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WO2024097639A1 - Anticorps se liant à hsa et protéines de liaison et leurs utilisations - Google Patents

Anticorps se liant à hsa et protéines de liaison et leurs utilisations Download PDF

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Publication number
WO2024097639A1
WO2024097639A1 PCT/US2023/078159 US2023078159W WO2024097639A1 WO 2024097639 A1 WO2024097639 A1 WO 2024097639A1 US 2023078159 W US2023078159 W US 2023078159W WO 2024097639 A1 WO2024097639 A1 WO 2024097639A1
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Prior art keywords
amino acid
acid sequence
seq
cdr
antibody
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English (en)
Inventor
Anuradha YAMMANURU
Zhen Zhang
Graham FARRINGTON
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ModernaTx Inc
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ModernaTx Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates generally to antibodies and binding fragments thereof, that specifically bind Human Serum Albumin (HSA), humanized versions thereof, and nucleic acids encoding the same.
  • the present disclosure further relates to methods of producing the disclosed antibodies, binding domains, proteins (e.g., purified anti-HSA binding proteins or chimeric molecules comprising such binding proteins), and nucleic acid molecules encoding such binding protein, as well as medical applications and treatments utilizing the disclosed antibodies, binding domains, proteins, and nucleic acid molecules.
  • HSA Human Serum Albumin
  • the present disclosure further relates to methods of producing the disclosed antibodies, binding domains, proteins (e.g., purified anti-HSA binding proteins or chimeric molecules comprising such binding proteins), and nucleic acid molecules encoding such binding protein, as well as medical applications and treatments utilizing the disclosed antibodies, binding domains, proteins, and nucleic acid molecules.
  • Monoclonal antibodies have been highly successful as stand-alone drugs in many therapeutic areas, however glycosylation, higher molecular weight, and their limited capacity to reach certain epitopes, has led efforts towards engineering Attorney Docket No.: 45817-0161WO1 smaller ( ⁇ 50kD) therapeutic molecules with multi-specific arms of antibody fragments. Due to their smaller size however, these molecules have a shorter systemic residence time due to their clearance through glomerular filtration, and other physiological factors, such as protease degradation. This results in a reduced therapeutic window, which requires repeated dosing to sustain the therapeutic efficiency of the drug molecule. The same short half-life applies to other smaller protein therapeutics.
  • half-life extension moiety as an arm in these molecules has been used to enable longer serum half-life.
  • the various methods of half-life extension include: increasing the dynamic radius of the molecules by fusion with polyethylene glycol; fusion to unstructured proteins like XTEN; or engineering molecules for efficient Neonatal Fc receptor (FcRn)-mediated recycling via fusion with Fc or albumin that enables recycling the therapeutic molecules back into the blood stream.
  • FcRn Neonatal Fc receptor
  • FcRn Neonatal Fc receptor
  • FcRn Neonatal Fc receptor
  • FcRn Neonatal Fc receptor
  • albumin that enables recycling the therapeutic molecules back into the blood stream.
  • HSA Human Serum Albumin
  • biologics and other therapeutics e.g., small molecules. This is based on the fact that HSA is abundant in blood serum and has a half-life of 19 days.
  • Binding of biologics to HSA protects them from endosomal degradation via FcRn-mediated recycling of HSA.
  • antibodies that bind to HSA have also been used for half-life extension.
  • Nanobodies, which are ⁇ 12-14 kD VHH fragments of heavy chain antibodies from Camelid have also been used to link a 22nM nanobody with anti- IL6R nanobody to enable a serum half-life of 6d in cynomolgus monkeys.
  • HSA-binding single domain antibodies that also bind to domain II of HSA (HSA DII) and are cross-reactive to cynomolgus serum albumin (CSA).
  • CSA cynomolgus serum albumin
  • present disclosure provides, among other things, antibodies, binding domains, and related proteins that bind Human Serum Albumin (HSA) and nucleic Attorney Docket No.: 45817-0161WO1 acids encoding the same.
  • Such antibodies, binding domains, and binding proteins are useful for improved drug delivery and for extending the half-life of biologics and other therapeutics.
  • the present disclosure provides a binding molecule comprising a single-domain antibody that binds both human serum albumin (HSA) and cynomolgus serum albumin (CSA).
  • HSA human serum albumin
  • CSA cynomolgus serum albumin
  • the single-domain antibody binds to human serum albumin domain 2 (HSA DII).
  • HSA DI or HSA DIII human serum albumin domain 1 or 3
  • the single-domain antibody can bind HSA at acidic pH.
  • the single-domain antibody has an affinity for HSA of less than 25 nM.
  • the single-domain antibody has an affinity for HSA of 0.01 nM to 25 nM.
  • the single-domain antibody comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% ,or at least 99% identical to any one of the sequences set forth in SEQ ID NOs.: 1-11, 13 or 17.
  • the single-domain antibody has an amino acid sequence that differs (i.e., amino acids are substituted) from any one of the sequences set forth in SEQ ID NOs.: 1-11, 13 or 17 at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 15, 16, 7, 18, 19, or 20 framework positions. In some cases, 1, 2, 3, 4, 5, 67, or 8 of framework positions 14, 83, 84, 108, 37, 44, 45, and 47 (numbering based on Kabat) are not substituted.
  • framework position (numbered according to Kabat) 37 is either a F, V, or I; position 44 is E or G; position 45 is R or L; and position 47 is G, W, Y, or F.
  • the single-domain antibody comprises the VHH-CDR1, VHH-CDR2, and VHH-CDR3 of any one of the sequences set forth in SEQ ID NOs.: 1-11, 13 or 17.
  • the VHH-CDR1, VHH-CDR2, and VHH-CDR3 are based on any one of the Kabat, Chothia, enhanced Chothia, Contact or IMGT CDR definitions.
  • the single-domain antibody comprises an amino acid sequence that is at least 85%, at least 86, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% ,or at least 99% identical to any one of the sequences set forth in SEQ ID NOs.: 2, 6, or 7.
  • the single-domain antibody has an amino acid sequence that differs from any one of the sequences set forth in SEQ ID NOs.: 2, 6, or 7 at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 15, 16, 7, 18, 19, or 20 framework positions.
  • framework position (numbered according to Kabat) 37 is either a F, V, or I; position 44 is E or G; position 45 is R or L; and position 47 is G, W, Y, or F.
  • the single-domain antibody comprises the VHH-CDR1, VHH-CDR2, and VHH-CDR3 of any one of the sequences set forth in SEQ ID NOs.: 2, 6, or 7.
  • the VHH-CDR1, VHH- CDR2, and VHH-CDR3 are based on any one of the CDR definitions known in the art, e.g., Kabat, Chothia, enhanced Chothia, Contact, or IMGT CDR definitions.
  • the single-domain antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs.: 88-117.
  • the single-domain antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs.: 118-147 or 168-173.
  • the single-domain antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs.: 148-167.
  • the binding molecule comprises a second single-domain antibody that binds a different antigen.
  • the different antigen is a tumor associated antigen.
  • the single-domain antibody and the second single-domain antibody are connected via a linker (e.g., a peptide or chemical linker).
  • the linker is a glycine serine linker.
  • the linker is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 5 to 10, 5 to 15, 5 to 20, 5 to 25, or 5 to 50 amino acids in length.
  • the single-domain antibody is at the N-terminus of the binding molecule. In some cases, the single-domain antibody is at the C-terminus of the binding molecule. In some Attorney Docket No.: 45817-0161WO1 instances, the single-domain antibody may have 1, 2, 3, 4, or 5 additional amino acids at the N- and/or C- terminus of the single-domain antibody. Such additional sequences can be added to reduce or prevent an immune response.
  • the present disclosure provides a single-domain antibody that specifically binds human serum albumin (HSA), human serum albumin domain 2 (HSA DII), or cynomolgus serum albumin (CSA) and comprises the following complementarity-determining regions (CDRs): (a) a CDR-1 comprising the amino acid sequence GRTYVPYT (SEQ ID NO: 26), a CDR-2 comprising the amino acid sequence TNSGGST (SEQ ID NO: 27), and a CDR-3 comprising the amino acid sequence AIAEGLGVYREEYLYDY (SEQ ID NO: 28); (b) a CDR-1 comprising the amino acid sequence GRTYVPYT (SEQ ID NO: 26), a CDR-2 comprising the amino acid sequence TNSGGST (SEQ ID NO: 27), and a CDR-3 comprising the amino acid sequence AVAEGLGVYREEYLYDY (SEQ ID NO: 29); (c) a CDR-1 comprising the amino acid sequence GRTFSRYA (SEQ ID NO:
  • the antibody specifically binds HSA. In some instances, the antibody specifically binds HSA DII. In some instances, the antibody specifically binds CSA. In some instances, the antibody specifically binds HSA, HSA DII, and CSA. Attorney Docket No.: 45817-0161WO1 In some instances, the antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 1-25 or 88-173. In some instances, the antibody comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 1-25 or 88-173.
  • the antibody comprises a heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 1-25 or 88-173. In some instances, the antibody binds HSA, HSA DII, or CSA with a K D of 20 nM or less.
  • the antibody binds HSA, HSA DII, or CSA with a KD of 10 nM or less, optionally wherein the antibody binds HSA, HSA DII, or CSA with a KD of 5 nM or less, optionally wherein the antibody binds HSA, HSA DII, or CSA with a K D of 1 nM or less, optionally wherein the antibody binds HSA, HSA DII, or CSA with a KD of 0.5 nM or less, optionally wherein the antibody binds HSA, HSA DII, or CSA with a K D of 0.1 nM or less, optionally wherein the antibody binds HSA, HSA DII, or CSA with a KD of 0.05 nM or less, optionally wherein the antibody binds HSA, HSA DII, or CSA with a KD of 0.01 nM or less.
  • the present disclosure provides a conjugate comprising a biologic and a single-domain antibody described herein.
  • the biologic and the single-domain antibody of the conjugate are covalently bound to one another as part of a single polypeptide chain.
  • the biologic and the single-domain antibody of the conjugate are connected via a chemical linker.
  • the biologic of the conjugate is selected from an antibody, a cytokine, a growth factor, an enzyme, a polypeptide, a protein, a carbohydrate, and a nucleic acid.
  • the conjugate when administered to a human subject, possesses a longer circulating half-life relative to the corresponding biologic that is not conjugated to the single-domain antibody.
  • the present disclosure provides an antibody or binding protein that specifically binds human serum albumin (HSA), human serum albumin domain 2 (HSA DII), or cynomolgus serum albumin (CSA) and comprises a heavy chain Attorney Docket No.: 45817-0161WO1 comprising: (a) a CDR-1 comprising the amino acid sequence GRTYVPYT (SEQ ID NO: 26), a CDR-2 comprising the amino acid sequence TNSGGST (SEQ ID NO: 27), and a CDR-3 comprising the amino acid sequence AIAEGLGVYREEYLYDY (SEQ ID NO: 28); (b) a CDR-1 comprising the amino acid sequence GRTYVPYT (SEQ ID NO: 26), a CDR-2 comprising the amino acid sequence TNSGGST (SEQ ID NO: 27), and a CDR-3 comprising the amino acid sequence AVAEGLGVYREEYLYDY (SEQ ID NO: 29); (c) a CDR-1 comprising the amino acid sequence GRTYVPY
  • the antibody or binding protein specifically binds HSA. In some instances, the antibody or binding protein specifically binds HSA DII. In some instances, the antibody or binding protein specifically binds CSA. In some instances, the antibody or binding protein specifically binds HSA, HSA DII, and CSA. In some instances, the antibody or binding protein comprises heavy chain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 1-25 or 88-173. In some instances, the antibody or binding protein comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 1-25 or 88-173.
  • the antibody or binding protein comprises heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 1-25 or 88-173. Attorney Docket No.: 45817-0161WO1 In some instances, the antibody or binding protein binds HSA, HSA DII, or CSA with a K D of 20 nM or less.
  • the antibody or binding protein binds HSA, HSA DII, or CSA with a KD of 10 nM or less, optionally the antibody or binding protein binds HSA, HSA DII, or CSA with a KD of 5 nM or less, optionally the antibody or binding protein binds HSA, HSA DII, or CSA with a KD of 1 nM or less, optionally the antibody or binding protein binds HSA, HSA DII, or CSA with a K D of 0.5 nM or less, optionally the antibody or binding protein binds HSA, HSA DII, or CSA with a KD of 0.1 nM or less, optionally the antibody or binding protein binds HSA, HSA DII, or CSA with a K D of 0.05 nM or less, optionally the antibody or binding protein binds HSA, HSA DII, or CSA with a KD of 0.01 nM or less.
  • the antibody or binding protein is a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multi- specific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab’)2 molecule, a tandem scFv (taFv), or a fusion protein.
  • scFv single-chain Fv molecule
  • the antibody or binding protein is a fusion protein comprising a biologic conjugated, either directly or indirectly, to the antibody, antigen-binding fragment, or binding protein.
  • the antibody or binding protein is a multi-specific antibody comprising at least one binding domain that specifically binds to an antigen other than HSA, HSA DII, or CSA.
  • Attorney Docket No.: 45817-0161WO1 the present disclosure provides a nucleic acid encoding a single-domain antibody, a conjugate, or an antibody or binding protein disclosed herein.
  • the nucleic acid is an mRNA.
  • the nucleic acid comprises, in the 5’-to-3’ direction: (a) a 5’ cap structure; (b) a 5’ untranslated region (UTR); (c) an open reading frame encoding the single-domain antibody, conjugate, antibody, antigen binging fragment, or binding protein, wherein the open reading frame consists of nucleosides is selected from the group consisting of (i) uridine or a modified uridine, (ii) cytidine or a modified cytidine, (iii) adenosine or a modified adenosine, and (iv) guanosine or a modified guanosine; (d) a 3’ UTR; and (e) a 3’ tailing sequence of linked nucleosides.
  • the nucleic acid comprises an open reading frame of nucleosides selected from the group consisting of (i) a modified uridine, (ii) cytidine, (iii) adenosine, and (iv) guanosine.
  • the nucleic acid comprises one or more modified uridines selected from 1-methylpseudouridine, pseudouridine, pyridin-4-one ribonucleoside, 5- aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl-uridine, 5-halo- uridine, 3-methyl-uridine, 5-methoxy-uridine, uridine 5-oxyacetic acid, uridine 5- oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thio- uridine, 5-aminomethyl-2-thio-uridine, 5-methyla
  • the nucleic acid comprises a modified uridine that is 1- methylpseudouridine.
  • the nucleic acid comprises one or more modified cytidines selected from 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl-cytidine, 5-halo- cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza- pseudoisocytidine
  • the nucleic acid comprises one or more modified adenosines selected from 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7- deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7- deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2- methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6- isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, 2-methylthio-N6-isopenten
  • the nucleic acid comprises one or more modified guanosines selected from inosine, 1-methyl-inosine, wyosine, methylwyosine, 4- demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl- queuosine, 7-cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6- thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7- methyl-inosine, 7
  • the nucleic acid comprises a 3’ tailing sequence of linked nucleosides, selected from a poly-adenylate (polyA) tail or a polyA-G quartet. In some instances, the 3’ tailing sequence of linked nucleosides is a polyA tail. In some instances, the nucleic acid comprises a 5’ cap structure selected from Cap0, Cap1, ARCA, inosine, 1-methyl-guanosine, 2′fluoroguanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, or 2- azidoguanosine.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a single domain antibody, a conjugate, an antibody, antigen- binding fragment, or binding protein, or a nucleic acid disclosed herein.
  • the pharmaceutical composition additionally comprises one or more pharmaceutically acceptable carriers, diluents, excipients, or any combination thereof.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles encapsulating the nucleic acid.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles having a mean particle size of from 80 nm to 160 nm.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles having a polydispersity index (PDI) of from 0.02 to 0.2 and/or a lipid:nucleic acid ratio of from 10 to 20.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles comprising a neutral lipid, a cationic lipid, a polyethyleneglycol (PEG) lipid, and/or a sterol.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles comprising 1,2-distearoyl-sn-glycero-3-phosphocholine.
