shielding the target cell (e.g., a genetically modified target cell expressing a therapeutic transgene) from being targeted from the immune system of the host organism or subject harboring the target cell.
  • the present disclosure provides methods for reducing blood loss at a hemorrhage site in a subject, the method comprising administering to a hemorrhage site in a subject an effective amount of a subject ELP composition, as disclosed herein, to reduce blood loss at the hemorrhage site, wherein an enzyme that can cleave the enzymatic cleavage site of the ELP is present in or around the hemorrhage site.
  • the LCST of the second ELP domain may be less than the LCST of the first ELP domain, such that, upon contact of the ELP with the enzyme present in or around the hemorrhage site, the ELP is cleaved thus enabling the second ELP domain to undergo a phase transition and form a physical barrier at the hemorrhage site to reduce bleeding loss.
  • the enzymatic cleavage of the subject ELP is a protease cleavage site.
  • the protease cleavage site is a cleavage site for a protease of the clotting cascade.
  • Suitable clotting cascade protease cleavage sites include, without limitation, a thrombin cleavage site, a factor Xa cleavage site, a factor VIIa cleavage site, a factor IXa cleavage site, a factor XIa cleavage site, a factor XIIa cleavage site, a kallikrein cleavage site, or a plasmin cleavage site, each as described herein.
  • the enzyme that can cleave said cleavage site and is present in or around the hemorrhage site may be a thrombin, a factor Xa, a factor VIIa, a factor IXa, a factor XIa, a factor XIIa, a kallikrein, or a plasmin.
  • the hemorrhage site is an external hemorrhage site.
  • the hemorrhage site is an internal hemorrhage site.
  • the hemorrhage site, whether external or internal, may be at the site of a wound.
  • the wound may be a surgical wound or a trauma wound (e.g., a surgical or traumatic soft tissue wound).
  • the present methods may find use in reducing blood loss at a hemorrhage site in a subject under coagulopathic conditions.
  • coagulopathic conditions it is meant a condition where clot formation in a subject is impaired (i.e., coagulopathy).
  • Coagulopathy may include one or more of a subset of conditions, such as dilution, hypothermia and acidosis.
  • the wounds may vary in severity, ranging from superficial skin wounds to wounds involving the laceration or rupture of a major artery or vein (e.g., the femoral artery).
  • the administering comprises applying a subject ELP composition to the external hemorrhage site.
  • the subject ELP composition may be applied via a substrate that is directly applied to the external hemorrhage wound.
  • the substrate may be a sterile sponge, gauze, bandage, compression bandage, pillow (e.g., to facilitate application to a head wound), sleeve (e.g., for covering a wound on a limb), and Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 the like.
  • the ELP composition may permeate at least a portion of an absorbent substrate (e.g., a portion of a sponge, gauze, or bandage).
  • the ELP composition may be directly to the external hemorrhage site via a swab, spray, or aerosol.
  • the substrate material may be composed of a natural fiber or polymer (e.g., cotton, silk, wool).
  • the substrate material may be composed of a synthetic fiber or polymer (e.g., rayon or polyester).
  • the administering comprises intravenously administering the ELP composition to the subject.
  • the methods comprise intravenously administering the ELP composition to a subject having an internal hemorrhage site. Said methods may include locating an appropriate intravenous site on the subject and injecting a subject ELP composition into the subject at the intravenous site. Upon injection of the ELP composition into the subject, the ELP composition may circulate through the circulatory system of the subject until cleaved by the corresponding enzyme present in or around the hemorrhage site.
  • the present disclosure provides methods for delivering a therapeutic agent to a target site in a subject, said methods comprising administering a subject ELP composition, as disclosed herein, to a subject, wherein an enzyme that can cleave the enzymatic cleavage site of the ELP is present in or around the target site.
  • the LCST of the second ELP domain may be less than the LCST of the first ELP domain, such that, upon contact of the ELP with the enzyme present in or around the target site, the ELP is cleaved thus enabling the second ELP domain to undergo a phase transition and assemble in or around the target site (e.g., in some cases the second ELP domain may coat the target site).
  • the ELP may serve as a targeted and/or controlled release vehicle for a payload polypeptide or conjugates payload molecule as described herein.
  • the enzymatic cleavage site of the ELP may be a protease cleavage site.
  • the corresponding enzyme present in or around the target site is the corresponding protease.
  • the administering comprises intravenously administering the ELP composition to the subject. Said methods may include locating an appropriate intravenous site on the subject and injecting a subject ELP composition into the subject at the intravenous site.
  • the ELP composition may circulate through the circulatory system of the subject until cleaved by the corresponding enzyme present in or around the target site.
  • the administering may comprise intramuscular injection, intradermal injection, or subcutaneous injection of the ELP composition in a subject.
  • the ELP composition may diffuse into the circulatory system of the subject and circulate throughout the body until cleaved by the corresponding enzyme present in or around the target site.
  • the injection site may be at or near the target site.
  • the ELP composition may diffuse into the surrounding tissue, which may encompass the target site, until cleaved by the corresponding enzyme present in or around the target site.
  • the target site may be a tumor.
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre- cancerous and cancerous cells and tissues.
  • the tumor may be a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor, and the like.
  • the enzymatic cleavage site of the ELP may be a protease cleavage site.
  • the corresponding enzyme present in or around the target site is the corresponding protease.
  • the protease cleavage site is a matrix metalloproteinase cleavage site.
  • the controlled remodeling of the extracellular matrix (ECM) is necessary for the growth, invasion of surrounding tissue, and metastasis of cancerous tumors.
  • ECM extracellular matrix
  • Matrix metalloproteinases can degrade ECM material and are known to play a role in tumor growth. It is also known that many tumors over-express MMPs.
  • Suitable MMP cleavage sites for use in an ELP of the present methods include, without limitation, a MMP-1 cleavage site, a MMP-13 cleavage site, a MMP-11 cleavage site, a MMP-12 cleavage site, a MMP-7 cleavage site, a MMP-26 cleavage site, a MMP-2 cleavage site, a MMP-9 cleavage site, a MMP-14 cleavage site, a MMP-16 cleavage site, a MMP- 17 cleavage site, a MMP-24 cleavage site, and a MMP-25 cleavage site, as described herein.
  • the subject ELP may comprise a payload polypeptide (e.g., a payload polypeptide fused to the second ELP domain via a linker).
  • the payload polypeptide may comprise a single-chain antibody comprising variable domains derived from Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, Dusigitumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab, Lucatumumab, Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumum
  • the payload polypeptide may comprise a cytokine, such as Interleukin-1, Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-7, Interleukin-9, Interleukin-11, Interleukin-12, Interleukin-13, Interleukin-15 , Interleukin-18, Interleukin-21 , Interleukin-23, Interleukin-27, TGF- ⁇ , IFN- ⁇ , TNF- ⁇ , PD-L1, 4- 1BBL, OX40L, ICOSLG, or variants thereof.
  • a cytokine such as Interleukin-1, Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-7, Interleukin-9, Interleukin-11, Interleukin-12, Interleukin-13, Interleukin-15 , Interleukin-18, Interleukin-21 , Inter
  • the subject ELP may be conjugated to a payload molecule (e.g., a payload molecule conjugated to the second ELP domain).
  • the payload molecule may be a microtubule inhibitor as disclosed herein, such as an auristatin, a maytansinoid, or a derivatives thereof.
  • the payload molecule may be a DNA-damaging agent.
  • the DNA-damaging agent may be a calicheamicin, a pyrrolobenzodiazepine, a topoisomerase inhibitor, or an alkylating agent as disclosed herein.
  • Tissue Scaffolds The present disclosure provides methods for producing a tissue scaffold at site of injury in a subject, the methods comprising administering a subject ELP composition to a subject, wherein an enzyme that can cleave the enzymatic cleavage site of the ELP is present in or around the site of injury.
  • the LCST of the second ELP domain may be less than the LCST Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 of the first ELP domain, such that, upon contact of the ELP with the enzyme present in or around the site of injury, the ELP is cleaved thus enabling the second ELP domain to undergo a phase transition and assemble a tissue scaffold that facilitates regrowth of tissue at the site of injury.
  • the administering comprises applying a subject ELP composition to the site of injury.
  • the subject ELP composition may be applied directly to the site of injury via a syringe, swab, spray, or aerosol.
  • the administering comprises intramuscular injection, intradermal injection, or subcutaneous injection of the ELP composition in a subject.
  • the ELP composition may diffuse into the circulatory system of the subject and circulate throughout the body until cleaved by the corresponding enzyme present in or around the site of injury.
  • the injection site may be at or near the site of injury.
  • kits comprising: a) a subject ELP as disclosed herein; and b) a pharmaceutical excipient.
  • the subject ELP may comprise a payload polypeptide (e.g., a payload polypeptide fused to the second ELP domain via linker).
  • the subject ELP may be conjugated to a payload molecule (e.g., a payload molecule conjugated to the second ELP domain).
  • the subject ELP may be in liquid form.
  • the subject ELP may be lyophilized.
  • the lyophilized ELP may be resuspended in the pharmaceutically acceptable excipient (e.g., when the pharmaceutically acceptable excipient is in liquid form).
  • the pharmaceutically acceptable excipient may comprise a pharmaceutically acceptable salt. Suitable salts for use in a present composition include, without limitation, sodium chloride, sodium citrate, sodium benzoate, sodium phosphates (e.g., phosphate buffered saline), potassium phosphate, potassium citrate, calcium carbonate, and calcium phosphate, or combinations thereof.
  • the pharmaceutically acceptable excipient may comprise a pharmaceutically acceptable sugar or polyol.
  • Suitable sugars or polyols for use Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 in a present composition include, without limitation, sucrose, trehalose, mannose, mannitol, raffinose, lactitol, sorbitol and lactobionic acid, glucose, maltulose, iso-maltulose, lactulose, maltose, lactose, iso-maltose, maltitol, palatinit, stachyose, melezitose, dextran, or combinations thereof.
  • the pharmaceutically acceptable excipient may comprise an amino acid(s).
  • Suitable amino acids for use in a present composition include, without limitation, glycine, arginine, lysine, and glutamine.
  • the pharmaceutically acceptable excipient may comprise water in combination with any other of the compounds described above.
  • a subject kit may further comprise one or more containers for storing the ELP and/ or the pharmaceutically acceptable excipient.
  • the size of the container may depend on the volume of the ELP (in lyophilized or liquid form) to be held in the container.
  • the container may be configured to hold an amount of a subject ELP and/or pharmaceutically acceptable excipient, ranging from 0.1 mg to 1000 mg, such as from 0.1 mg to 900 mg, such as from 0.1 mg to 800 mg, such as from 0.1 mg to 700 mg, such as from 0.1 mg to 600 mg, such as from 0.1 mg to 500 mg, such as from 0.1 mg to 400 mg, or 0.1 mg to 300 mg, or 0.1 mg to 200 mg, or 0.1 mg to 100 mg, 0.1 mg to 90 mg, or 0.1 mg to 80 mg, or 0.1 mg to 70 mg, or 0.1 mg to 60 mg, or 0.1 mg to 50 mg, or 0.1 mg to 40 mg, or 0.1 mg to 30 mg, or 0.1 mg to 25 mg, or 0.1 mg to 20 mg, or 0.1 mg to 15 mg, or 0.1 mg to 10 mg, or 0.1 mg to 5 mg, or 0.1 mg to 1 mg, or 0.1 mg to 0.5 mg.
  • 0.1 mg to 1000 mg such as from 0.1 mg to 900 mg, such as from
  • the container is configured to hold an amount of ELP (in lyophilized or liquid form) and/or pharmaceutically acceptable excipient, ranging from 0.1 g to 10 g, or 0.1 g to 5 g, or 0.1 g to 1 g, or 0.1 g to 0.5 g.