  • the pharmaceutical composition comprises a plurality of lipid nanoparticles comprising a compound of Formula (I).
  • the pharmaceutical Attorney Docket No.: 45817-0161WO1 composition comprises a plurality of lipid nanoparticles comprising PEG 2000 dimyristoyl glycerol.
  • the pharmaceutical composition comprises a sterol selected from cholesterol, adosterol, agosterol A, atheronals, avenasterol, azacosterol, blazein, cerevisterol, colestolone, cycloartenol, daucosterol, 7-dehydrocholesterol, 5- dehydroepisterol, 7-dehydrositosterol, 20 ⁇ ,22R-dihydroxycholesterol, dinosterol, epibrassicasterol, episterol, ergosterol, ergosterol, fecosterol, fucosterol, fungisterol, ganoderenic acid, ganoderic acid, ganoderiol, ganodermadiol, 7 ⁇ -hydroxycholesterol, 22R-hydroxycholesterol, 27
  • the pharmaceutical composition comprises cholesterol.
  • the present disclosure provides a host cell comprising a single domain antibody, a conjugate, an antibody, antigen-binding fragment, or binding protein, or a nucleic acid disclosed herein.
  • the host cell is a eukaryotic cell.
  • the host cell is a mammalian cell.
  • the host cell is a CHO cell or HEK cell.
  • the present disclosure provides a method of extending half- life of a therapeutic molecule, comprising conjugating the therapeutic molecule to a single domain antibody or an antibody or binding protein disclosed herein.
  • the therapeutic molecule is a biologic.
  • the biologic is selected from an antibody, a cytokine, a growth factor, an enzyme, a polypeptide, a protein, a carbohydrate, and a nucleic acid.
  • the therapeutic molecule is conjugated via a peptide bond.
  • the therapeutic molecule is conjugated via a chemical linker.
  • a therapeutic molecule and a single-domain antibody, antibody or binding protein described herein are encoded by a single RNA, such that Attorney Docket No.: 45817-0161WO1 the therapeutic molecule and the single-domain antibody, antibody or binding protein expressed as a fusion protein when the RNA is translated.
  • the present disclosure provides a method of treating a disease or condition, comprising administering to a subject with a disease or condition a conjugate disclosed herein.
  • the subject is a human.
  • the disease or condition is selected from a cancer, an autoimmune condition, an inflammatory condition, or an infection.
  • the present disclosure provides a kit comprising (i) a single domain antibody, a conjugate, an antibody, antigen-binding fragment, binding protein, a nucleic acid, or a pharmaceutical composition disclosed herein, and (ii) a package insert instructing a user of the kit to administer the single-domain antibody, conjugate, antibody, antigen-binding fragment, binding protein, nucleic acid, or pharmaceutical composition to a subject in need thereof.
  • the disclosure features a polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds both human serum albumin (HSA) and cynomolgus monkey serum albumin (CSA).
  • the VHH binds domain II of HSA and the VHH comprises an amino acid sequence (i) that is at least 85%, identical to any one of the sequences set forth in SEQ ID NOs.: 88 to 173; and/or (ii) with a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of any one of the sequences set forth in SEQ ID NOs: 2, 6, or 7.
  • the VHH also has one or both of the following properties: (i) affinity of 0.01 nM to 25 nM for HSA; (ii) binds HSA at acidic pH.
  • the disclosure relates to a polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds both human serum albumin (HSA) and cynomolgus monkey serum albumin (CSA).
  • VHH comprises an amino acid sequence that is at least 85%, identical, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence Attorney Docket No.: 45817-0161WO1 of any one of SEQ ID NOs.: 88 to 173.
  • the VHH binds domain II of HSA.
  • the disclosure provides a polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds both human serum albumin (HSA) and cynomolgus monkey serum albumin (CSA).
  • VHH comprises an amino acid sequence with a VHH-CDR1, a VHH-CDR2, and a VHH-CDR3 of any one of the sequences set forth in SEQ ID NOs: 2, 6, or 7.
  • the VHH binds domain II of HSA.
  • the VHH-CDR1, VHH-CDR2, and VHH-CDR3 are based on any one of the Kabat, Chothia, enhanced Chothia, contact, IMGT or Aho definitions.
  • the VHH CDR1, VHH CDR2, and VHH CDR3 comprise the corresponding amino acid sequences of the VHH CDRs of any one of VHH2, VHH6, or VHH7 set forth in Table I, II, III, IV, or V in the Detailed Description.
  • the VHH is humanized.
  • one, two, three, or four of positions 37, 44, 45, and 47 are humanized.
  • one, two, three, or four of positions 37, 44, 45, and 47 are not humanized.
  • positions 37 and/or 47 are not humanized.
  • positions 44 and/or 45 are humanized.
  • position 37 of the VHH is F, V, or I; position 44 of the VHH is E or G; position 45 of the VHH is R or L; or position 47 of the VHH is G, W, Y, or F, wherein the positions are based on Kabat numbering.
  • one, two, three, four, five, six, seven, or eight of positions 14, 37, 44, 45, 47, 83, 84, and 108 are not humanized.
  • the polypeptide comprises an amino acid sequence that is identical to any one of SEQ ID NOs.: 88 to 173.
  • the polypeptide comprises a second VHH that binds to a different antigen.
  • the different antigen is a tumor associated antigen.
  • the different antigen is an antigen on a T cell. In other cases, the different antigen is an antigen on a NK cell.
  • the second VHH is linked to the C-terminus of the VHH via a peptide linker. In certain cases, the second VHH is linked to the N-terminus of the VHH via a peptide linker.
  • the linker is G4S (SEQ ID NO:176) or (G4S)3 (SEQ ID NO:178) .
  • the polypeptide further comprises comprising a human Ig Fc domain and optionally a human Ig hinge domain.
  • the human Ig hinge if present is at the N terminus of the human Ig Fc domain.
  • the human Ig is a human IgG1, human IgG2, human IgG3, or human IgG4.
  • the human Ig domain comprises the human IgG4PAA hinge and Fc domain.
  • the disclosure features a polypeptide that binds two, or at least two, different epitopes or antigens.
  • the polypeptide comprises a means for binding HSA (e.g. HSA DII) and CSA.
  • the means for binding HSA and CSA comprises the three VHH CDRs of a VHH set out in any one of Tables I-IV or Table 8. In some cases, the means for binding HSA and CSA comprises a VHH set out in any one of Tables 20 to 22.
  • the polypeptide also comprises a binding polypeptide that binds a different antigen (different from HSA and CSA).
  • the different antigen can be an antigen on a T cell or a N K cell.
  • the binding polypeptide that binds a different antigen is a VHH.
  • the binding polypeptide that binds a different antigen is a Fab. In some instances, the binding polypeptide that binds a different antigen is a scFv.
  • the means for binding HAS and CSA is linked to the binding polypeptide that binds a different Attorney Docket No.: 45817-0161WO1 antigen via a peptide linker (e.g., a glycine linker, a serine linker, a glycine-serine linker).
  • the linker comprises a sequence set forth in SEQ ID NO:177. In other cases, the linker comprises a sequence set forth in SEQ ID NO: 176 or 178.
  • the disclosure also features a pharmaceutical composition comprising a polypeptide described above and a pharmaceutically acceptable carrier.
  • the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a means for binding HSA (e.g. HSA DII) and CSA.
  • the means for binding HSA and CSA comprises a VHH comprising the three VHH CDRs of a VHH set out in any one of Tables I-IV or Table 8.
  • the means for binding HSA and CSA comprises a VHH set out in any one of Tables 20 to 22.
  • nucleic acid or nucleic acids encoding a polypeptide described herein, a vector or vectors comprising such nucleic acids, and a host cell comprising the nucleic acid(s) or vector(s).
  • the host cell is a mammalian cell.
  • the host cell is a CHO cell, a 293 cell, a 3T3 cell, or a COS cell.
  • the disclosure provides methods of making a polypeptide described above by culturing the host cell under conditions that facilitate expression of the polypeptide, and isolating the polypeptide.
  • the isolated polypeptide is formulated as a sterile pharmaceutical composition.
  • the disclosure features a polynucleotide comprising an mRNA comprising: (i) a 5' UTR; (ii) an open reading frame (ORF) encoding a polypeptide described herein; (iii) a stop codon; and (iv) a 3' UTR.
  • the mRNA comprises a microRNA (miR) binding site.
  • the microRNA is expressed in an immune cell of hematopoietic lineage or a cell that expresses TLR7 and/or TLR8 and secretes pro-inflammatory cytokines and/or chemokines.
  • the microRNA binding site is for a microRNA selected from miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27, miR-26a, or any combination thereof.
  • the microRNA binding site is for a microRNA selected from miR126-3p, miR- 142-3p, miR-142-5p, miR-155, or any combination thereof.
  • the microRNA binding site is located in the 3' UTR of the mRNA.
  • the mRNA comprises a 5' terminal cap.
  • the 5' terminal cap comprises a Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof
  • the mRNA comprises a poly-A region.
  • the poly-A region is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 nucleotides in length, or at least about 100 nucleotides in length. In other cases, the poly-A region is about 10 to about 200, about 20 to about 180, about 50 to about 160, about 70 to about 140, or about 80 to about 120 nucleotides in length. In some instances, the mRNA comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof.
  • the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil ( ⁇ ), N1-methylpseudouracil (m1 ⁇ ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4’-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof.
  • at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are N1-methylpseudouracils.
  • the open reading frame consists of nucleosides selected from the group consisting of (i) uridine or a modified uridine, (ii) cytidine or a modified cytidine, (iii) adenosine or a modified adenosine, and (iv) guanosine or a modified guanosine.
  • the modified uridine is 1-methylpseudouridine.
  • the mRNA comprises a 5’terminal cap comprising Cap1 and a poly-A region 100 nucleotides in length. In some cases, all uracils of the polynucleotide are N1-methylpseudouracils.
  • the disclosure features a pharmaceutical composition comprising a polynucleotide described above, and a delivery agent.
  • the delivery agent comprises a lipid nanoparticle.
  • the lipid nanoparticle has a mean particle size of from 80 nm to 160 nm.
  • the lipid nanoparticle has a polydispersity index (PDI) of from 0.02 to 0.2 and/or a lipid:nucleic acid ratio of from 10 to 20.
  • the lipid nanoparticle comprises a neutral lipid, an ionizable amino lipid, a polyethyleneglycol (PEG) lipid, and/or a sterol.
  • the lipid nanoparticle comprises a neutral lipid that is 1,2-distearoyl-sn-glycero- 3-phosphocholine. In some cases, the lipid nanoparticle comprises an ionizable amino lipid. In certain cases, the lipid nanoparticle comprises a PEG lipid that is PEG 2000 dimyristoyl glycerol or OL56.
  • the lipid nanoparticle comprises a sterol that is cholesterol, adosterol, agosterol A, atheronals, avenasterol, azacosterol, blazein, cerevisterol, colestolone, cycloartenol, daucosterol, 7-dehydrocholesterol, 5- dehydroepisterol, 7-dehydrositosterol, 20 ⁇ ,22R-dihydroxycholesterol, dinosterol, epibrassicasterol, episterol, ergosterol, ergosterol, fecosterol, fucosterol, fungisterol, ganoderenic acid, ganoderic acid, ganoderiol, ganodermadiol, 7 ⁇ -hydroxycholesterol, 22R-hydroxycholesterol, 27-hydroxycholesterol, inotodiol, lanosterol, lathosterol, lichesterol, lucidadiol, lumisterol, oxycholesterol,
  • the disclosure features a kit comprising (i) a polypeptide, a pharmaceutical composition, or a polynucleotide described above, and (ii) a package insert instructing a user of the kit to administer the polypeptide, pharmaceutical composition, or polynucleotide to a human subject in need thereof.
  • a kit comprising (i) a polypeptide, a pharmaceutical composition, or a polynucleotide described above, and (ii) a package insert instructing a user of the kit to administer the polypeptide, pharmaceutical composition, or polynucleotide to a human subject in need thereof.
  • FIG.2 shows gating scheme for Llama B cell sorting by FACS. Llama Lymphocytes were first gated, followed by singlet and live cell gating. CSA+ sdAb+ B cells which formed ⁇ 0.05% of the total live B cells were sorted into 96 well plates at 2-3 cells/well.
  • FIG.3 shows Venn diagram to summarize results of binding assay by BLI, using sdAb-his plasmid-expressed expi293 culture supernatants.12 clones were positive for all three antigens.
  • FIG.5 shows SEC-MAL analysis of purified his-tagged single domain antibodies (sdAbs). The SEC profile of the majority of the clones that showed binding to the serum albumins showed single homogenous peaks with >95% purity.
  • FIG.6 shows V5- and FLAG-tagged sdAb design of the constructs prepared for in vivo PK studies. The figure discloses SEQ ID NOS 208, 212-213, 208, 212 and 214, respectively, in order of appearance.
  • FIG.7 shows an SDS-PAGE of non-reduced samples of V5 or FLAG-tagged sdAb proteins selected and purified for in vivo PK studies.
  • FIG.8 shows SEC-MAL analysis of purified V5 or FLAG-tagged sdAbs.
  • FIG.9 shows the study design for evaluation of sdAb clones in vivo. The dosing and bleeding regime for each group of mice described in Table 9 are shown.
  • FIGs.10A-B shows in vivo PK profiles, determined by ELISA, of: A) V5- tagged clones VHH3, VHH1, VHH5, VHH7, VHH9, and VHH11 at 750X dilution; and B) FLAG-tagged clones VHH2, VHH4, VHH6, VHH13, VHH17, and VHH10 at 800X dilution.
  • FIG.11 shows SPR interaction profiles of sdAbs with HSA, CSA, and HSA DII.
  • FIG.12 shows a dendrogram to demonstrate sequence similarity of the 5 shortlisted clones.
  • FIG.13 shows in vivo PK data from humanized mice (HSA+ huFcRn+ of the 5 selected clones.
  • FIG.14 shows in vitro binding affinity measurements measured using Biacore at pH 7.4.
  • FIG.15 shows SDS-PAGE of protein expression of the 80 humanized variants generated of the 3 short-listed clones.
  • FIG.16 shows SEC-MAL analysis of selected purified VHH2 IGHV311*05 and VHH2 GHV323*02 humanized sdAbs and parental clone (VHH2 WT).
  • FIG.17 shows SEC-MAL analysis of selected purified VHH7 IGHV311*05 humanized sdAb and parental clone (VHH7).
  • FIG.18 shows SEC-MAL analysis of selected purified VHH6 IGHV311*05 and VH6 GHV323*02 humanized sdAbs and parental clone (VHH6 WT).
  • HSA Human Serum Albumin
  • VHH heavy chain variable domains
  • CDRs complementarity determining regions
  • the disclosure also provides nucleic acids encoding the disclosed proteins, and methods of using such antibodies, antigen-binding fragments, binding proteins, and nucleic acids.
  • the antibodies are single domain antibodies (e.g., a VHH).
  • the antibodies are full-length antibodies that include a pair of heavy chains and a pair of light chains, each containing a variable domain and a constant region.
  • the antibodies are single-domain antibodies (sdAbs) or single chain Fv (scFv) molecules, among other antigen-binding fragments described herein.
  • the antibodies are bispecific antibodies (i.e., engagers) that bind to HSA and another antigenic target.
  • compositions and methods of the disclosure exhibit a series of beneficial biochemical properties.
  • VHH domains described herein and antibodies and antigen-binding fragments are capable of binding HSA, HSA DII, and CSA with high affinity.
  • the antibodies and antigen-binding fragments may extend the half-life of biologics and other therapeutic compounds.
  • the term “about” refers to a stated numerical term and a value that is no more than 10% above or below the value being described.
  • the term “about 5 nM” indicates disclosure of both the stated value of 5 nM and a range of from 4.5 nM to 5.5 nM.
  • HSA antibody refers to an antibody or fragment thereof that specifically binds to, or is immunologically reactive with, human serum albumin (HSA), human serum albumin domain 2 (HSA DII), or Attorney Docket No.: 45817-0161WO1 cynomolgus serum albumin (CSA).
  • HSA binding protein or “anti-HSA binding protein” refers to any protein comprising at least one domain (such as a VHH domain disclosed herein) that specifically binds to or is immunologically reactive with HSA.
  • a “HSA binding protein” or “anti-HSA binding protein” includes, for example, anti-HSA antibodies (both monospecific and bispecific), and other constructs that bind to HSA.
  • antibody refers to an immunoglobulin molecule, or a molecule having an immunoglobulin-like scaffold, that specifically binds to, or is immunologically reactive with, a particular antigen.
  • antibody includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including, but not limited to, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri-, quad-, and multispecific antibodies, diabodies, triabodies, and tetrabodies), and single-domain antibodies (sdAb), including the VHH sdAbs disclosed herein.
  • the VHH sdAbs disclosed herein may be incorporated into antibody constructs, which may optionally comprise additional antibody binding specificities.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full- length antibody.
  • the antibody fragments can be, e.g., a single-domain antibody (sdAb) also known as a nanobody or VHH, Fab, F(ab’)2, Fab Fv, scFv, SMIP, diabody, a triabody, an affibody, an aptamer, or recombinant fragments thereof.
  • sdAb single-domain antibody
  • sdAb also known as a nanobody or VHH, Fab, F(ab’)2, Fab Fv, scFv, SMIP, diabody, a triabody, an affibody, an aptamer, or recombinant fragments thereof.
  • binding fragments encompassed by the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including V H and V L domains; (vi) a dAb fragment (Ward et al., Nature 341:544- 546, 1989), which consists of a VH domain; (vii) a dAb which consists of a VH or a VL Attorney Docket No.: 45817-0161WO1 domain; (viii) an isolated complement
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426 (1988), and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)).
  • scFv single chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art.
  • biological refers to a medicinal preparation that is created by biological processes rather than chemical synthesis.
  • Exemplary biologics include certain vaccines, antibodies, cell preparations, tissue preparations, recombinant proteins, nucleic acids, cytokines, growth factors, enzymes, peptides, proteins, carbohydrates, or combinations thereof.
  • Biologics also include biosimilar molecules (or biosimilars), which are molecular entities that are structurally similar to and have no clinically meaningful differences in terms of safety, purity, and potency from known biologics.
  • bispecific antibodies refers to monoclonal, often human or humanized antibodies that have binding specificities for at least two different antigens.
  • Bispecific HSA antibodies of the invention may have binding specificities that are directed towards HSA and any other antigen, e.g., for a cell- surface protein, receptor, receptor subunit, or tissue-specific antigen.
  • a bispecific antibody may also be an antibody or antigen-binding fragment thereof that includes two separate antigen-binding domains (e.g., two scFvs joined by a linker). The scFvs may bind the same antigen or different antigens.
  • the term “engager” may be used interchangeably with “bispecific antibody.”
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • multispecific antibodies refers to monoclonal, often human or humanized antibodies that exhibit affinity for more than one target antigen, for example, bispecific antibodies.
  • Multispecific HSA antibodies of the invention may have binding specificities that are directed towards HSA and any other antigen(s), e.g., for a cell-surface protein, receptor, receptor subunit, or tissue-specific antigen.
  • a multispecific antibody may also be an antibody or antigen-binding fragment thereof that includes multiple separate antigen-binding domains (e.g., two scFvs joined by a linker). The scFvs may bind the same antigen or different antigens.
  • Multispecific antibodies can have structures similar to full immunoglobulin molecules and include Fc regions, for example IgG Fc regions.
  • Such structures can include, but not limited to, IgG-Fv, IgG-(scFv) 2 , DVD-Ig, (scFv) 2 -(scFv) 2 -Fc and (scFv) 2 -Fc-(scFv) 2 .
  • the scFv can be attached to either the N-terminal or the C- terminal end of either the heavy chain or the light chain.
  • antibody fragments can be components of multi-specific molecules without Fc regions, based on fragments of IgG or DVD or scFv.
  • Exemplary multi-specific molecules that lack Fc regions and into which antibodies or antibody fragments can be incorporated include scFv dimers (diabodies), trimers (triabodies) and tetramers (tetrabodies), Fab dimers (conjugates by adhesive polypeptide or protein domains) and Fab trimers (chemically conjugated), are described by Hudson and Souriau, 2003, Nature Medicine 9:129-134; Attorney Docket No.: 45817-0161WO1 incorporated herein by reference.
  • chimeric antibody refers to an antibody having portions of its sequence derived from at least two different sources, such as variable domain sequences (e.g., CDR sequences) derived from an immunoglobulin of one source organism, such as rat or mouse, and constant regions derived from an immunoglobulin of a different organism (e.g., a human, another primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, member of the bovidae family (such as cattle, bison, buffalo, elk, and yaks, among others), cow, sheep, horse, bison, llama, camel, or shark among others).
  • variable domain sequences e.g., CDR sequences
  • constant regions derived from an immunoglobulin of a different organism e.g., a human, another primate, pig, goat, rabbit, hamster, cat, dog, guinea pig, member of the bovidae family (such as cattle, bison, buffalo, e
  • CDR complementarity determining region
  • variable domains can be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions.
  • the invention includes antibodies comprising modifications in these hybrid hypervariable positions.
  • the variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a ⁇ -sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see, Kabat et al., Sequences of Proteins of Attorney Docket No.: 45817-0161WO1 Immunological Interest (National Institute of Health, Bethesda, Md. (1987); incorporated herein by reference). As used herein, numbering of immunoglobulin amino acid residues is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.
  • the terms “conservative mutation,” “conservative substitution,” “conservative amino acid substitution,” and the like refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and/or steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in Table 1 below.
  • conjugate refers to a compound formed by the chemical bonding of a reactive functional group of one molecule with an appropriately reactive functional group of another molecule. Conjugates may additionally be produced, e.g., as two polypeptide domains covalently bound to one another as part of a single polypeptide chain that is synthesized by the translation of a single RNA transcript encoding both polypeptides in frame with one another.
  • the term “construct” refers to a fusion protein containing a first polypeptide domain bound to a second polypeptide domain.
  • the polypeptide domains may each independently be anti-HSA single chain polypeptides, for instance, as described herein.
  • the first polypeptide Attorney Docket No.: 45817-0161WO1 domain may be covalently bound to the second polypeptide domain, for instance, by way of a linker, such as a peptide linker or a disulfide bridge, among others.
  • exemplary linkers that may be used to join the polypeptide domains of a HSA construct include, without limitation, those that are described in Leriche et al., Bioorg. Med.
  • the term “derivatized antibodies” refers to antibodies that are modified by a chemical reaction so as to cleave residues or add chemical moieties not native to an isolated antibody. Derivatized antibodies can be obtained by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by addition of known chemical protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein.
  • any of a variety of chemical modifications can be carried out by known techniques, including, without limitation, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. using established procedures. Additionally, the derivative can contain one or more non-natural amino acids, e.g., using amber suppression technology (see, e.g., US Patent No.6,964,859; incorporated herein by reference).
  • diabodies refers to bivalent antibodies comprising two polypeptide chains, in which each polypeptide chain includes VH and VL domains joined by a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of VH and VL domains on the same peptide chain. This configuration forces each domain to pair with a complementary domain on another polypeptide chain so as to form a homodimeric structure.
  • triabodies refers to trivalent antibodies comprising three peptide chains, each of which contains one VH domain and one VL domain joined by a linker that is exceedingly short (e.g., a linker composed of 1-2 amino acids) to permit intramolecular association of VH and VL domains within the same peptide chain.
  • linker that is exceedingly short (e.g., a linker composed of 1-2 amino acids) to permit intramolecular association of VH and VL domains within the same peptide chain.
  • peptides configured in this way typically trimerize so as to position the V H and V L domains of neighboring peptide chains Attorney Docket No.: 45817-0161WO1 spatially proximal to one another to permit proper folding (see, Holliger et al., Proc. Natl. Acad. Sci.
  • endogenous describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).
  • a particular organism e.g., a human
  • a particular location within an organism e.g., an organ, a tissue, or a cell, such as a human cell.
  • exogenous describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted there from.
  • framework region or “FW region” includes amino acid residues that are adjacent to the CDRs.
  • FW region residues may be present in, for example, human antibodies, rodent-derived antibodies (e.g., murine antibodies), humanized antibodies, primatized antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), single-chain antibody fragments (e.g., scFv fragments), antibody domains, and bispecific antibodies, among others.
  • fusion protein refers to a protein that is joined via a covalent bond to another molecule.
  • a fusion protein can be chemically synthesized by, e.g., an amide-bond forming reaction between the N-terminus of one protein to the C-terminus of another protein.
  • a fusion protein containing one protein covalently bound to another protein can be expressed recombinantly in a cell (e.g., a eukaryotic cell or prokaryotic cell) by expression of a polynucleotide encoding the fusion protein, for example, from a vector or the genome of the cell or as a nucleic acid molecule administered in a delivery vehicle.
  • a fusion protein may contain one protein that is covalently bound to a linker, which in turn is covalently bound to another molecule.
  • linkers that can be used for the formation of a fusion protein include peptide-containing linkers, such as those that contain naturally Attorney Docket No.: 45817-0161WO1 occurring or non-naturally occurring amino acids.
  • Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582 (2012)).
  • heterospecific antibodies refers to monoclonal (e.g., human or humanized) antibodies that have binding specificities for at least two different antigens.
  • monoclonal antibodies e.g., human or humanized antibodies that have binding specificities for at least two different antigens.
  • the recombinant production of heterospecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537 (1983)). Similar procedures are disclosed, e.g., in WO 93/08829, U.S. Pat.
  • Heterospecific antibodies can include Fc mutations that enforce correct chain association in multi-specific antibodies, as described by Klein et al., mAbs 4(6):653- 663 (2012); incorporated herein by reference.
  • the term “human antibody” refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, C L , C H domains (e.g., CH1, CH2, CH3), hinge, (VL, VH)) is substantially non-immunogenic in humans, with only minor sequence changes or variations.
  • a human antibody can be produced in a human cell (e.g., by recombinant expression), or by a non-human animal or a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes.
  • a human antibody when a human antibody is a single-chain antibody, it can include a linker peptide that is not Attorney Docket No.: 45817-0161WO1 found in native human antibodies.
  • an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. See, U.S. Patent Nos.4,444,887 and 4,716,111; and PCT publications WO 1998/46645; WO 1998/50433; WO 1998/24893; WO 1998/16654; WO 1996/34096; WO 1996/33735; and WO 1991/10741; incorporated herein by reference. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • humanized antibodies refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, or immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2, VHH or other target-binding subdomains of antibodies), which contain minimal sequences derived from non-human immunoglobulin.
  • a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin. All or substantially all of the FRs may also be those of a human immunoglobulin sequence.
  • the humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.
  • Fc immunoglobulin constant region
  • Methods of antibody humanization are known in the art. See, e.g., Reichmann et al., Nature 332:323-7 (1988); U.S. Patent Nos: 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 to Queen et al; EP239400; PCT publication WO 91/09967; U.S. Patent No.5,225,539; EP592106; and EP519596; the disclosure of each of which is incorporated herein by reference.
  • lipid nanoparticle refers to a transfer vehicle including one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG- modified lipids).
  • lipids e.g., cationic lipids, non-cationic lipids, and PEG- modified lipids.
  • Exemplary lipid nanoparticles are formulated to deliver one or more mRNA to one or more target cells.
  • suitable lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides).
  • Lipid nanoparticles may contain a cationic lipid, or a lipid species with a net positive charge at a selected pH (e.g., physiological pH), to encapsulate and/or enhance the delivery of mRNA into the target cells.
  • a selected pH e.g., physiological pH
  • the terms “messenger RNA” or “mRNA” refer to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo.
  • the basic components of an mRNA molecule include a coding region, a 5’UTR, a 3’UTR, a 5’ cap, and a poly-A tail.
  • modified messenger RNA or “modified mRNA” refer to mRNA polynucleotides that include naturally occurring and/or non-naturally occurring modifications, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage, or to the phosphodiester backbone).
  • Non-natural modified nucleotides may be introduced during synthesis of post-synthesis of the polynucleotides to achieve desired functions or properties.
  • the modifications may be present on an internucleoside linkage, purine or pyrimidine base, or sugar.
  • nucleic acid includes any compound containing a continuous segment of nucleosides joined by way of one or more internucleoside linkages (e.g., polymers of nucleosides linked by way of phosphodiester bonds).
  • nucleic acids include ribonucleic acids (RNA), deoxyribonucleic acids (DNA), threose nucleic acids (TNA), glycol nucleic acids (GNA), peptide nucleic Attorney Docket No.: 45817-0161WO1 acids (PNA), locked nucleic acids (LNA), or hybrids thereof.
  • Nucleic acids also include RNAi inducers, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNAs, tRNAs, RNAs that induce triple spiral formation, aptamers, vectors, and the like.
  • the nucleic acid is one or more modified messenger RNAs (modified mRNAs).
  • modified mRNAs modified messenger RNAs
  • percent (%) sequence identity As used herein, the terms “percent (%) sequence identity,” “percent (%) identity,” and the like, with respect to a reference polynucleotide or polypeptide sequence, is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST.
  • the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, is calculated as: 100 multiplied by (the fraction X/Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B.
  • nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B
  • percent sequence identity of A to B will not equal the percent sequence identity of B to A.
  • primary antibody refers to an antibody comprising framework regions from primate-derived antibodies and other regions, such as CDRs and/or constant regions, from antibodies of a non-primate source. Methods for producing primatized antibodies are known in the art. See, e.g., U.S. Patent Nos.5,658,570; 5,681,722; and 5,693,780; incorporated herein by reference.
  • a primatized antibody or antigen-binding fragment thereof described herein can be produced by inserting the CDRs of a non-primate antibody or antigen- binding fragment thereof into an antibody or antigen-binding fragment thereof that contains one or more framework regions of a primate.
  • the term “operatively linked” in the context of a polynucleotide fragment is intended to mean that the two polynucleotide fragments are joined such that the amino acid sequences encoded by the two polynucleotide fragments remain in-frame.
  • the term “pharmacokinetic profile” refers to the absorption, distribution, metabolism, and clearance of a therapeutic agent (e.g., a polypeptide, such as an anti-HSA antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct of the disclosure) over time following administration of the drug to a patient.
  • a therapeutic agent e.g., a polypeptide, such as an anti-HSA antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct of the disclosure
  • regulatory sequence includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation, e.g., of antibody chain genes.
  • scFv refers to a single-chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain.
  • ScFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (VL) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) and the variable region of an antibody heavy chain (V H ) (e.g., CDR- Attorney Docket No.: 45817-0161WO1 H1, CDR-H2, and/or CDR-H3) separated by a linker.
  • VL antibody light chain
  • V H variable region of an antibody heavy chain
  • the linker that joins the VL and V H regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids.
  • linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (e.g., linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment (e.g., hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (e.g., a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites).
  • linkers containing D-amino acids e.g., hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues
  • hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating
  • VL and VH domains of a scFv molecule can be derived from one or more antibody molecules.
  • variable regions of the scFv molecules described herein can be modified such that they vary in amino acid sequence from the antibody molecule from which they were derived.
  • nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and/or framework residues).
  • mutations are made to CDR amino acid residues to optimize antigen binding using art recognized techniques. ScFv fragments are described, for example, in WO 2011/084714; incorporated herein by reference.