  • ELP in lyophilized or liquid form
  • pharmaceutically acceptable excipient ranging from 0.1 g to 10 g, or 0.1 g to 5 g, or 0.1 g to 1 g, or 0.1 g to 0.5 g.
  • the container may be configured to hold a volume (e.g., a volume of a liquid) ranging from 0.1 ml to 1000 ml, such as from 0.1 ml to 900 ml, or 0.1 ml to 800 ml, or 0.1 ml to 700 ml, or 0.1 ml to 600 ml, or 0.1 ml to 500 ml, or 0.1 ml to 400 ml, or 0.1 ml to 300 ml, or 0.1 ml to 200 ml, or 0.1 ml to 100 ml, or 0.1 ml to 50 ml, or 0.1 ml to 25 ml, or 0.1 ml to 10 ml, or 0.1 ml to 5 ml, or 0.1 ml to 1 ml, or 0.1 ml to 0.5 ml.
  • a volume e.g., a volume of a liquid
  • the container is configured to hold a volume ranging from 0.1 ml to 200 ml.
  • Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes.
  • Containers can be formed from a variety of suitable materials, including glass or plastic.
  • the container may be composed of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREX TM ), fused quartz glass, fused silica glass, and the like.
  • suitable materials for the containers include plastics, such as, but not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethers (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkanes (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), and the like.
  • PTFE polytetrafluoroethylene
  • PFE perfluoroethers
  • FEP fluorinated ethylene propylene
  • PFA perfluoroalkoxy alkanes
  • PET polyethylene terephthalate
  • PE polyethylene
  • PEEK polyetheretherketone
  • the container may be sealed. That is, the container may include a seal that substantially prevents the contents of the container from exiting the container. The seal of the container may also substantially prevent other substances from entering the container.
  • the seal may be a water-tight seal that substantially prevents liquids from entering or exiting the container, or may be an air-tight seal that substantially prevents gases from entering or exiting the container.
  • the seal is a removable or breakable seal, such that the contents of the container may be exposed to the surrounding environment when so desired, e.g., if it is desired to remove a portion of the contents of the container.
  • the seal is made of a resilient material to provide a barrier (e.g., a water-tight and/or air-tight seal) for retaining a sample in the container.
  • Particular types of seals include, but are not limited to, films, such as polymer films, caps, etc., depending on the type of container.
  • Suitable materials for the seal include, for example, rubber or polymer seals, such as, but not limited to, silicone rubber, natural rubber, styrene butadiene rubber, ethylene-propylene copolymers, polychloroprene, polyacrylate, polybutadiene, polyurethane, styrene butadiene, and the like, and combinations thereof.
  • a container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the kit may further comprise a syringe, needle, or cannula.
  • the kit may further comprise a substrate material.
  • the substrate material may be a sterile sponge, gauze, bandage, compression bandage, pillow, sleeve, and the like.
  • the substrate may be any permeable or semi-permeable substrate that would enable absorption of the subject ELP into at least a portion of the substrate when the ELP is applied to the substrate.
  • the substrate material may be composed of a natural fiber or polymer (e.g., cotton, silk, wool).
  • the substrate material may be composed of a synthetic fiber or polymer (e.g., rayon or polyester).
  • kits comprising: a) a subject expression vector comprising a nucleic acid encoding a subject ELP as disclosed herein; b) an endonuclease; and c) a ligase.
  • a subject expression vector may comprise convenient restriction sites (i.e., restriction endonuclease sites) located near a promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest.
  • the restriction site present in the expression vector is for a restriction endonuclease that generates cohesive (or “sticky”) ends, including but not limited to, AscI, Aval, BamHI, BclI, BglII, BstEI, Bst, BI, BstYI, Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 EcoRI, MluI, NarI, NheI, NotI, PstI, PvuI, SacI, SalI, SpeI, StyI, XbaI, XhoI and XmaI.
  • the restriction site present in expression vector is for a restriction endonuclease that generates blunt ends, including but not limited to, EcoRV, FspI, NaeI, NruI, PvuII, SmaI, SnaBI, and StuI.
  • the endonuclease may be restriction endonuclease that generates cohesive (or “sticky”) ends, including but not limited to, AscI, Aval, BamHI, BclI, BglII, BstEI, Bst, BI, BstYI, EcoRI, MluI, NarI, NheI, NotI, PstI, PvuI, SacI, SalI, SpeI, StyI, XbaI, XhoI and XmaI.
  • AscI AscI, Aval, BamHI, BclI, BglII, BstEI, Bst, BI, BstYI, EcoRI, MluI, NarI, NheI, NotI, PstI, PvuI, SacI, SalI, SpeI, StyI, XbaI, XhoI and XmaI.
  • a subject kit may include instructions for applying a subject ELP composition to a substrate (e.g., a gauze) for application to a hemorrhage site in a subject.
  • a subject kit may include instructions for cloning a transgene into the expression vector comprising a nucleic acid encoding a subject ELP.
  • a suitable medium or substrate e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like.
  • An isothermally phase-separable elastin-like polypeptide comprising: i) a first ELP domain having a first lower critical solution temperature (LCST); and ii) a second ELP domain having a second LCST that is different from the first LCST, wherein the first and second ELP domains are separated by an enzymatic cleavage site.
  • LCST lower critical solution temperature
  • S23-364 2.
  • the ELP of Clauses 1 or 2 wherein the first and second ELP domain each comprises one or more pentapeptide repeats. 4.
  • each pentapeptide repeat comprises an amino acid sequence VPGXG (SEQ ID NO:1), wherein X is any amino acid other than proline. 5.
  • each pentapeptide repeat comprises an amino acid sequence VPGXG (SEQ ID NO:2), wherein X is selected from valine, alanine, glycine, lysine, histidine, serine, and glutamate. 6.
  • the ELP of Clause 5 wherein: i) The first ELP domain comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5), VPGAG (SEQ ID NO:6), VPGGG (SEQ ID NO:7), VPGKG (SEQ ID NO:8), VPGSG (SEQ ID NO:9), and VPGEG (SEQ ID NO:10); and ii) The second ELP domain comprises one or more pentapeptide repeats, each pentapeptide repeat comprising an amino acid sequence selected from: VPGVG (SEQ ID NO:5) and VPGHG (SEQ ID NO:11). 7.
  • the ELP of any one of clauses 4-9 wherein: i) the first ELP domain comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:12; and Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 ii) the second ELP domain comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:13. 11.
  • the ELP of any one of clauses 4-10 wherein: i) the first ELP domain comprises the amino acid sequence set forth in SEQ ID NO:12; and ii) the second ELP domain comprises the amino acid sequence set forth in SEQ ID NO:13. 12.
  • the ELP of Clause 14, wherein the protease cleavage site is selected from: a TEV protease cleavage site, an enterokinase cleavage site a thrombin cleavage site, a factor Xa cleavage site, a factor VIIa cleavage site, a factor IXa cleavage site, a factor XIa cleavage site, a factor XIIa cleavage site, a kallikrein cleavage site, a plasmin cleavage site, a furin cleavage site, a matrix metalloproteinase cleavage site, a caspase cleavage site, a paracaspase cleavage site, a granzyme B cleavage site, and a proline-endopeptidase cleavage site.
  • the first and second linkers each comprise an amino acid sequence comprising: a) the amino acid sequence set forth in SEQ ID NO: 14; or b) the amino acid sequence set forth in SEQ ID NO: 15, wherein n is an integer equal to or greater than 1.
  • the ELP of Clause 19 wherein the payload polypeptide comprises a single-chain antibody, a cytokine, a growth factor, a peptide hormone, or a therapeutic peptide.
  • 23 The ELP of any one of clauses 1-22, wherein the ELP further comprises an N- terminal leader domain.
  • 32. A cell comprising the expression vector of Clause 31. 33. The cell of Clause 32, wherein the cell is a eukaryotic cell. 34. The cell of Clause 33, wherein the eukaryotic cell is a mammalian cell. 35. The cell of Clause 34, wherein the mammalian cell is a human cell. 36. The cell of any one of clauses 32-35, wherein the cell is in vivo. 37.
  • An ELP composition comprising: a) an ELP of any one of clauses 1-28; and b) a pharmaceutically acceptable excipient. 38.
  • a method for assembling a cell-protective biopolymer on the surface of a target cell comprising: contacting the surface of a target cell with an ELP of any one of clauses 1-25 to assemble a cell-protective biopolymer, wherein the surface of the target cell comprises an enzyme that can cleave the enzymatic cleavage site of the ELP.
  • a method for assembling a cell-protective biopolymer on the surface of a target cell comprising: Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 expressing an ELP of any one of clauses 1-25 in the target cell to assemble a cell- protective biopolymer on the surface of said cell, wherein the ELP is secreted by the target cell, and wherein the target cell comprises an enzyme that can cleave the enzymatic cleavage site of the ELP.
  • the target cell comprises the expression vector of Clause 30 or 31. 41 The method of Clause 39 or 40, wherein the enzyme is present on the surface of the target cell. 42.
  • a method for reducing blood loss at a hemorrhage site in a subject comprising: administering to a hemorrhage site in a subject an effective amount of an ELP composition according to clause 37 to reduce blood loss at the hemorrhage site, wherein an enzyme that can cleave the enzymatic cleavage site of the ELP is present in or around the hemorrhage site.
  • an enzyme that can cleave the enzymatic cleavage site of the ELP is present in or around the hemorrhage site.
  • a method for delivering a therapeutic agent to a target site in a subject comprising: administering an ELP composition according to Clause 37 to a subject, wherein an enzyme that can cleave the enzymatic cleavage site of the ELP is present in or around the target site.
  • the administering comprises intravenously administering the ELP composition to the subject.
  • the administering comprises intramuscular injection, intradermal injection, or subcutaneous injection of the ELP composition in a subject.
  • the method of Clause 58, wherein the enzymatic cleavage site of the ELP is a protease cleavage site.
  • the method of Clause 59, wherein protease cleavage site is a matrix metalloproteinase cleavage site.
  • 61. A method for producing a tissue scaffold at a site of injury in a subject, the method comprising: administering an ELP composition according to Clause 37 to a subject, wherein an enzyme that can cleave the enzymatic cleavage site of the ELP is present in or around the site of injury.
  • the method of Clause 61, wherein the administering comprises applying the ELP composition to the site of injury.
  • the administering comprises intramuscular injection, intradermal injection, or subcutaneous injection of the ELP composition in a subject.
  • 64. A kit comprising: a) an ELP according to any one of clauses 1-28; and b) a pharmaceutical excipient.
  • 65. The kit of Clause 64, wherein the ELP is lyophilized.
  • 66. The kit of Clauses 64 or 65, wherein the kit further comprises a substrate material.
  • the substrate material comprises a gauze. 68.
  • kit of any one of Clauses 64-67, wherein the kit further comprises a syringe. 69.
  • a kit comprising: a) an expression vector according to Clause 30; b) an endonuclease; and c) a ligase.
  • the following examples are provided by way of illustrations and not by way of limitation.
  • EXAMPLES Example 1 Discovery of non-immunogenic biopolymers to protect genetically altered cells Goals & Objectives: This research seeks to discover biopolymers that protect genetically modified cells from immune responses.
  • Protective biopolymers will combine polypeptides that mimic the extracellular matrix (ECM) with protease-guided mechanisms that activate material assembly in the local cellular environment.
  • ECM extracellular matrix
  • proteases typically degrade the ECM
  • proteases can also induce material assembly by exposing functional peptide domains.
  • thrombin cleaves fibrinogen to guide the formation of fibrin clots during blood coagulation.
  • Biopolymer assembly will be driven using elastin-like polypeptides (ELPs), which mimic the ECM and elicit minimal immune responses.
  • ELPs have drawn interest as thermally sensitive biopolymers due to their “inverse” phase behavior. Specifically, ELPs are soluble in water at low temperatures and become insoluble upon heating above a lower critical solution temperature (LCST, FIG.1A).