  • single-domain antibody As used herein, the terms “single-domain antibody,” “sdAb,” “nanobody,” and “VHH antibody” are used interchangeably to refer to a single-chain antibody fragment that contains only a single heavy-chain variable domain. Unlike a traditional, full-length antibody, which includes heavy chains and light chains, each containing a corresponding variable domain (i.e., a heavy chain variable domain, VH, and a light chain variable domain, V L ) having three CDRs, a single-domain antibody Attorney Docket No.: 45817-0161WO1 only includes one heavy-chain variable domain having a total of three CDRs (referred to herein as VHH CDR-H1, VHH CDR-H2, and VHH CDR-H3).
  • the phrase “specifically binds” refers to a binding reaction which is determinative of the presence of an antigen in a heterogeneous population of proteins and other biological molecules that is recognized, e.g., by an antibody or antigen-binding fragment thereof, with particularity.
  • An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a K D of less than 100 nM.
  • an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen via the antigen binding domain with a K D of up to 100 nM (e.g., between 1 pM and 100 nM).
  • An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a K D of greater than 100 nM (e.g., greater than 500 nm, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM) for that particular antigen or epitope thereof.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate.
  • the terms “subject” and “patient” refer to an organism that receives treatment (e.g., by administration of an HSA polypeptide, such as an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct described herein) for a particular disease or condition.
  • an HSA polypeptide such as an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct described herein
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovidae family (such as cattle, bison, buffalo, and yaks, among others), sheep, and horses, among others.
  • a patient that may be treated using the compositions and methods described herein may have an established disease, in which case the patient has been diagnosed as having the disease and has Attorney Docket No.: 45817-0161WO1 shown symptoms of the disease for a prolonged period of time (e.g., over the course of days, weeks, months, or years).
  • a patient may be symptomatic for a particular disease, but has yet to be diagnosed with the disease by a physician.
  • compositions and methods described herein include those that have been diagnosed as having a disease or disorder, and may or may not be showing symptoms of the disease as of yet.
  • transfection refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran transfection and the like.
  • treat or “treatment” refer to therapeutic treatment, in which the object is to inhibit or slow down (lessen) an undesired physiological change or disorder.
  • Beneficial or desired clinical results of treatment include, without limitation, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Those in need of treatment include those already having the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be inhibited.
  • serum albumin refers to a protein found in blood.
  • HSA is important in regulation of plasma colloid osmotic pressure and acts as a carrier protein for a wide range of endogenous molecules including hormones, fatty acids, and metabolites, as well as exogenous molecules such as drugs and biologics.
  • HSA is composed of three homologous domains, numbered I, II, and III. Each domain is grouped into subdomains A and B that possess common structural motifs. The two principal regions responsible for ligand-binding to HSA are known as Sudlow's Site I and II, located in subdomain IIA and IIIA, respectively.
  • variable region CDR includes amino acids in a CDR or complementarity determining region as identified using sequence or structure-based methods.
  • CDR or complementarity determining region refers to the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem.252:6609-6616 (1977) and Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91 -3242 (1991); by Chothia et al., J. Mol.
  • the term “CDR” is a CDR as defined by Kabat based on sequence comparisons.
  • the term “vector” includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, an RNA vector, virus or other suitable replicon (e.g., viral vector).
  • a variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994/11026; incorporated herein by reference. Expression vectors described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell.
  • vectors that can be used for the expression of antibodies include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
  • Other useful vectors for expression of antibodies, antibody fragments, and/or binding proteins contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5’ and 3’ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • IRS internal ribosomal entry site
  • the expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector.
  • a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.
  • VH refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab.
  • References to “VL” refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.
  • Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity.
  • Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain of a native antibody has at the amino terminus a variable domain (V H ) followed by a number of constant domains. Each light chain of a native antibody has a variable domain at the amino terminus (V L ) and a constant domain at the carboxy terminus.
  • alkyl As used herein, the term “alkyl,” “alkyl group,” or “alkylene” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted.
  • the notation “C1-14 alkyl” means an optionally substituted Attorney Docket No.: 45817-0161WO1 linear or branched, saturated hydrocarbon including 1-14 carbon atoms. Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups.
  • alkenyl means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted.
  • C 2-14 alkenyl means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond.
  • An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds.
  • C18 alkenyl may include one or more double bonds.
  • a C 18 alkenyl group including two double bonds may be a linoleyl group.
  • an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.
  • alkynyl As used herein, the term “alkynyl,” “alkynyl group,” or “alkynylene” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted.
  • the notation “C2-14 alkynyl” means an optionally substituted linear or branched hydrocarbon including 2- 14 carbon atoms and at least one carbon-carbon triple bond.
  • An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds.
  • C 18 alkynyl may include one or more carbon-carbon triple bonds.
  • an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.
  • the term “carbocycle” or “carbocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty Attorney Docket No.: 45817-0161WO1 membered rings.
  • C3-6 carbocycle means a carbocycle including a single ring having 3-6 carbon atoms.
  • Carbocycles may include one or more carbon- carbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl groups).
  • Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2 dihydronaphthyl groups.
  • cycloalkyl as used herein means a non-aromatic carbocycle and may or may not include any double or triple bond.
  • carbocycles described herein refers to both unsubstituted and substituted carbocycle groups, i.e., optionally substituted carbocycles.
  • heterocycle or “heterocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom.
  • Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen membered rings.
  • Heterocycles may include one or more double or triple bonds and may be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl groups).
  • heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups.
  • heterocycloalkyl as used herein means a non-aromatic heterocycle and may or may not include any double or triple bond. Unless otherwise specified, heterocycles described herein refers to both unsubstituted and substituted heterocycle groups, i.e., optionally substituted heterocycles.
  • heteroalkyl refers respectively to an alkyl, alkenyl, alkynyl group, as defined herein, which further comprises one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus) wherein the one or more heteroatoms is inserted between adjacent carbon atoms within the parent carbon chain and/or one or more heteroatoms is inserted between a carbon atom and the parent molecule, i.e., between the point of Attorney Docket No.: 45817-0161WO1 attachment.
  • heteroatoms e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus
  • heteroalkyls, heteroalkenyls, or heteroalkynyls described herein refers to both unsubstituted and substituted heteroalkyls, heteroalkenyls, or heteroalkynyls, i.e., optionally substituted heteroalkyls, heteroalkenyls, or heteroalkynyls.
  • a “biodegradable group” is a group that may facilitate faster metabolism of a lipid in a mammalian entity.
  • a biodegradable group may be selected from the group consisting of, but is not limited to, -C(O)O-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, an aryl group, and a heteroaryl group.
  • an “aryl group” is an optionally substituted carbocyclic group including one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups.
  • heteroaryl group is an optionally substituted heterocyclic group including one or more aromatic rings.
  • heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted.
  • M and M' can be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole, and thiazole. In the Formulas herein, M and M' can be independently selected from the list of biodegradable groups above.
  • aryl or heteroaryl groups described herein refers to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.
  • Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified.
  • R is an alkyl or alkenyl group, as defined herein.
  • the substituent groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein.
  • a C 1-6 alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.
  • Compounds of the disclosure that contain nitrogens can be converted to N- oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to afford other compounds of the disclosure.
  • an oxidizing agent e.g., 3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides
  • N-hydroxy compounds can be prepared by oxidation of the parent amine by an oxidizing agent such as m CPBA.
  • nitrogen-containing compounds are also considered, when allowed by valency and structure, to cover both the compound as shown and its N- hydroxy (i.e., N-OH) and N-alkoxy (i.e., N-OR, wherein R is substituted or unsubstituted C 1 -C 6 alkyl, C 1 -C 6 alkenyl, C 1 -C 6 alkynyl, 3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.
  • N-OH N-hydroxy
  • N-alkoxy i.e., N-OR, wherein R is substituted or unsubstituted C 1 -C 6 alkyl, C 1 -C 6 alkenyl, C 1 -C 6 alkynyl, 3-14-membered carbocycle or 3-14-membered heterocycle
  • an antibody or binding protein comprising the three CDRs of any one VHH listed in Table 7 below.
  • the three VHH-CDRs can be based on any one CDR definition known in the art. In some cases, the three VHH- CDRs are based on the Kabat definition. In other cases, the three VHH-CDRs are based on the Chothia definition. In yet other cases, the three VHH-CDRs are based on the enhanced Chothia definition. In certain cases, the three VHH-CDRs are based on the Contact definition. In further cases, cases, the three VHH-CDRs are based on the IMGT definition.
  • the Kabat CDRs of VHH2, VHH6, and VHH7 are provided below in Table I. KABAT below in Table II. Attorney Docket No.: 45817-0161WO1 VHH Name CHOTHIA CDR1 CDR2 CDR3
  • the enhanced Chothia CDRs of VHH2, VHH6, and VHH7 are provided below in Table III.
  • Enhanced CHOTHIA For example, the enhanced Contact CDRs of VHH2, VHH6, and VHH7 are provided below in Table IV.
  • IMGT VHH N CDR1 CDR2 CDR3 the disclosure provides an antibody or binding protein having one, two, or three of the CDRs described in Table 8, below.
  • an anti-HSA antibody or binding protein of the disclosure is an antibody (e.g., a VHH or bispecific antibody) or antigen- binding fragment thereof (e.g., a scFv) having one or more of the following CDRs: (a) a CDR1 having the amino acid sequence selected from the group consisting of: GRTYVPYT (SEQ ID NO: 26); GRTFSRYA (SEQ ID NO: 30); Attorney Docket No.: 45817-0161WO1 GRTFVPYT (SEQ ID NO: 33); GRAFSSYT (SEQ ID NO: 35); GRTFSIYT (SEQ ID NO: 38); GRTFVIYT (SEQ ID NO:41); GRTFSSYV (SEQ ID NO: 44); GRTFSTYR (SEQ ID NO: 47); GRTFSSYA (SEQ ID NO: 50); GRALSAASGRAFSRYA (SEQ ID NO: 53); GITFSNYA (SEQ ID NO: 58
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH1, shown below (CDR sequences shown in bold): QVQLVESGGGFVQTGGSLRVSCKASGRTYVPYTMGWFRQAPGKEREFVATI TNSGGSTSYGESVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAIAEGLG VYREEYLYDYWGQGTQVTVSS (VHH1, SEQ ID NO: 1).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least Attorney Docket No.: 45817-0161WO1 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 1.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 1.
  • the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 1.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2, shown below (CDR sequences shown in bold): QVQLVESGGGFVQAGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREFVATI TNSGGSTSYGESVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAVAEGL GVYREEYLYDYWGQGTQVTVSS (VHH2, SEQ ID NO: 2).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 2.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 2.
  • the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 2.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH3, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREFVGVI SWNGGTTSYADSVKGRFTISRENARNTVYLQMNSLKPEDTAVYYCAAAWD LGVRNGEYKYDYWGQGTQVTVSS (VHH3, SEQ ID NO: 3).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 3.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 3.
  • the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 3.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH4, shown below (CDR sequences shown in bold): QVQLVESGGGFVQAGGSLRLSCAASGRTFVPYTMGWFRQAPGKEREFVATI TRSGGSTSYGESVKGRFTISRDNAENTMYLQMNSLKPEDTAVYYCAVAEGL GVYREEYLYDYWGQGTQVTVSS (VHH4, SEQ ID NO: 4).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 4.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 4.
  • the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 4.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH5, shown below (CDR sequences shown in bold): QVQLVESGGGFVQAGGSLRLSCAASGRAFSSYTMGWFRQAPGKEREFVATI TRSSGSTLYGESVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCAVAEGL GRYREEYLYDYWGQGTQVTVSS (VHH5, SEQ ID NO: 5).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 5.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6, shown below (CDR sequences shown in bold): QVQLVESGGGFVQAGDSLRLSCAASGRTFSIYTMGWFRQAPGKEREF VATITHSGGSTSYRGSVKGRFTISRDNAKNTVYLQMNSLKPTDTAVYYCAV AEGAGIYREDYLYDYWGQGTQVTVSS (VHH6, SEQ ID NO: 6).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the Attorney Docket No.: 45817-0161WO1 amino acid sequence of SEQ ID NO: 6.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 6.
  • the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 6.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7, shown below (CDR sequences shown in bold): QVQLVESGGGFVQAGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVATI THSSSSTSYGESVKGRFTISRDNAKNTVYLQMNSLKPTDTAVYYCAIAEGAG VYREDYLYDYWGQGTQVTVSS (VHH7, SEQ ID NO: 7).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 7.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH8, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVQAGGSLRLSCTASGRTFSSYVMGWFRQAPGKEREFVAAV GWSGTSTGYADSVKDRFTISRDNAKNTVYLQMDSLKSEDTAVYYCAADRD RAWSGRYYPNWYEYDYWGQGTQVTVSS (VHH8, SEQ ID NO: 8).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 8.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH9, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGGSLRLSCAASGRTFSTYRMGWFRQAPGKEREFVATV SGSGYSPYYGDSVKGRFTISRDNAKNMVYLQMNSLKPEDTAIYFCAAKTDG LWGQVLPIHYDVWGQGTQVTVSS (VHH9, SEQ ID NO: 9).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 9.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 9.
  • the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 9.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH10, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKERESVARV SRSGLTTYYADSVKGRFTISRDSAKNTVYLQMDSLKPEDTAVYYCAAETDY TRLLSQHEVRYWGQGTQVTVSS (VHH10, SEQ ID NO: 10).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 10.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH11, shown below (CDR sequences shown in bold): QVQLVESGGGLVQSGGSLRLSCAASGRALSAASGRAFSRYAMAWFRQAPG KERNFVAAISSSAGRTSYADSVKGRFTISRDNTKNTAYLQMNSLKPEDTAVY YCAAGGFSGTFSALGNDVDYDYWGQGTQVTVSS (VHH11, SEQ ID NO: 11).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the Attorney Docket No.: 45817-0161WO1 amino acid sequence of SEQ ID NO: 11.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 11.
  • the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 11.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH12, shown below (CDR sequences shown in bold): QVQLIESGGGFVQAGGSLRLSCKASGRTYVPYTMGWFRQAPGKEREFVATIT NSGGSTSYGESVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAVAEGLG VYREEYLYDYWGQGTQVTVSS (VHH12, SEQ ID NO: 12).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 12.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH13, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVQAEGSLRLSCTISGRTFSSYALGWFRQAPGKERESVARVS RSGLTTYYEDFVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCAAETDYT RLLSQREIRYWGQGTQVTVSS (VHH13, SEQ ID NO: 13).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 13.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 13.
  • the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 13.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH14, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGGSLRLSCAASGITFSNYAMGWFRQAPGKERESVAAIS RSGRNTYYADSVKGRFAMSRENAKKTAYLQMNSLKPEDTAVYYCAADYY GLGDLRGSEYDYWGQGTQVTVSS (VHH14, SEQ ID NO: 14).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 14.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH15, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGFRLDYYVIGWFRQAPGKEREGVSCIS STDRVTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATNCDGT TGWDDYWGQGTQVTVSS (VHH15, SEQ ID NO: 15).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 15.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH16, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQVPGKEREFVASI SWGGGVTYYADSVKGRFTISRGNSKNTVYLQMNSLKPEDTAVYYCAASPPE YSDYLEGNEFDYWGQGTQVTVSS (VHH16, SEQ ID NO: 16).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the Attorney Docket No.: 45817-0161WO1 amino acid sequence of SEQ ID NO: 16.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 16.
  • the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 16.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH17, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGGSLRLSCAASGVTFSNYAMGWFRQAPGKAREFVAAI SRGDNIYYTESVKGRFIISRDNAKKTLYLHMNSLKPEDTAVYYCAADYYGL GSSQWPDYEYWGQGTQVTVSS (VHH17, SEQ ID NO: 17).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 17.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH18, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQVPGKEREFVASI SWGGGVTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAASPPE YSDYLEGNEYDYWGQGTQVTVSS (VHH18, SEQ ID NO: 18).
  • VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 18.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH19, shown below (CDR sequences shown in bold): QVQVVESGGGLAQAGGSLRLSCAASGITFSNYAMGWFRQAPGKEREFVGAI SRGSNKYYGDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAADYYG LGSSQWPDYEYRGQGTQVTVSS (VHH19, SEQ ID NO: 19).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 19.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH20, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMAWFRQVPGKEREFVASV SWGGGVTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAASPPE YSDYLEGNEYDYWGQGTQVTVSS (VHH20, SEQ ID NO: 20).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 20.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH21, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGDSLRLSCAASGLTFSNYAVGWFRQAPGNEREFIATIS RGGRTYYADSVKGRSTISRDNAKKTVFLQMNSLKPEDTAAYYCAADYYGL GSSRTGEYEYWGQGTQVIVSS (VHH21, SEQ ID NO: 21).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the Attorney Docket No.: 45817-0161WO1 amino acid sequence of SEQ ID NO: 21.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 21.
  • the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 21.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH22, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGESLRLSCVVFGSTLRFNDMGWYRQAPGKQRELVASI SPGGNTNSADSVKGRFTISRNNDKNTVYLQMNNLKPEDTAVYYCNFVTFRG IGSNNFWGQGTQVTVSS (VHH22, SEQ ID NO: 22).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 22.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH23, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLLQAGGSLRLSCVMSGGTFRWYAMGWFRQAPGKEREFVAT LNRRGDTTYYADSVKGRFAISRDDAKNTVYLQMNSLKPEDTAVYYCAADY YGLGSRSPYEYEYWGQGTQVTVSS (VHH23, SEQ ID NO: 23).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 23.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH24, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGGSLRLSCAASGRTENTYAMGWFRQAPGKEREFVAAI SWSGALTYYQDSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCAASYPK YISDYERGATYEYWGQGTQVTVSS (VHH24, SEQ ID NO: 24).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 24.
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH25, shown below (CDR sequences shown in bold): QVQLVESGGGLVQAGGSLRLSCVASGGTFSDYVMGWFRQAPGKEREFVAAI SWTTSTYYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCTAKSGT YYYQERRNWRNYDYWGQGTQVTVSS (VHH25, SEQ ID NO: 25).
  • the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody or binding protein contains a VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody or binding protein contains a VHH domain having the amino acid sequence of SEQ ID NO: 25.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H1, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTYVPYTMAWFRQAPGKELEWVSTI TNSGGSTSYGESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H1, SEQ ID NO: 88).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 88.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 88.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 88. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H2, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKELEWVSTI TNSGGSTSYGESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H2, SEQ ID NO: 89).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 89.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H3, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVKPGGSLRLSCAASGRTYVPYTMAWFRQAPGKEREWVSTI TNSGGSTSYGESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H3, SEQ ID NO: 90).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 90.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H4, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTYVPYTMAWFRQAPGKELEWVSTI TNSGGSTSYGESVKGRFTISRDNAENSLYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H4, SEQ ID NO: 91).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 91.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H5, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTYVPYTMAWFRQAPGKELEWVSTI TNSGGSTSYGESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H5, SEQ ID NO: 92).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 92.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H6, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREWVSTI TNSGGSTSYGESVKGRFTISRDNAENTLYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H6, SEQ ID NO: 93).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 93.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 93.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 93. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H7, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREWVSTI TNSGGSTSYGESVKGRFTISRDNAENTVYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H7, SEQ ID NO: 94).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 94.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H8, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREWVATI TNSGGSTSYGESVKGRFTISRDNAENTLYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H8, SEQ ID NO: 95).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 95.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H9, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREFVSTI TNSGGSTSYGESVKGRFTISRDNAENTLYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H9, SEQ ID NO: 96).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 96.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H10, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREFVSTI TNSGGSTSYGESVKGRFTISRDNAENTLYLQMNSLRPEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H10, SEQ ID NO: 97).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 97.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H11, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREFVATI TNSGGSTSYGESVKGRFTISRDNAENTLYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H11, SEQ ID NO: 98).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 98.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 98.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 98. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H12, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREFVSTI TNSGGSTSYGESVKGRFTISRDNAENTVYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H12, SEQ ID NO: 99).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 99.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H13, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREFVATI TNSGGSTSYGESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H13, SEQ ID NO: 100).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 100.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H14, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREWVATI TNSGGSTSYGESVKGRFTISRDNAENTLYLQMNSLRPEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H14, SEQ ID NO: 101).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 101. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 101. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 101.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H15, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREWVATI TNSGGSTSYGESVKGRFTISRDNAENTVYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H15, SEQ ID NO: 102).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 102. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 102. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 102.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H16, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKELEFVATI TNSGGSTSYGESVKGRFTISRDNAENTVYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS(VHH2-H16, SEQ ID NO: 103).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 103.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 103.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 103. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H17, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTYVPYTMAWFRQAPGKEREFVATI TNSGGSTSYGESVKGRFTISRDNAENTVYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H17, SEQ ID NO: 104).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 104. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 104.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H18, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREFVATI TNSGGSTSYGESVKGRFTISRDNAENSVYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H18, SEQ ID NO: 105).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 105. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 105. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 105.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H19, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREWVSTI TNSGGSTSYGESVKGRFTISRDNAENTVYLQMNSLRPEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H19, SEQ ID NO: 106).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 106. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 106. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 106.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H20, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREFVATI TNSGGSTSYGESVKGRFTISRDNAENTVYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H20, SEQ ID NO: 107).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 107.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H21, shown below (CDR sequences shown in bold): QVQLVQSGGGLVQSGGSLRLSCAASGRTYVPYTMAWVRQAPGKGLEWVST ITNSGGSTSYGESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H21, SEQ ID NO: 108).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 108.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 108.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 108. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H22, shown below (CDR sequences shown in bold): EVQLVESGGGLVKPGGSLRLSCAASGRTYVPYTMAWIRQAPGKGLEWVSTI TNSGGSTSYGESVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H22, SEQ ID NO: 109).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 109. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 109. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 109.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H23, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVKLLESGGGLVQPGGSLRLSCSASGRTYVPYTMAWVRQAPGKGREYVSTI TNSGGSTSYGESVKGRFTISRDNSKNTVYLQMSSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H23, SEQ ID NO: 110).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 110. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 110.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H24, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCSASGRTYVPYTMAWVRQAPGKEREYVSTI TNSGGSTSYGESVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H24, SEQ ID NO: 111).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 111. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 111. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 111.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H25, shown below (CDR sequences shown in bold): EVQLLESGGGLVQPGGSLRLSCAASGRTYVPYTMAWVRQAPGKEREFVSTI TNSGGSTSYGESVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTTVTVSS (VHH2-H25, SEQ ID NO: 112).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 112. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 112.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H26, shown below (CDR sequences shown in bold): QVQLVESGGGVVQPGRSLRLSCAASGRTYVPYTMAWVRQAPGKEREFVATI TNSGGSTSYGESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAEGL GVYREEYLYDYWGQGTLVTVSS (VHH2-H26, SEQ ID NO: 113).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 113.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 113.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 113. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H27, shown below (CDR sequences shown in bold): EVQLLESGGGLVQPGGSLRLSCAASGRTYVPYTMAWFRQAPGKGREFVSTIT NSGGSTSYGESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAEGLG VYREEYLYDYWGQGTQVTVSS (VHH2-H27, SEQ ID NO: 114).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 114.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H28, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 EVQLLESGGGLVQPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREFVATI TNSGGSTSYGESVKGRFTISRDNSENTLYLQMNSLRPEDTAVYYCAVAEGLG VYREEYLYDYWGQGTQVTVSS (VHH2-H28, SEQ ID NO: 115).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 115. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 115.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H29, shown below (CDR sequences shown in bold): EVQLLESGGGFVQPGGSLRLSCAASGRTYVPYTMAWFRQAPGKEREFVSTIT NSGGSTSYGESVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVAEGLG VYREEYLYDYWGQGTQVTVSS (VHH2-H29, SEQ ID NO: 116).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 116. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 116. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 116.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH2-H30, shown below (CDR sequences shown in bold): EVQLLESGGGFVQPGGSLRLSCKASGRTYVPYTMAWFRQAPGKEREFVATIT NSGGSTSYGESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAEGLG VYREEYLYDYWGQGTQVTVSS (VHH2-H30, SEQ ID NO: 117).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 117. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 117. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 117.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H1, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKELEFVSTIT HSGGSTSYRGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H1, SEQ ID NO: 118).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 118.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 118.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 118. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H2, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKELEFVATIT HSGGSTSYRGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H2, SEQ ID NO: 119).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 119.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H3, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKELEFVSTIT HSGGSTSYRGSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H3, SEQ ID NO: 120).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 120. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 120.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H4, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKELEFVSTIT HSGGSTSYRGSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H4, SEQ ID NO: 121).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 121. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 121.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H5, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVSTIT HSGGSTSYRGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H5, SEQ ID NO: 122).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 122. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 122.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H6, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H6, SEQ ID NO: 123).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 123.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 123.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 123. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H7, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVSTIT HSGGSTSYRGSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H7, SEQ ID NO: 124).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 124. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 124.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H8, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVSTIT HSGGSTSYRGSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H8, SEQ ID NO: 125).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 125. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 125. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 125.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H9, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVSTIT HSGGSTSYRGSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAVAEGA GIYREDYLYDYWGQGTLVTVSS (VHH6-H9, SEQ ID NO: 126).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 126. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 126. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 126.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H10, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H10, SEQ ID NO: 127).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 127.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H11, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H11, SEQ ID NO: 128).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 128.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 128.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 128. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H12, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAVAEGA GIYREDYLYDYWGQGTLVTVSS (VHH6-H12, SEQ ID NO: 129).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 129. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 129. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 129.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H13, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H13, SEQ ID NO: 130).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 130. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 130. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 130.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H14, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H14, SEQ ID NO: 131).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 131. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 131. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 131.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H15, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVSTIT HSGGSTSYRGSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H15, SEQ ID NO: 132).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 132. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 132. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 132.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H16, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNAKNTLYLQMNSLRATDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H16, SEQ ID NO: 133).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 133.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 133.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 133. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H17, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNSKNTVYLQMNSLRATDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H17, SEQ ID NO: 134).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 134. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 134. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 134.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H18, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVSTIT HSGGSTSYRGSVKGRFTISRDNAKNTVYLQMNSLRATDTAVYYCAVAEGA GIYREDYLYDYWGQGTLVTVSS (VHH6-H18, SEQ ID NO: 135).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 135.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 135.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 135.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H19, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H19, SEQ ID NO: 136).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 136. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 136.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H20, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNAKNTVYLQMNSLRATDTAVYYCAVAEGA GIYREDYLYDYWGQGTLVTVSS (VHH6-H20, SEQ ID NO: 137).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 137.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H21, shown below (CDR sequences shown in bold): QVQLVESGGGVVQPGKSLRLSCVASGRTFSIYTMGWVRQAPGKGLEWVATI THSGGSTSYRGSVKGRFTVSRDNSKNTLYLQMNSLRPEDTAVYYCAVAEGA GIYREDYLYDYWGQGTLVTVSS (VHH6-H21, SEQ ID NO: 138).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 138.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 138.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 138. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H22, shown below (CDR sequences shown in bold): QVQLVESGGGVVQPGRSLRLSCAASGRTFSIYTMGWVRQAPGKGLEWVATI THSGGSTSYRGSVKGRFTISRDNSKNTLYLQMNTLRPEDTAVYYCAVAEGA GIYREDYLYDYWGQGTLVTVSS (VHH6-H22, SEQ ID NO: 139).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 139. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 139. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 139.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H23, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVDSGGGLVQPGGSLRLSCAASGRTFSIYTMGWVRQAPGKGREWVSTI THSGGSTSYRGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAEGA GIYREDYLYDYWGQGTQVTVSS (VHH6-H23, SEQ ID NO: 140).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 140.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H24, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFSIYTMGWIRQAPGKGREWVSTIT HSGGSTSYRGSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTTVTVSS (VHH6-H24, SEQ ID NO: 141).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 141. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 141. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 141.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H25, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCSASGRTFSIYTMGWVRQAPGKEREYVSTIT HSGGSTSYRGSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCAVAEGAGI YREDYLYDYWGQGTLVTVSS (VHH6-H25, SEQ ID NO: 142).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 142. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 142. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 142.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H26, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMAWVRQAPGKEREFVSTIT HSGGSTSYRGSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H26, SEQ ID NO: 143).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 143.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 143.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 143. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H27, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGESLRLSCAASGRTFSIYTMAWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTQVTVSS (VHH6-H27, SEQ ID NO: 144).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 144. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 144. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 144.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H28, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 EVQLVESGGGFVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKGREFVSTIT HSGGSTSYRGSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAVAEGAGI YREDYLYDYWGQGTQVTVSS (VHH6-H28, SEQ ID NO: 145).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 145. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 145. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 145.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H29, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKGREFVSTIT HSGGSTSYRGSVKGRFTISRDNSKNTLYLQMNSLRPTDTAVYYCAVAEGAGI YREDYLYDYWGQGTQVTVSS (VHH6-H29, SEQ ID NO: 146).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 146. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 146. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 146.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H30, shown below (CDR sequences shown in bold): EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVSTIT HSGGSTSYRGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTQVTVSS (VHH6-H30, SEQ ID NO: 147).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 147. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 147. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 147.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H31, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVSTIT HSGGSTSYRGSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H31, SEQ ID NO: 168).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 168.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 168.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 168. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H32, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGDSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVSTIT HSGGSTSYRGSVKGRFTISRDNSKNTVYLQMNSLRATDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H32, SEQ ID NO: 169).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 169. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 169. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 169.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H33, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H33, SEQ ID NO: 170).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 170.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H34, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGDSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNSKNTVYLQMNSLRATDTAVYYCAVAEGAG IYREDYLYDYWGQGTLVTVSS (VHH6-H34, SEQ ID NO: 171).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 171.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 171.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 171.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H35, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGGSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAVAEGA GIYREDYLYDYWGQGTLVTVSS (VHH6-H35, SEQ ID NO: 172).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 172. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 172.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH6-H36, shown below (CDR sequences shown in bold): QVQLVESGGGLVQPGDSLRLSCAASGRTFSIYTMGWFRQAPGKEREFVATIT HSGGSTSYRGSVKGRFTISRDNAKNTVYLQMNSLRATDTAVYYCAVAEGA GIYREDYLYDYWGQGTLVTVSS (VHH6-H36, SEQ ID NO: 173).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 173.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 173.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 173. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H1, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKGLEFVSTIT HSSSSTSYGESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H1, SEQ ID NO: 148).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 148. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 148.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H2, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKELEFVSTIT HSSSSTSYGESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H2, SEQ ID NO: 149).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 149. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 149. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 149.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H3, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKGREFVSTIT HSSSSTSYGESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H3, SEQ ID NO: 150).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 150. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 150. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 150.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H4, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKGLEFVSTIT HSSSSTSYGESVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H4, SEQ ID NO: 151).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 151.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H5, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVSTIT HSSSSTSYGESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H5, SEQ ID NO: 152).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 152.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 152.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 152. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H6, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVATIT HSSSSTSYGESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H6, SEQ ID NO: 153).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 153. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 153. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 153.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H7, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVSTIT HSSSSTSYGESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H7, SEQ ID NO: 154).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 154. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 154. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 154.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H8, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVSTIT HSSSSTSYGESVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H8, SEQ ID NO: 155).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 155. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 155. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 155.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H9, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVSTIT HSSSSTSYGESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H9, SEQ ID NO: 156).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 156. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 156.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H10, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVATIT HSSSSTSYGESVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H10, SEQ ID NO: 157).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 157.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 157.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 157. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H11, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVATIT HSSSSTSYGESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H11, SEQ ID NO: 158).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 158. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 158.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H12, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVATIT HSSSSTSYGESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H12, SEQ ID NO: 159).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 159. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 159. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 159.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H13, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVATIT HSSSSTSYGESVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H13, SEQ ID NO: 160).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 130. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 160. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 160.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H14, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVATIT HSSSSTSYGESVKGRFTISRDNAKNSVYLQMNSLRPEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H14, SEQ ID NO: 161).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 161.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 161.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 161.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H15, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVSTIT HSSSSTSYGESVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H15, SEQ ID NO: 162).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 162.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 162.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 162. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H16, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVATIT HSSSSTSYGESVKGRFTISRDNAKNTLYLQMNSLRATDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H16, SEQ ID NO: 163).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 163.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 163.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 163.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H17, shown below (CDR sequences shown in bold): Attorney Docket No.: 45817-0161WO1 QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVATIT HSSSSTSYGESVKGRFTISRDNAKNSVYLQMNSLRATDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H17, SEQ ID NO: 164).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 164. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 164. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 164.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H18, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVSTIT HSSSSTSYGESVKGRFTISRDNAKNTVYLQMNSLRATDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H18, SEQ ID NO: 165).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 165. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 165. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 165.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H19, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVATIT HSSSSTSYGESVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H19, SEQ ID NO: 166).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 166. In some embodiments, the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 166. In some embodiments, the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 166.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of VHH, VHH7-H20, shown below (CDR sequences shown in bold): QVQLVESGGGLVKPGGSLRLSCAASGRTFVIYTMGWFRQAPGKEREFVATIT HSSSSTSYGESVKGRFTISRDNAKNTVYLQMNSLRATDTAVYYCAIAEGAGV YREDYLYDYWGQGTLVTVSS (VHH7-H20, SEQ ID NO: 167).