  • the LCST can be tuned from 10 to 90 °C by modifying the repetitive polypeptide sequence (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO:1), in which Xaa can be any Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 amino acid except proline and the sequence is repeated n times.
  • the LCST is typically lowered by increasing hydrophobicity, molecular weight, and/or protein concentration.
  • a class of protease-cleavable ELPs that are water soluble at physiological temperature (37 °C) in their full-length form and are cleaved into water soluble and insoluble components upon exposure to a specific protease are proposed (FIG.1B). Insoluble components will assemble into coatings that protect cells. Moreover, coatings can assemble at the surfaces of genetically altered mammalian cells that display the specific protease. Specifically, mixed charged/hydrophobic ELPs are soluble up to 45 °C under most physiological solution conditions, whereas hydrophobic-only ELPs are soluble up to 35 °C. A mixed charge/hydrophobic ELP will be soluble upon delivery to cells at physiological conditions (37 °C).
  • proteases expressed at the cell surface will trigger cleavage and subsequent insoluble ELP assembly into a protective coating (FIG. 1C).
  • the molecular design for cleavage-induced assembly defies conventional wisdom in polypeptide design, in which low molecular- weight ELPs are typically more water soluble than high molecular- weight ELPs.
  • Example 2 Encoding Cellular Self-Protective Biomaterials into Therapeutic Gene Delivery Systems Numerous genetic disorders can be treated using gene replacement therapy – the reintroduction of missing genes into patients’ affected cells. However, proteins expressed from such genes will be recognized as “non-self,” and cells expressing these proteins will be inevitably attacked by the immune system. This immune response leads to reduced therapeutic efficiency or, worse, serious adverse effects.
  • biomaterials can provide physical barriers to protect genetically modified cells from immune responses.
  • Self-protective biomaterials will integrate recombinant proteins that mimic the extracellular matrix (ECM) with protease-dependent mechanisms to activate material assembly in the local cellular environment.
  • ECM extracellular matrix
  • proteases typically degrade the ECM, the modular, orthogonal nature of proteolytic cleavage will be leveraged to guide ECM generation.
  • ECM-mimetic biomaterials will comprise a class of protease-cleavable elastin-like polypeptides (ELPs), which elicit minimal immune responses and will form protective barriers around protease-expressing cells.
  • ELPs are repetitive proteins with the pentapeptide sequence (VPGXG)n, where the guest residue X can be any amino acid except proline and the pentapeptide is repeated n times.
  • ELPs have drawn interest as thermally sensitive materials due to their lower critical solution temperature (LCST) in water.
  • ELPs are soluble in water at temperatures below the LCST and become insoluble upon heating above the LCST (FIG.1A). ELP phase behavior can be tuned from 10 to 90 °C by modifying the identity of guest residue X and repeat number n. Generally, the LCST is lowered by increasing hydrophobicity, molecular weight, and/or protein concentration.
  • a class of protease-cleavable ELPs that are water soluble in their full-length form and will be cleaved into water soluble and insoluble components upon exposure to proteases localized at cell surfaces is proposed (FIGs. 1B-1C).
  • Insoluble components will assemble into physical biomaterials around genetically altered mammalian cells that display a specific protease from Tobacco Etch Virus (TEV).
  • Protease-cleavable ELPs will have 3 key features: a charged domain (VPGEG)n (SEQ ID NO:108) that enhances ELP solubility, a cleavage site ENLYFQG that is recognized by TEV protease, and a hydrophobic domain (VPGVG)n (SEQ ID NO:109) that becomes insoluble upon cleavage.
  • VPGVG hydrophobic domain
  • VPGVG valine-only ELPs
  • VPGVG valine-only ELPs
  • VPGVG valine-only ELPs
  • TEV proteases expressed Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 at the cell surface will trigger cleavage and subsequent ELP phase separation into a protective biomaterial surrounding the cell.
  • TEV protease treatment is anticipated to lower the LCST of the cleavable ELP below physiological temperature (37 °C); if this goal is not achieved, valine residues will be substituted with more hydrophobic leucine residues to decrease the LCST after cleavage.
  • the optimized TEV-responsive ELP will be validated in cell culture.
  • HEK Human embryonic kidney cells will be genetically engineered to display TEV protease, and administer the ELP to them. ELP phase separation only in the presence of TEV protease is proposed.
  • the HEK cells will display a common antigen (e.g., OVA) during co-culture with T cells expressing the cognate T cell receptor. The extent to which the protease-guided ELP deposition reduces T cell cytotoxicity against the HEK cells will be assessed.
  • OVA common antigen
  • the HEK cells will be replaced with a hepatocyte cell line and the aforementioned experiments repeated.
  • Aim 2 Programmed secretion of ECM-mimetic materials by genetically altered therapeutic cells. ELPs secreted by cells must remain soluble throughout the entire secretion pathway, for which the unique biochemical features of different secretory compartments and the protease- responsive ELPs developed in Aim 1 will be leveraged.
  • ELPs Elastin-like polypeptides
  • Temperature remains the predominant stimulus for promoting ELP self-assembly, as these proteins become insoluble and aggregate above a sequence- dependent transition temperature.
  • the relatively constant human body temperature poses a significant challenge to temperature-driven ELP self-assembly with high spatiotemporal control.
  • Other stimuli that are typically used to induce ELP self-assembly, including pH and salt concentration, are likewise challenging to manipulate in vivo.
  • an ELP is presented that self-assembles when exposed to a biological stimulus (a protease) with the potential to function in isothermal environments such as genetically engineered cells (e.g. immune-protective biomaterials), blood vessels (e.g. “smart” hemostatic materials), and cancerous tumors (e.g.
  • the designed protease-responsive ELP is soluble at physiologically relevant temperatures; upon incubation with a protease, the ELP produces an insoluble ELP fragment that self assembles into a biomaterial (FIG.2A).
  • the ELP has a tobacco etch virus (TEV) protease recognition sequence between hydrophilic and hydrophobic ELP blocks, where hydrophilicity of each block was tuned by altering the amino acid, X, in the VPGXG (SEQ ID NO:1) repeats that comprise ELPs. This recognition sequence can be substituted with any other protease recognition sequence.
  • TSV tobacco etch virus
  • FIG. 2C demonstrates that the ELP remains soluble before cleavage, with the hydrophilic block acting as a solubility tag for the entire protein (see FIG.3C for additional controls).
  • FIG. 4 An example of MALDI-TOF mass spectrometry of a TEV protease-responsive ELP is shown in FIG. 4. It is proposed that this protease-driven ELP self-assembly behavior will be transferrable to other compatible protease-cut site pairings, which is a current research focus. In conclusion, we demonstrate isothermal ELP self-assembly driven by proteolytic cleavage of a soluble ELP. Example 3 Introduction In this work, a protease-responsive, “cleavable” ELP that produces a phase-separating ELP fragment upon isothermal proteolytic cleavage is reported.
  • the cleavable ELP consists of hydrophobic and hydrophilic ELP blocks separated by a protease cleavage site. With this design, the overall ELP hydrophilicity is regulated by proteolytic cleavage, thereby linking protease-driven biochemical changes to emergent phase behavior. ELP concentrations and temperatures amenable to protease-driven phase separation were identified by constructing a phase diagram of a cleavable ELP and its cleavage products. Cleavable ELP variants with responses to four unique proteases demonstrate generalizability of protease-driven phase separation of ELPs.
  • Cleavable ELPs comprise the soluble fragment ELP[V 1 A 8 G 7 ] 6 , a protease cleavage site as a linker, and the insoluble fragment ELP[V] 60 .
  • Protease-driven, isothermal phase separation is designed to occur in the temperature window in which the diblock ELP[V 1 A 8 G 7 ] 6 –ELP[V] 60 is soluble and the hydrophobic block ELP[V] 60 is insoluble.
  • Cleavable ELPs contain an N-terminal ELP[V 1 A 8 G 7 ] 6 hydrophilic block and C-terminal ELP[V] 60 hydrophobic block.
  • the soluble block includes 6 repeats of ELP[V 1 A 8 G 7 ] to allow for the detection of ELP blocks with distinct molecular weights using polyacrylamide gel electrophoresis (PAGE).
  • PAGE polyacrylamide gel electrophoresis
  • ELP variants were produced by changing the cleavage site to be recognized by tobacco etch virus protease (TEVP), thrombin (Thr), enterokinase (EK), or factor Xa (FXa) (FIG.5B).
  • TEVP tobacco etch virus protease
  • Thr thrombin
  • EK enterokinase
  • FXa factor Xa
  • ELP[V] 60 All ELPs contained an N-terminal leader sequence MGGWGP (SEQ ID NO:117) and a C-terminal trailer sequence WLE(H)6 (SEQ ID NO:118).
  • the tryptophan residues enable detection of full-length ELPs and cleavage products using Stain-Free PAGE gels.
  • Cleavable ELP gene assembly Briefly, cleavable ELP variants were cloned using both Golden Gate assembly and directional ligation. Gene sequences encoding repetitive ELPs were generated using a codon scrambling algorithm and manual adjustments to reduce mRNA folding near the translation initiation site. Golden Gate assembly overhangs were designed using the NEBridge Golden Gate online resource. First, each ELP block was assembled separately using Golden Gate assembly through BsmBI sites. Then, the blocks were combined and subcloned into a modified pET-22b(+) expression vector (SI) using directional ligation.
  • SI modified pET-22b(+) expression vector
  • annealed oligos (data not shown) encoding protease cleavage sites were inserted between the blocks in a second Golden Gate assembly reaction using BsaI.
  • DNA sequences for TEVP-, Thr-, EK-, and FXa-cleavable ELP variants were validated with whole-plasmid sequencing (Primordium Labs) and are reported in Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 the supplementary information. Plasmids encoding these four cleavable ELP variants, along with a precursor plasmid to generate variants with other protease cleavage sites, are deposited in Addgene with catalog numbers (data not shown).
  • ELP[V]60 gene synthesis was cloned using a single Golden Gate assembly reaction with BsmBI sites, followed by directional ligation into the modified pET-22b(+) expression vector.
  • the gene sequence was designed similarly to the variants described above.
  • the DNA sequence was validated by whole-plasmid sequencing and deposited in Addgene (data not shown).
  • the protein sequence is included in Supplementary Information. Protein expression, purification, and validation ELPs were produced by recombinant protein expression in the BLR(DE3) (Novagen) strain of Escherichia coli.
  • Cultures were harvested by centrifugation; were resuspended in 35 mL of lysis buffer (3 mM MgCl 2 , 1 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, 10 mM Tris, pH 7.5) and frozen overnight at -80 °C. Lysates were thawed and incubated at 4 °C with stirring for at least one hour with lysozyme (0.5–1 mg/mL lysate), DNase I (0.5–1 ⁇ L/10 mL lysate), and RNase A (spatula tip).
  • lysis buffer 3 mM MgCl 2 , 1 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, 10 mM Tris, pH 7.5
  • Lysates were thawed and incubated at 4 °C with stirring for at least one hour with lysozyme (0.5–1 mg/
  • Lysates were further processed with probe sonication, followed by lysate clarification via centrifugation (4 °C, 14635 ⁇ g, 1 hr).
  • ELPs were isolated with inverse transition cycling (ITC), with each ITC cycle comprising a hot spin followed by a cold spin.
  • ITC inverse transition cycling
  • solid NaCl was added to a final concentration of 1.5 M, and solutions were warmed to 37 °C for at least 2 hr and until all NaCl dissolved to drive selective coacervation of the ELP.
  • ELP-enriched pellets were obtained via centrifugation in a pre-heated centrifuge (37 °C, 14635 ⁇ g, 1 hr).