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Attorney Docket No.: 45817-0161WO1 the amino acid sequence of SEQ ID NO: 167.
  • the antibody or binding protein contains a humanized VHH domain having an amino acid sequence that is at least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of SEQ ID NO: 167.
  • the antibody or binding protein contains a humanized VHH domain having the amino acid sequence of SEQ ID NO: 167.
  • an antibody or antigen-binding fragment is a murine- specific antibody or antigen-binding fragment, e.g., the antibody or binding protein specifically binds the murine antigen.
  • an antibody or antigen- binding fragment is a rat-specific antibody or antigen-binding fragment, e.g., the antibody or binding protein specifically binds the rat antigen.
  • an antibody or antigen-binding fragment is a llama-specific antibody or antigen- binding fragment, e.g., the antibody or binding protein specifically binds the llama antigen.
  • an antibody or antigen-binding fragment is a human- specific antibody or antigen-binding fragment, e.g., the antibody or binding protein specifically binds the human antigen. In some embodiments, an antibody or antigen- binding fragment is human-specific even if the antibody or binding protein is not human or humanized.
  • the anti-HSA binding moiety is a VHH domain that is derived from a llama and binds to human HSA. Preferably, the anti-HSA binding moiety is humanized. In a preferred embodiment, the anti-HSA VHH domain binds to human and non-human primate HSA.
  • Multispecific antibodies in another aspect, provides multispecific antibodies, for example, bispecific antibodies (BsAbs), that may have binding specificities that are directed towards HSA and any other antigen, e.g., for a cell-surface protein, receptor, receptor subunit, or tissue-specific antigen, or other non-HSA antigen.
  • BsAbs bispecific antibodies
  • Multispecific antibodies typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen Attorney Docket No.: 45817-0161WO1 (i.e., HSA and any other antigen).
  • Each antigen-binding domain of a bispecific antibody can comprise a heavy chain variable domain (VH), a light chain variable domain (VL), or a VH and a VL.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH and VL a VH and a VL.
  • each antigen binding domain comprises at least one CDR that alone, or in combination with one or more additional CDRs and/or framework regions, specifically binds to a particular antigen (i.e., HSA, any other antigen).
  • the first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding fragment (i.e., bispecific ScFv) further bound to an Fc domain.
  • the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate Fc domain.
  • Bispecific antigen-binding fragments of the present disclosure may comprise two Fc domains that are each individually part of a separate antibody heavy chain.
  • the first and second Fc domains may be of the same sequence, or the Fc domains may have a mutation in the CH3 domain intended for the facilitation or ease of purification of heterodimeric (i.e., bispecific) molecules.
  • a multispecific antibody may also be an antibody or antigen-binding fragment thereof that includes at least two separate antigen-binding domains (e.g., two scFvs joined by a linker). The scFvs may bind the same antigen or different antigens.
  • a bispecific antibody can also comprise multiple chains.
  • a bispecific antibody may be an antibody or antigen-binding fragment thereof that includes a F(ab) with binding specificity directed towards a first antigen and a VHH domain with binding specificity directed towards a second antigen (e.g., HSA) joined by a linker.
  • multispecific antibodies of the present disclosure are secreted (e.g., released from a cell, for example, into the extracellular milieu).
  • Multispecific antibodies of the present disclosure can include any anti-HSA CDRs, or VHH domains described herein.
  • Multispecific antibodies of the present disclosure can comprise binding specificities that are directed towards HSA and any other antigen.
  • Any other antigen may be or comprise, for example, a cancer cell antigen/marker, an immune cell antigen (e.g., a T cell activation marker), a pathogenic antigen, or any other non-HSA antigen.
  • the disclosed multispecific antibodies may be produced by any means known in the art for producing multispecific antibodies, so long as the resulting multispecific antibody retains the functional characteristic of being able to specifically bind HSA and at least one other antigen.
  • the BsAbs may be created using the methods described in Labrijin et al., Proc. Natl. Acad. Sci. USA, 110(13):5145-50 (2013). Briefly, the two parental Abs, each containing single matched point mutations in the CH3 domains, are separately expressed and then mixed under reducing conditions in vitro. This separates the Abs into half-molecules, followed by reassembly, to form bispecific antibodies, and is compatible with large-scale manufacturing of bispecific antibodies. However, this is simply one exemplary method for making a multispecific antibody.
  • multispecific antibody formats or technologies may be used to make the multispecific antigen-binding molecules of the present disclosure.
  • an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antigen-binding molecule.
  • bispecific formats that can be used in the context of the present invention include, without limitation, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, Attorney Docket No.: 45817-0161WO1 IgGl/IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al.2012, mAbs 4:6, 1-11 , and references cited therein, for a review of the foregoing formats).
  • the disclosed multispecific antibodies can be made from or incorporate the CDRs or variable regions from polyclonal, monoclonal, chimeric, human, partially or fully humanized, and/or recombinant antibodies.
  • the “parent” antibodies for the disclosed multispecific antibodies are not particularly limited; however, they are preferably fully human or humanized.
  • the parent antibody can be a polyclonal antibody.
  • the parent antibody can be a monoclonal.
  • the parent antibody can be a human antibody.
  • Antibodies, antigen-binding fragments, or binding proteins of the disclosure may have an affinity for HSA, HSA DII, and/or CSA of, for example, from 1 nM to 100 nM (e.g., from 10 nM to 90 nM, from 20 nM to 80 nM, from 30 nM to 70 nM, from 40 nM to 60 nM, or about 50 nM).
  • antibodies, antigen- binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of from about 1 nM to about 100 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of from about 1 nM to about 90 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity HSA, HSA DII, and/or CSA of from about 1 nM to about 80 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of from about 1 nM to 60 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of from about 1 nM to 40 nM. In some embodiments, antibodies, antigen-binding Attorney Docket No.: 45817-0161WO1 fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of from about 1 nM to 20 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 100 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 95 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 90 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 85 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 80 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 75 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 70 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 65 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 60 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 55 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 50 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 45 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 40 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 35 nM. In some Attorney Docket No.: 45817-0161WO1 embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 30 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 25 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 20 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 15 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 10 nM.
  • antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 5 nM. In some embodiments, antibodies, antigen-binding fragments, or binding proteins of the disclosure have an affinity for HSA, HSA DII, and/or CSA of about 1 nM.
  • the specific binding of an antibody, antigen-binding fragments, or binding proteins described herein to HSA, HSA DII, and/or CSA can be determined by any of a variety of established methods.
  • the affinity can be represented quantitatively by various measurements, including the concentration of antibody or binding protein needed to achieve the equilibrium constant (K D ) of the antibody- , antigen-binding fragment-, or binding proteins - antigen complex dissociation.
  • K D which describes the interaction of HSA, HSA DII, and/or CSA with an antibody, antigen-binding fragment, or binding proteins described herein is the chemical equilibrium constant for the dissociation reaction of a antigen-antibody, – antigen-binding fragment, or – binding protein complex into solvent-separated antigen and antibody, antigen-binding fragment, or binding proteins that do not interact with one another.
  • Antibodies, antigen-binding fragments, or binding proteins described herein include those that specifically bind to HSA, HSA DII, and/or CSA with a KD value of less than 100 nM (e.g., less than 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 Attorney Docket No.: 45817-0161WO1 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM).
  • nM e.g., less than 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45
  • the antibodies, antigen-binding fragments, or binding proteins described herein specifically bind to HSA, HSA DII, and/or CSA with a KD value of less than 10 nM (e.g., less than 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM).
  • Antibodies, antigen-binding fragments, or binding proteins described herein can also be characterized by a variety of in vitro binding assays.
  • Examples of experiments that can be used to determine the KD or EC50 of an anti-HSA antibody or binding protein include, e.g., surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, ELISA-based assays, gene expression assays, and protein expression assays, among others.
  • ELISA represents a particularly useful method for analyzing antibody or binding protein activity, as such assays typically require minimal concentrations of binding domains (e.g., antibodies, antigen-binding fragments, binding proteins).
  • a common signal that is analyzed in a typical ELISA assay is luminescence, which is typically the result of the activity of a peroxidase conjugated to a secondary antibody that specifically binds a primary antibody (e.g., an anti-HSA antibody, antigen-binding fragment described herein).
  • Antibodies, antigen- binding fragments, or binding proteins described herein may bind HSA, HSA DII, and/or CSA and fragments thereof.
  • Antibodies, antigen-binding fragments, or binding proteins described herein may additionally bind isolated peptides derived from HSA, HSA DII, and/or CSA that structurally pre-organize various residues in a manner that simulates the conformation of the above fragments in the native protein.
  • this binding can be quantified, e.g., by analyzing the luminescence that occurs upon incubation of an HRP substrate (e.g., 2,2’-azino-di-3- ethylbenzthiazoline sulfonate) with an antigen-antibody, antigen-antigen-binding fragment, or antigen-binding protein complex bound to a HRP-conjugated secondary antibody.
  • an HRP substrate e.g., 2,2’-azino-di-3- ethylbenzthiazoline sulfonate
  • SPR surface plasmon resonance
  • the antigen is typically immobilized on a solid metallic surface which is treated in pulses with solutions of increasing concentrations of antibody, antigen-binding fragment, or binding proteins.
  • Antibody-, antigen-binding fragment-, or binding protein-antigen binding induces distortion in the angle of reflection of incident light at the metallic surface, and this change in refractive index over time as antibody or binding protein is introduced to the system can be fit to established regression models in order to calculate the association and dissociation rate constants of an antibody- or antigen-binding-fragment- or binding protein- antigen interaction.
  • Antibodies, antigen-binding fragments, or binding proteins described herein may exhibit high kon and low koff values upon interaction with HSA.
  • antibodies, antigen-binding fragments, or binding proteins described herein may exhibit k on values in the presence of HSA, HSA DII, and/or CSA of greater than 10 4 M -1 s -1 (e.g., 1.0 x 10 4 M -1 s -1 , 1.5 x 10 4 M -1 s -1 , 2.0 x 10 4 M -1 s -1 , 2.5 x 10 4 M -1 s -1 , 3.0 x Attorney Docket No.: 45817-0161WO1 10 4 M -1 s -1 , 3.5 x 10 4 M -1 s -1 , 4.0 x 10 4 M -1 s -1 , 4.5 x 10 4 M -1 s , 5.0 x 10 4 M -1 s , 5.5 x 10 4 M -1 s -1 , 6.0 x 10 4 M -1 s -1 , 6.5 x 10 4 M -1 s -1 , 7.0 x
  • Antibodies, antigen-binding fragments, or binding proteins described herein may exhibit low koff values when bound to HSA, HSA DII, and/or CSA.
  • antibodies, antigen-binding fragments, or binding proteins described herein may exhibit koff values of less than 10- 3 s -1 when complexed to HSA, HSA DII, and/or CSA (e.g., 1.0 x 10 -3 s -1 , 9.5 x 10 -4 s -1 , 9.0 x 10 -4 s -1 , 8.5 x 10 -4 s -1 , 8.0 x 10 -4 s -1 , 7.5 x 10 -4 s -1 , 7.0 x 10 -4 s -1 , 6.5 x 10 -4 s -1 , 6.0 x 10 -4 s -1 , 5.5 x 10 -4 s -1 , 5.0 x 10 -4 s -1 , 4.5 x 10 -4 s -1
  • Antibodies, antigen-binding fragments, or binding proteins described herein can include fully human, humanized, primatized, and chimeric antibodies that contain one or more of the CDR sequences shown in Table 8, below.
  • one strategy that can be used to design humanized antibodies, antigen-binding fragments, or binding proteins described herein is to align the sequences of the VH and/or VL of an antibody or binding protein (e.g., of the present disclosure) with the V H and/or V L of a consensus human antibody.
  • Consensus human antibody heavy chain and light chain sequences are known in the art (see, e.g., the “VBASE” human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91 -3242 (1991); Tomlinson et al., J. Mol. Biol.227:776-98 (1992); and Cox et al., Eur. J. Immunol.24:827-836 (1994); the disclosure of which is Attorney Docket No.: 45817-0161WO1 incorporated herein by reference).
  • variable domain framework residues and CDRs can be identified by sequence alignment (see, Kabat, supra).
  • this strategy can also be used to produce primatized anti-HSA antibodies, antigen-binding fragments, or binding proteins, as one can substitute, for example, one or more, or all, of the CDRs of a primate antibody consensus sequence with, for example, one or more, or all, of the CDRs of an antibody or binding protein of the disclosure.
  • Consensus primate antibody sequences known in the art (see, e.g., U.S. Patent Nos.5,658,570; 5,681,722; and 5,693,780; the disclosures of each of which are incorporated herein by reference).
  • US Patent No.6,054,297 identifies several instances when it may be advantageous to retain certain framework residues from a particular antibody heavy chain or light chain variable region in the resulting humanized antibody, antigen-binding fragment, or binding proteins.
  • framework residues may engage in non-covalent interactions with the antigen and thus contribute to the affinity of the antibody, antigen-binding fragment, or binding proteins for the target antigen.
  • individual framework residues may modulate the conformation of a CDR, and thus indirectly influence the interaction of the antibody, antigen-binding domain, or binding proteins with the antigen.
  • Certain framework residues may form the interface between VH and V L domains, and may therefore contribute to the global antibody, antigen-binding domain, or binding protein structure.
  • framework residues may constitute functional glycosylation sites (e.g., Asn-X-Ser/Thr) which may dictate antibody, antigen-binding domain, or binding protein structure and antigen affinity upon attachment to carbohydrate moieties.
  • the closest human germline sequence for each VHH can be selected and the CDRs and canonical residues grafted into the human framework region.
  • residues deemed important for VHH stability and function, based on previous humanization experiments can be back mutated from human to llama.
  • sequences may be designed with various human amino acids and/or llama back-mutations to screen for the clone that retains the biophysical properties, affinity, and potency of the parental molecule.
  • Antibodies described herein also include antibody fragments, Fab domains, F(ab’) molecules, F(ab’) 2 molecules, single-chain variable fragments (scFvs), tandem scFv fragments, diabodies, triabodies, dual variable domain immunoglobulins, multi- specific antibodies, bispecific antibodies, and heterospecific antibodies that contain one or more of the CDRs in Table 8, below, or a CDR having at least 85% sequence identity thereto (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Attorney Docket No.: 45817-0161WO1 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto).