  • Pellets were resuspended in 1 ⁇ phosphate-buffered saline (PBS, 137 mM NaCl) or ultrapure water and placed on a rotisserie rotator at 4 °C for at least 2 hr to solubilize the pellets. Insoluble contaminants were removed via centrifugation in a subsequent cold spin in a pre-chilled centrifuge (4 °C, 14635 ⁇ g, 1 hr). Two Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 additional ITC cycles were performed for cleavable ELP variants; in these subsequent cycles, 4.5 M NaCl solution was added to reach a final concentration of 1.5 M NaCl.
  • ELP[V] 60 was purified using three ITC cycles.
  • coacervation was induced by adding solid NaCl to a final concentration of 3 M for the first cycle and 4.5 M NaCl solution to reach 1.5 M NaCl for the following two cycles.
  • all ELPs were dialyzed against ultrapure water (Milli- Q, 18.2 M ⁇ -cm, 4 L, 4 °C, 5–6 cycles, at least 3 hr/cycle) in 14000 Da cutoff cellulose dialysis tubing. Samples were subsequently lyophilized to yield a white, fluffy material.
  • ELP variants yielded between 20–40 mg/L, and ELP[V] 60 yielded between 60–140 mg/L.
  • Successful expression of each ELP was validated with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, Protein and Nucleic Acid Facility, Stanford University).
  • Sample preparation Protein stock solutions were produced by weighing and resuspending lyophilized protein in buffer. Stock solutions were mixed overnight at 4 °C in a rotisserie rotator.
  • TEVP-cleavable ELP 200 ⁇ L of 30 ⁇ M TEVP-cleavable ELP in PBS were mixed with 8 ⁇ L of either TEVP (10 units/ ⁇ L) or PBS (negative control).
  • TEVP-only controls contained 200 ⁇ L of PBS and 8 ⁇ L TEVP.
  • OD350 was recorded every 5 minutes, with each reported measurement corresponding to the mean of 25 reads per well. Each measurement was preceded by 5 s of orbital shaking.
  • reaction products were analyzed using SDS-PAGE. A sample containing the TEVP-cleavable ELP and TEVP was cooled to 4 °C to resolubilize the phase- separated component. An aliquot was reserved for further analysis.
  • the insoluble phase was separated from the soluble Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 phase via centrifugation. Briefly, the remaining sample was heated to 37 °C in a thermocycler and centrifuged to separate the insoluble component (37 °C, 21100 ⁇ g, 15 mins). The supernatant was removed, and the pellet was dried upside down before resuspending it in an equal volume of PBS at 4 °C. Samples were analyzed using a 12% SDS-PAGE Stain-Free gel under reducing conditions following the manufacturer’s instructions (Bio-Rad).
  • Coomassie Brilliant Blue staining of samples was unsuccessful due to poor visualization of the hydrophilic cleavage product (FIG. 10). Poor visualization of the hydrophilic ELP cleavage product likely arises from its low content of basic and aromatic residues that are typically bound by Coomassie Brilliant Blue. Unsuccessful staining of ELP fragments with Coomassie Brilliant Blue has been previously reported. Turbidimetry Transition temperatures of ELP[V] 60 , TEVP-cleavable ELP, and cleavage products were determined using turbidimetry.
  • Cleavage products were obtained by incubating 100 ⁇ M TEVP- cleavable ELP with TEVP (10 units/ ⁇ L) at a 60:1 volume ratio for at least 3 hours at 34 °C with 150 rpm orbital shaking. Temperature-dependent OD 350 was measured in a 1 mm path length quartz cuvette that was capped to prevent evaporation using a Jasco J-815 spectrometer. Samples were placed in the pre-cooled sample chamber and equilibrated for one minute before starting the measurement. The sample chamber temperature was increased from 20 °C to 55 °C by 1 °C/min, and OD350 was measured every 0.5 °C.
  • transition temperature was determined by calculating the inflection point of the temperature- dependent OD 350 curve as detailed in the Supplementary Information. Transition temperatures are reported as the average and standard deviation of triplicate measurements.
  • Combinatorial screening of isothermal phase separation Generalizability of protease-driven phase separation of ELPs was demonstrated via time- resolved turbidimetry with cleavable ELP variants.
  • Cleavable ELP variant stock solutions were prepared as described above using either PBS for reactions with TEVP or a general reaction buffer (20 mM Tris, 100 mM NaCl, 2 mM CaCl2, pH 8.0) for reactions with thrombin, factor Xa, and enterokinase.
  • the plate reader sample chamber and 30 ⁇ M cleavable ELP solutions were equilibrated at 37 °C for at least 30 minutes prior to starting measurements.
  • Enzymes (6 ⁇ L TEVP at 10 units/ ⁇ L, 3 ⁇ L thrombin at 1 unit/ ⁇ L, 3 ⁇ L factor Xa at 1 mg/mL, 3 ⁇ L enterokinase at 16 units/ ⁇ L) were added to a second 96-well plate held at 4 °C to preserve enzymatic activity. Solutions containing 30 ⁇ M cleavable ELP variants (150 ⁇ L) were transferred to the enzyme 96- well plate, and the entire plate was placed in the plate reader.
  • OD350 was taken every 10 minutes, Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 with each reported measurement corresponding to the mean of 25 reads per well. Each measurement was preceded by 5 s of orbital shaking. Reaction products were analyzed with SDS- PAGE Stain-Free gels (Bio-Rad). Results and Discussion Isothermal phase separation of TEVP-cleavable ELP Incubation of TEVP-cleavable ELP with TEVP at 37 °C led to isothermal phase separation. Isothermal phase separation was monitored with time-resolved turbidimetry during a 3-hour reaction between TEVP-cleavable ELP and TEVP (FIG.6A).
  • Lane 1 contained the full-length TEVP- cleavable ELP, which appeared as a single high molecular weight band near 75 kDa.
  • the expected protein molecular weight of 64.0 kDa was validated with MALDI-TOF MS (FIG.12A).
  • ELPs and other proline-rich proteins tend to exhibit anomalous electrophoretic migration that results in a higher apparent molecular weight than the true molecular weight. This anomalous migration likely stems from conformations introduced by high proline content.
  • Lane 2 contained the TEVP and cleavable ELP reaction mixture, which appeared as three distinct bands between 25 kDa and 50 kDa.
  • lane 5 contained only the hydrophilic ELP block Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 and TEVP.
  • Lane 6 contained recombinant ELP[V] 60 to validate the depletion of the hydrophobic ELP block from the supernatant. Overall, this endpoint analysis demonstrates that increased turbidity emerges due to phase separation of the hydrophobic ELP[V]60 cleavage product.
  • Thermodynamic behavior of TEVP-cleavable ELP reveals conditions for protease-driven, isothermal phase separation
  • phase behaviors of TEVP- cleavable ELP and its cleavage products were mapped using temperature-dependent turbidimetry.
  • the transition temperatures of TEVP-cleavable ELP were 4 to 5 °C higher than its cleavage products at all concentrations investigated (FIG.7).
  • transition temperatures reflect those of the ELP[V] 60 hydrophobic block, consistent with its selective phase separation at 30 ⁇ M and 37 °C (FIG.6B, FIG.16A-16B).
  • transition temperature of recombinant ELP[V] 60 likely arises from the hydrophobicity of the leader sequence tag (MGGWGP) (SEQ ID NO:117), which is not included in the ELP[V] 60 cleavage product sequence (data not shown).
  • MGGWGP leader sequence tag
  • Time-resolved turbidimetry of 30 ⁇ M TEVP-cleavable ELP incubated with TEVP at 32 °C did not result in substantial phase separation. Therefore, the suppression of transition temperatures in the recombinant ELP[V] 60 generated an incorrect boundary between regions I and II in the TEVP-cleavable ELP phase diagram.
  • SDS-PAGE resolved a high intensity band corresponding to the cleavable ELP as well as two lower intensity bands that migrated similarly to the intended cleavage products.
  • the lower molecular weight bands suggest that off-target cleavage occurred within the ELP’s protease cleavage sequence.
  • the proteases with off-target reactivity are trypsin-like proteases, which promiscuously cleave after lysine or arginine. This activity is consistent with off-target cleavage occurring within the cleavage sequence (Scheme 1), thereby producing the intended ELP cleavage products.
  • Off-target cleavage activity is relatively weak, as indicated by the low intensity of cleavage product bands and high intensity of starting material bands.
  • off-target reactivity did not result in elevated turbidity (FIG.8).
  • Turbidity of the two off-target cleavable ELP–protease pairings nearly matched that of ELP-only controls, indicating that no ELP phase separation occurred in these reactions.
  • phase separation of the hydrophobic cleavage product only occurred above a critical concentration of approximately 10 ⁇ M for the TEVP-cleavable ELP variant (FIG. 7).
  • At least one third of the initial 30 ⁇ M TEVP-cleavable ELP must be converted to its cleavage products to observe phase separation.
  • Low conversion of cleavable ELPs in off-target reactions resulted in prohibitively low concentrations of hydrophobic cleavage products to drive phase separation.
  • higher levels of off-target reactivity might produce unintended phase separation responses; however, off-target reactivity could be leveraged beneficially to engineer broad-spectrum protease-cleavable ELPs that respond to families of similar proteases.
  • Protease-responsive “cleavable” ELPs were designed by placing a protease cleavage site between hydrophobic and hydrophilic ELP blocks.
  • the hydrophilic ELP block enhances the solubility of the hydrophobic ELP block, thereby producing a temperature window in which protease-driven phase separation proceeds during an isothermal reaction.
  • the hydrophilic block sequence design space is vast: hydrophilic ELP blocks containing polar or charged guest residues Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 can increase the solubility of hydrophobic ELPs, as can hydrophilic tags or protein domains beyond ELPs.
  • protease-driven ELP phase separation will benefit from systematic understanding of the role of the hydrophilic block sequence on the temperature window for protease-driven ELP phase separation.
  • Analysis of 11,430 recombinant protein production experiments reveals that protein yield is tunable by synonymous codon changes of translation initiation sites.
  • DOI: 10.1039/B909777E Attorney Docket No.: STAN-2151WO Stanford No.: S23-364 46. Trabbic ⁇ Carlson, K.; Meyer, D. E.; Liu, L.; Piervincenzi, R.; Nath, N.; LaBean, T.; Chilkoti, A. Effect of protein fusion on the transition temperature of an environmentally responsive elastin ⁇ like polypeptide: a role for surface hydrophobicity? Protein Eng., Des. Sel.2004, 17 (1), 57- 66. DOI: 10.1093/protein/gzh006 47. Waugh, D. S. An overview of enzymatic reagents for the removal of affinity tags.
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above.
  • a range includes each individual member.
  • a group having 1-3 articles refers to groups having 1, 2, or 3 articles.
  • a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

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Abstract

La présente divulgation concerne des polypeptides de type élastine (ELP) à phases séparables isothermiquement, les ELP comprenant : i) un premier domaine ELP ayant une première température de solution critique inférieure (LCST) ; et ii) un second domaine ELP ayant une seconde LCST qui est différente de la première LCST, les premier et second domaines ELP étant séparés par un site de clivage enzymatique. La présente divulgation concerne également des conjugués des ELP divulgués, des acides nucléiques codant pour les ELP, des cellules comprenant les acides nucléiques codant pour les ELP, des procédés d'utilisation des ELP, et des kits associés.
PCT/US2024/049779 2023-10-04 2024-10-03 Polypeptides de type élastine à phases séparables isothermiquement, compositions et leurs procédés d'utilisation Pending WO2025076213A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110039776A1 (en) * 2006-09-06 2011-02-17 Ashutosh Chilkoti Fusion peptide therapeutic compositions
US20230000952A1 (en) * 2008-06-27 2023-01-05 Duke University Therapeutic agents comprising elastin-like peptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110039776A1 (en) * 2006-09-06 2011-02-17 Ashutosh Chilkoti Fusion peptide therapeutic compositions
US20230000952A1 (en) * 2008-06-27 2023-01-05 Duke University Therapeutic agents comprising elastin-like peptides

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