  • Anti-HSA antibodies, antigen-binding fragments, or binding proteins described herein can be prepared by any of a variety of established techniques. For instance, an anti-HSA antibody or antigen-binding fragment described herein can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell.
  • a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the desired antibody chain(s), antigen-binding fragments, and/or additional binding protein domains (e.g., transmembrane domains, hinge domains).
  • additional binding protein domains e.g., transmembrane domains, hinge domains.
  • the light and/or heavy chains of an antibody or an antigen-binding fragment can be expressed in the host cell and, optionally, secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered.
  • Standard recombinant DNA methodologies are used to obtain antibody heavy chain genes, light chain genes, and binding protein domains and to incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), Current Protocols in Molecular Biology (Ausubel et al., eds., Greene Publishing Associates (1989), and in U.S. Patent No.4,816,397; the disclosures of each of which are incorporated herein by reference.
  • Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell).
  • Viral genomes are particularly useful vectors for gene delivery Attorney Docket No.: 45817-0161WO1 because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration.
  • viral vectors examples include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., Measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein- Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox).
  • adenovirus e.g., Ad5, Ad26, Ad
  • viruses useful for delivering polynucleotides encoding antibody light and heavy chains or antibody fragments or binding proteins described herein include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV- BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
  • murine leukemia viruses include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses.
  • vectors are described, for example, in McVey et al., (U.S. Patent. No.5,801,030); the disclosures of each of which are incorporated herein by reference.
  • Non-viral vectors such as plasmids
  • plasmids include, but are not limited to prokaryotic and eukaryotic vectors (e.g., yeast- and bacteria-based plasmids), as well as plasmids for expression in mammalian cells.
  • Methods of introducing the vectors into a host cell and isolating and purifying the Attorney Docket No.: 45817-0161WO1 expressed protein are also well known in the art (e.g., Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989)).
  • Genome editing techniques In addition to viral vectors, a variety of additional methods have been developed for the incorporation of genes, e.g., those encoding antibody light and heavy chains, single-domain antibodies, single-chain variable fragments (scFvs), tandem scFvs, Fab domains, F(ab’)2 domains, diabodies, and triabodies, among others, into the genomes of target cells for antibody, antigen-binding fragment, and/or binding protein expression.
  • One such method that can be used for incorporating polynucleotides encoding anti-HSA antibodies, antigen-binding fragments, or binding proteins into prokaryotic or eukaryotic cells includes the use of transposons.
  • Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by excision sites at the 5’ and 3’ positions. Once a transposon has been delivered into a cell, expression of the transposase gene commences and results in active enzymes that cleave the gene of interest from the transposon. This activity is mediated by the site-specific recognition of transposon excision sites by the transposase. In some embodiments, these excision sites may be terminal repeats or inverted terminal repeats.
  • the gene of interest can be integrated into the genome of a prokaryotic or eukaryotic cell by transposase-catalyzed cleavage of similar excision sites that exist within nuclear genome of the cell.
  • This allows the gene encoding an anti-HSA antibody or antigen-binding fragment or binding protein described herein to be inserted into the cleaved nuclear DNA at the excision sites, and subsequent ligation of the phosphodiester bonds that join the gene of interest to the DNA of the prokaryotic or eukaryotic cell genome completes the incorporation process.
  • the transposon is a retrotransposon, such that the gene encoding the antibody or binding protein is first transcribed to an RNA product and then reverse- transcribed to DNA before incorporation in the prokaryotic or eukaryotic cell genome.
  • exemplary transposon systems include the piggybac transposon (described in detail in Attorney Docket No.: 45817-0161WO1 WO 2010/085699) and the sleeping beauty transposon (described in detail in US20050112764); the disclosures of each of which are incorporated herein by reference.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the CRISPR/Cas system consists of palindromic repeat sequences within plasmid DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci.
  • Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas9 nuclease to this site.
  • highly site-specific cas9-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings cas9 within close proximity of the target DNA molecule is governed by RNA:DNA hybridization.
  • RNA:DNA hybridization As a result, one can theoretically design a CRISPR/Cas system to cleave any target DNA molecule of interest.
  • Additional genome editing techniques that can be used to incorporate polynucleotides encoding antibodies, antigen-binding fragments, or binding proteins described herein into the genome of a prokaryotic or eukaryotic cell include the use of ARCUS TM meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA.
  • Single-chain meganucleases can thus be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations. These single-chain nucleases have been described extensively, e.g., in U.S. Patent Nos.8,021,867 and 8,445,251; the disclosures of each of which are incorporated herein by reference.
  • polynucleotide sequence elements to express an anti-HSA antibodies, antigen-binding fragments, or binding proteins described herein, polynucleotides encoding partial or full-length light and heavy chains, e.g., polynucleotides that encode a one or more of the CDR sequences of an antibody or binding protein described herein, can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • Polynucleotides encoding the light chain gene and the heavy chain of an anti-HSA antibody or binding protein can be inserted into separate vectors, or, optionally, both polynucleotides can be incorporated into the same expression vector using established techniques described herein or known in the art.
  • the recombinant expression vectors described herein may carry regulatory sequences that control the expression of the antibody chain genes or binding protein domains in a host cell.
  • the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed or the level of expression of protein desired.
  • suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • Viral regulatory elements, and sequences thereof are described in detail, for instance, in U.S. Patent No. 5,168,062, U.S. Patent No.4,510,245, and U.S.
  • Patent No.4,968,615 the disclosures of each of which are incorporated herein by reference.
  • the recombinant expression vectors described herein can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • a selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patents Nos.4,399,216, 4,634,665 and 5,179,017).
  • the selectable marker gene confers resistance to cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, to a host cell into which the vector has been introduced.
  • cytotoxic drugs such as G418, puromycin, blasticidin, hygromycin or methotrexate
  • Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR ” host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • the expression vector(s) containing polynucleotides encoding the heavy and light chains can be transfected into a host cell by standard techniques.
  • Exemplary mammalian host cells for expressing the recombinant antibodies, antigen-binding fragments, or binding proteins described herein include Chinese Hamster Ovary (CHO cells) (including DHFR CHO cells, described in Urlaub and Chasin (1980, Proc. Natl. Acad. Sci. USA 77:4216-4220), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, 293 cells (e.g., expi293), and SP2/0 cells.
  • Chinese Hamster Ovary CHO cells
  • DHFR CHO cells described in Urlaub and Chasin (1980, Proc. Natl. Acad. Sci. USA 77:4216-4220
  • a DHFR selectable marker e.g., as described in Kaufman and Sharp (1982, Mol. Biol. 159:601-621
  • Additional cell types that may be useful for the expression of antibodies, antigen-binding fragments, or binding proteins include bacterial cells, such as BL- 21(DE3) E. Coli cells, which can be transformed with vectors containing foreign DNA according to established protocols.
  • Additional eukaryotic cells that may be useful for expression of antibodies, antigen-binding fragments, or binding proteins include yeast cells, such as auxotrophic strains of S. cerevisiae, which can be transformed and selectively grown in incomplete media according to established procedures known in the art.
  • the antibodies When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies, antigen-binding fragments, or binding proteins can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. Also included herein are methods in which the above procedure is varied according to established protocols known in the art.
  • an anti-HSA antibody or antigen-binding fragment described herein can be purified by any method known in the art, such as a method useful for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for HSA, HSA DII, and/or CSA after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for HSA, HSA DII, and/or CSA after Protein A or Protein G selection, and sizing column chromatography
  • centrifugation e.g., centrifugation after Protein A or Protein G selection, and sizing column chromatography
  • differential solubility e.g., differential solubility, or by any other standard technique for the purification of proteins.
  • the anti-HSA antibodies described herein or fragments thereof can be fuse
  • an anti-HSA single-chain or single-domain antibody can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology (Work and Burdon, eds., Elsevier, 1980); incorporated herein by reference), or by gel filtration chromatography, such as on a Superdex TM 75 column (Pharmacia Biotech AB, Uppsala, Sweden).
  • an anti-HSA antibody or antigen-binding fragment of the disclosure or a multispecific antibody comprising the same is conjugated to a second molecule, e g., to extend the half-life of the anti-HSA antibody or antigen- binding fragment in vivo.
  • a second molecule e g., to extend the half-life of the anti-HSA antibody or antigen- binding fragment in vivo.
  • Such molecules that can extend half-life of the anti-HSA antibody or antigen-binding fragment are described below, and include polyethylene glycol (PEG), among others.
  • Anti-HSA antibodies and fragments thereof can be conjugated to these half-life extending molecules at, e.g., the N-terminus or C- terminus of a light and/or heavy chain of the antibody using any one of a variety of conjugation strategies known in the art.
  • pairs of reactive functional groups that can be used to covalently tether an anti-HSA antibody or fragment thereof Attorney Docket No.: 45817-0161WO1 to a half-life extending or other molecule include, without limitation, thiol pairs, carboxylic acids and amino groups, ketones and amino groups, aldehydes and amino groups, thiols and alpha,beta-unsaturated moieties (such as maleimides or dehydroalanine), thiols and alpha-halo amides, carboxylic acids and hydrazides, aldehydes and hydrazides, and ketones and hydrazides.
  • Anti-HSA antibodies can be conjugated to various molecules for the purpose of improving the half-life, solubility, and stability of the protein in aqueous solution.
  • examples of such molecules include polyethylene glycol (PEG), murine serum albumin (MSA), bovine serum albumin (BSA), and human serum albumin (HSA), among others.
  • PEG polyethylene glycol
  • MSA murine serum albumin
  • BSA bovine serum albumin
  • HSA human serum albumin
  • one can conjugate an anti-HSA antibody or antigen- binding fragment to carbohydrate moieties in order to evade detection of the antibody antigen-binding fragment by the immune system of the patient receiving treatment. This process of hyperglycosylation reduces the immunogenicity of therapeutic proteins by sterically inhibiting the interaction of the protein with B cell receptors in circulation.
  • anti-HSA antibodies, antigen-binding fragments, or binding proteins can be conjugated to molecules that prevent clearance from human serum and improve the pharmacokinetic profile of the antibodies, antigen-binding fragments, or binding proteins.
  • Serum albumin is a globular protein that is the most abundant blood protein in mammals. Serum albumin is produced in the liver and constitutes about half of the blood serum proteins. It is monomeric and soluble in the blood. Some of the most crucial functions of serum albumin include transporting hormones, fatty acids, and other proteins in the body, buffering pH, and maintaining osmotic pressure needed for proper distribution of bodily fluids between blood vessels and body tissues. In some embodiments, serum albumin is MSA or HSA.
  • MSA or HSA is joined to the N- or C-terminus of an antibody or antigen-binding fragment of the disclosure described herein to increase the serum half-life of the antibody or antigen- binding fragment.
  • MSA or HSA can be joined, either directly or through a linker, to the N- or C-terminus of an antibody or antigen-binding fragment of the disclosure.
  • an antibody or antigen-binding fragment described herein is Attorney Docket No.: 45817-0161WO1 fused to the N- or C-terminus of a serum albumin through genetic or chemical means, e.g., chemical conjugation.
  • the MSA has the amino acid sequence of UniProt ID NO: Q546G4 (SEQ ID NO: 174), or an amino acid sequence that is at least 85% identical (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 174, shown below: MKWVTFLLLLFVSGSAFSRGVFRREAHKSEIAHRYNDLGEQHFKGLV LIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLF GDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERP EAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILT
  • fusion proteins containing anti-HSA antibodies, antigen-binding fragments, or binding proteins can be expressed recombinantly from a cell (e.g., a eukaryotic cell or prokaryotic cell). This can be accomplished, for example, by incorporating a polynucleotide encoding the fusion protein into the genome of a cell (e.g., using techniques described herein or known in the art).
  • a cell e.g., a eukaryotic cell or prokaryotic cell.
  • antibodies and fragments thereof described herein can be joined to a half-life extending molecule by forming a covalent bond between the antibody and a linker.
  • linker can then be subsequently conjugated to another molecule, or the linker can be conjugated to another molecule prior to ligation to the anti-HSA antibody or antigen-binding fragment.
  • linkers that can be used for the formation of a conjugate include polypeptide linkers, such as those that contain naturally occurring or non-naturally occurring amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker, as these residues are not present in naturally-occurring proteins and are thus more resistant to degradation by endogenous proteases.
  • Fusion proteins containing polypeptide linkers can be made using chemical synthesis techniques, such as those described herein, or through recombinant expression of a polynucleotide encoding the fusion protein in a cell (e.g., a prokaryotic or eukaryotic cell).
  • Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic Attorney Docket No.: 45817-0161WO1 conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med.
  • an anti-HSA antibody or antigen-binding fragment of the disclosure is conjugated to a second molecule or therapeutic, e g., to extend the half-life of the second molecule or therapeutic in vivo.
  • Anti-HSA antibodies and fragments thereof can be conjugated to other molecules or therapeutics at, e.g., the N- terminus or C-terminus of a light and/or heavy chain of the antibody using any one of a variety of conjugation strategies known in the art.
  • pairs of reactive functional groups that can be used to covalently tether an anti-HSA antibody or fragment thereof to another molecule or therapeutic include, without limitation, thiol pairs, carboxylic acids and amino groups, ketones and amino groups, aldehydes and amino groups, thiols and alpha,beta-unsaturated moieties (such as maleimides or dehydroalanine), thiols and alpha-halo amides, carboxylic acids and hydrazides, aldehydes and hydrazides, and ketones and hydrazides.
  • Anti-HSA antibodies can be conjugated to various molecules for the purpose of improving the half-life, solubility, and stability of the molecule or therapeutic in aqueous solution.
  • anti-HSA antibody or antigen-binding fragment of the disclosure is conjugated to any one of a peptide, polypeptide (e.g. growth factor CIBP2, antimicrobial cyclic peptides), a protein, an enzyme or polypeptide (e.g. iduronate-2-sulfatase (IDS), acid beta-glucosidase (GCase), serine proteases, growth factors), an antibody or a fragment operable to bind a target epitope (e.g.
  • a peptide e.g. growth factor CIBP2, antimicrobial cyclic peptides
  • a protein e.g. iduronate-2-sulfatase (IDS), acid beta-glucosidase (GCase), serine proteases, growth factors
  • IDS iduronate-2-sulfatase
  • GCase acid beta-glucosidase
  • serine proteases e.g.
  • anti- microbial antibodies anti-inflammatory antibodies, intrabodies, BBB-crossing antibodies, neurodegeneration targets antibodies, ion channel targeting antibodies for pain, imaging, diagnostic, affinity purification reagents, anti-cancer targets, checkpoint inhibitors, GPCR targeting antibodies), or combinations thereof, in which Attorney Docket No.: 45817-0161WO1 both the antibody or an antigen-binding fragment and the rest of the conjugate remain functional for their intended purpose.
  • Anti-HSA antibodies, antigen-binding fragments, or binding proteins can be covalently appended directly to another molecule or therapeutic by chemical conjugation as described.
  • fusion proteins containing anti-HSA antibodies, antigen-binding fragments, or binding proteins can be expressed recombinantly from a cell (e.g., a eukaryotic cell or prokaryotic cell). This can be accomplished, for example, by incorporating a polynucleotide encoding the fusion protein into the genome of a cell (e.g., using techniques described herein or known in the art). As such, a fusion protein can be synthesized by the translation of a single RNA transcript encoding both domains in frame with one another.
  • antibodies and fragments thereof described herein can be joined to a second molecule or therapeutic by forming a covalent bond between the antibody and a linker.
  • linker can then be subsequently conjugated to another molecule or theraputic, or the linker can be conjugated to another molecule or therapeutic prior to ligation to the anti-HSA antibody or antigen-binding fragment.
  • linkers that can be used for the formation of a conjugate include polypeptide linkers, such as those that contain naturally occurring or non-naturally occurring amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker, as these residues are not present in naturally-occurring proteins and are thus more resistant to degradation by endogenous proteases.
  • Fusion proteins containing polypeptide linkers can be made using chemical synthesis techniques, such as those described herein, or through recombinant expression of a polynucleotide encoding the fusion protein in a cell (e.g., a prokaryotic or eukaryotic cell).
  • Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582 (2012)).
  • nucleic acid molecules of the disclosure may include one or more alterations.
  • a nucleotide, nucleoside, or polynucleotide such as the nucleic acids of the invention (e.g., an mRNA or an oligonucleotide)
  • alteration or, as appropriate, “alternative” refer to alteration with respect to A, G, U or C ribonucleotides.
  • the alterations may be various distinct alterations.
  • the coding region, the flanking regions, and/or the terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide alterations.
  • an alternative polynucleotide introduced to a cell may exhibit reduced degradation in the cell, as compared to an unaltered polynucleotide.
  • the polynucleotides can include any useful alteration, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage, or to the phosphodiester backbone).
  • alterations are present in each of the sugar and the internucleoside linkage.
  • Alterations according to the present invention may be alterations of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs) (e.g., the substitution of the 2’OH of the ribofuranosyl ring to 2’H), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof. Additional alterations are described herein.
  • nucleic acid molecule introduced into the cell may be degraded intracellularly.
  • degradation of a nucleic acid molecule may be preferable if precise timing of protein production is desired.
  • the invention provides an alternative nucleic acid molecule containing a degradation domain, which is capable of being acted on in a directed manner within a cell.
  • the polynucleotides can optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors, etc.).
  • the polynucleotides may include one or more messenger RNAs (mRNAs) having one or more alternative nucleoside or nucleotides (i.e., mRNA molecules).
  • the polynucleotides may include one or more oligonucleotides having one or more alternative nucleoside or nucleotides.
  • a composition of the invention includes an mRNA and/or one or more oligonucleotides having one or more alternative nucleoside or nucleotides. Modified nucleic acids According to Aduri et al., (Aduri, R.
  • nucleosides including 1-methyladenosine, 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine, 2-methyladenosine, 2-O-ribosylphosphate adenosine, N6-methyl-N6- threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6- hydroxynorvalylcarbamoyladenosine, 1,2-O-dimethyladenosine,
  • Nucleosides containing modified sugars The alternative nucleosides and nucleotides (e.g., building block molecules), which may be incorporated into a polynucleotide (e.g., RNA or mRNA, as described herein), can be altered on the sugar of the ribonucleic acid.
  • a polynucleotide e.g., RNA or mRNA, as described herein
  • the 2′ hydroxyl group (OH) can be modified or replaced with a number of different substituents.
  • Exemplary substitutions at the 2′-position include, but are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy; optionally Attorney Docket No.: 45817-0161WO1 substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C 3-8 cycloalkoxy; optionally substituted C 6-10 aryloxy; optionally substituted C 6-10 aryl- C1-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), - O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1
  • Exemplary, non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar.
  • Attorney Docket No.: 45817-0161WO1 Alterations on the nucleobase The present disclosure provides for alternative nucleosides and nucleotides.
  • nucleoside is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • organic base e.g., a purine or pyrimidine
  • nucleotide is defined as a nucleoside including a phosphate group.
  • Exemplary non-limiting alterations include an amino group, a thiol group, an alkyl group, a halo group, or any described herein.
  • a nucleic acid of the invention includes one or more 2’-OMe nucleotides, 2’-methoxyethyl nucleotides (2’-MOE nucleotides), 2’-F nucleotide, 2’-NH2 nucleotide, 2’fluoroarabino nucleotides (FANA nucleotides), locked nucleic acid nucleotides (LNA nucleotides), or 4’-S nucleotides.
  • the alternative nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, and guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or alternative nucleotides including non- standard or alternative bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures.
  • non-standard base pairing is the base pairing between the alternative nucleotide inosine and adenine, cytosine, or uracil.
  • the alternative nucleosides and nucleotides can include an alternative nucleobase.
  • nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil.
  • nucleobase found in DNA Attorney Docket No.: 45817-0161WO1 include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleobases can be altered or wholly replaced to provide polynucleotide molecules having enhanced properties (e.g., resistance to nucleases and stability), and these properties may manifest through disruption of the binding of a major groove binding partner.
  • the alternative nucleobase is an alternative uracil.
  • nucleobases and nucleosides having an alternative uracil include pseudouridine ( ⁇ ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio- 5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine (chm 5 U
  • the nucleic acid is modified to contain 1- methylpseudouridine (m 1 ⁇ ) in lieu of uridine at each instance.
  • the alternative nucleobase is an alternative cytosine.
  • Exemplary nucleobases and nucleosides having an alternative cytosine include 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl- cytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl- cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 1-methyl
  • the alternative nucleobase is an alternative adenine.
  • Exemplary nucleobases and nucleosides having an alternative adenine include 2- Attorney Docket No.: 45817-0161WO1 amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro- purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido- adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7- deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1-methyl-adenosine (m 1 A), 2-methyl-adenine (m 2 A), N6
  • the alternative nucleobase is an alternative guanine.
  • Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl- wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o 2 yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza- guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7- deaza-guanosine (
  • the nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine, or pyrimidine analog.
  • the nucleobase can each be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine.
  • the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5- uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted
  • each letter refers to the representative base and/or derivatives thereof (e.g., A includes adenine or adenine analogs (e.g., 7-deaza adenine)).
  • the polynucleotides of the invention contain 5- methoxy-uracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and Attorney Docket No.: 45817-0161WO1 cytosines.
  • the polynucleotides of the invention contain 5- methoxy-uracil, uracil, 5-trifluoromethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uracil, uracil, 5-hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uracil, uracil, 5-bromo-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the invention contain 5- methoxy-uracil, uracil, 5-iodo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-methoxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-phenyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-fluoro-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, N4-acetyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-formyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-aminoallyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5-methoxy-uracil, uracil, 5-carboxy-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the invention contain 1-methyl- pseudouracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl- pseudouracil, uracil, 5-trifluoromethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1- methyl-pseudouracil, uracil, 5-hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-bromo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-iodo-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-methoxy-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-phenyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-fluoro-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, N4-acetyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-formyl-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouracil, uracil, 5-aminoallyl-cytosine, and cytosine as the only uracils and cytosines.
  • the polynucleotides of the invention Attorney Docket No.: 45817-0161WO1 contain 1-methyl-pseudouracil, uracil, 5-carboxy-cytosine, and cytosine as the only uracils and cytosines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-hydroxymethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-bromo-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-iodo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-ethyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-phenyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-ethnyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uridine, uridine, N4-methyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-fluoro-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uridine, uridine, N4-acetyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-formyl-cytidine, and cytidine as the only uridines and Attorney Docket No.: 45817-0161WO1 cytidines. In some embodiments, the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-aminoallyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain 5- methoxy-uridine, uridine, 5-carboxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl- pseudouridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1- methyl-pseudouridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-hydroxymethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-bromo-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-iodo-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-ethyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-phenyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-ethnyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, N4-methyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-fluoro-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, N4-acetyl-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the Attorney Docket No.: 45817-0161WO1 invention contain 1-methyl-pseudouridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-formyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-aminoallyl-cytidine, and cytidine as the only uridines and cytidines. In some embodiments, the polynucleotides of the invention contain 1-methyl-pseudouridine, uridine, 5-carboxy-cytidine, and cytidine as the only uridines and cytidines.
  • the polynucleotides of the invention contain the uracil of one of the nucleosides of Table 3 and uracil as the only uracils. In other embodiments, the polynucleotides of the invention contain a uridine of Table 3 and uridine as the only uridines.
  • the polynucleotides of the invention contain a cytidine of Table 4 and cytidine as the only cytidines.
  • Table 4 – Exemplary cytosine containing nucleosides Nucleoside Name ⁇ -thio-ctidine Attorney Docket No.: 45817-0161WO1 Nucleoside Name N4-Bz-cytidine Attorney Docket No.: 45817-0161WO1 Nucleoside Name 2’-fluor-N4-Bz-cytidine ’
  • Alterations on the internucleoside linkage The alternative nucleotides, which may be incorporated into a polynucleotide molecule, can be altered on the internucleoside linkage (e.g., phosphate backbone).
  • Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent.
  • the alternative nucleosides and nucleotides can include the wholesale replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein.
  • Examples of alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be altered by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
  • the alternative nucleosides and nucleotides can include the replacement of one or more of the non-bridging oxygens with a borane moiety (BH3), sulfur (thio), methyl, ethyl and/or methoxy.
  • two non-bridging oxygens at the same position can be replaced with a sulfur (thio) and a methoxy.
  • the replacement of one or more of the oxygen atoms at the ⁇ position of the phosphate moiety is provided to confer stability (such as against exonucleases and endonucleases) to RNA and DNA through the unnatural Attorney Docket No.: 45817-0161WO1 phosphorothioate backbone linkages.
  • Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. While not wishing to be bound by theory, phosphorothioate linked polynucleotide molecules are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
  • an alternative nucleoside includes an alpha-thio- nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine ( ⁇ -thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-pseudouridine).
  • alpha-thio- nucleoside e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine ( ⁇ -thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-pseu
  • the polynucleotides of the invention can include a combination of alterations to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more alterations described herein. Synthesis of polynucleotides
  • the polynucleotide molecules for use in accordance with the invention may be prepared according to any useful technique, as described herein.
  • the alternative nucleosides and nucleotides used in the synthesis of polynucleotide molecules disclosed herein can be prepared from readily available starting materials using the following general methods and procedures.
  • product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography (e.g., high performance liquid (HPLC) or thin layer chromatography).
  • spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography (e.g., high performance liquid (HPLC) or thin layer chromatography).
  • spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography (e.g., high performance liquid (HPLC) or
  • the reactions of the processes described herein can be carried out in suitable solvents, which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out (i.e., temperatures which can range from the solvent’s freezing temperature to the solvent’s boiling temperature). A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.
  • Resolution of racemic mixtures of alternative polynucleotides or nucleic acids can be carried out by any of numerous methods known in the art.
  • An example method includes fractional recrystallization using a “chiral resolving acid” which is an optically active, salt-forming organic acid.
  • Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids.
  • Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., Attorney Docket No.: 45817-0161WO1 dinitrobenzoylphenylglycine).
  • Suitable elution solvent composition can be determined by one skilled in the art.
  • Alternative nucleosides and nucleotides e.g., building block molecules
  • nucleosides and nucleotides can be prepared according to the synthetic methods described in Ogata et al., J. Org. Chem.74:2585-2588 (2009); Purmal et al., Nucl.
  • polynucleotide includes one or more alternative nucleosides or nucleotides, the polynucleotides of the invention may or may not be uniformly altered along the entire length of the molecule.
  • nucleotide e.g., purine or pyrimidine, or any one or more or all of A, G, U, C
  • nucleotide may or may not be uniformly altered in a polynucleotide of the invention, or in a given predetermined sequence region thereof.
  • all nucleotides X in a polynucleotide of the invention (or in a given sequence region thereof) are altered, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
  • nucleotide analogs or other alteration(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased.
  • An alteration may also be a 5′ or 3′ terminal alteration.
  • the polynucleotide may contain from 1% to 100% alternative nucleosides, nucleotides, or internucleoside linkages (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e.
  • any one or more of A, G, U or C) or any intervening percentage e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to Attorney Docket No.: 45817-0161WO1 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 90% to 95%, from 90% to 9
  • the remaining percentage is accounted for by the presence of A, G, U, or C.
  • the remaining percentage necessary to total 100% is accounted for by the corresponding natural nucleoside, nucleotide, or internucleoside linkage.
  • the remaining percentage necessary to total 100% is accounted for by a second alternative nucleoside, nucleotide, or internucleoside linkage.
  • Messenger RNA The present invention features compositions including one or more mRNAs, where each mRNA encodes a polypeptide (e.g., an anti-HSA antibody or antigen- binding fragment described herein).
  • Exemplary mRNAs of the disclosure each include (i) a 5’-cap structure; (ii) a 5’-UTR; (iii) an open reading frame encoding the polypeptide; (iv) a 3’-untranslated region (3’-UTR); and (v) a poly-A region.
  • the mRNA includes from about 30 to about 3,000 (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,000, from 1,500 to Attorney Docket No.: 45817-0161WO1 2,500, from 1,500
  • mRNA: 5’-cap The 5′-cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
  • CBP mRNA Cap Binding Protein
  • the cap further assists the removal of 5′ proximal introns removal during mRNA splicing.
  • Endogenous mRNA molecules may be 5′-end capped generating a 5′-ppp-5′- triphosphate linkage between a terminal guanosine cap residue and the 5′-terminal transcribed sense nucleotide of the mRNA.
  • This 5′-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue.
  • nucleotides may be used during the capping reaction.
  • a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with ⁇ -thio-guanosine nucleotides according to the manufacturer’s instructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap.
  • Additional alternative guanosine nucleotides may be used such as ⁇ -methyl-phosphonate and seleno-phosphate nucleotides.
  • Additional alterations include, but are not limited to, 2′-O-methylation of the ribose sugars of 5′-terminal and/or 5′-anteterminal nucleotides of the mRNA (as Attorney Docket No.: 45817-0161WO1 mentioned above) on the 2′-hydroxyl group of the sugar ring.
  • Multiple distinct 5′-cap structures can be used to generate the 5′-cap of a nucleic acid molecule, such as an mRNA molecule.
  • 5’-cap structures include those described in International Patent Publication Nos. WO2008/127688, WO2008/016473, and WO2011/015347, each of which is incorporated herein by reference in its entirety.
  • Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5′-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non- enzymatically) or enzymatically synthesized and/linked to a nucleic acid molecule.
  • the Anti-Reverse Cap Analog (ARCA) cap contains two guanosines linked by a 5′-5′-triphosphate group, wherein one guanosine contains an N7 methyl group as well as a 3′-O-methyl group (i.e., N7,3′-O-dimethyl-guanosine-5′- triphosphate-5′-guanosine (m 7 G-3′mppp-G; which may equivalently be designated 3′ O-Me-m7G(5')ppp(5')G)).
  • N7,3′-O-dimethyl-guanosine-5′- triphosphate-5′-guanosine m 7 G-3′mppp-G; which may equivalently be designated 3′ O-Me-m7G(5')ppp(5')G
  • the 3′-O atom of the other, unaltered, guanosine becomes linked to the 5′-terminal nucleotide of the capped nucleic acid molecule (e.g., an mRNA or mmRNA).
  • the N7- and 3′-O-methlyated guanosine provides the terminal moiety of the capped nucleic acid molecule (e.g., mRNA or mmRNA).
  • Another exemplary cap is mCAP, which is similar to ARCA but has a 2′-O- methyl group on guanosine (i.e., N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′- guanosine, m 7 Gm-ppp-G).
  • the cap is a dinucleotide cap analog.
  • the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in US Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety.
  • Attorney Docket No.: 45817-0161WO1 the cap analog is a N7-(4-chlorophenoxyethyl) substituted dicnucleotide form of a cap analog known in the art and/or described herein.
  • Non-limiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)- G(5’)ppp(5’)G and a N7-(4-chlorophenoxyethyl)-m 3’-O G(5’)ppp(5’)G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 21:4570-4574 (2013); the contents of which are herein incorporated by reference in its entirety).
  • a cap analog of the present invention is a 4-chloro/bromophenoxyethyl analog. While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts remain uncapped. This, as well as the structural differences of a cap analog from endogenous 5′-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.
  • Nucleic acids of the invention may also be capped post-transcriptionally, using enzymes.5’ cap structures produced by enzymatic capping may enhance binding of cap binding proteins, increase half-life, reduce susceptibility to 5′ endonucleases and/or reduce 5′ decapping, as compared to synthetic 5′-cap structures known in the art (or to a wild-type, natural or physiological 5′-cap structure).
  • recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme can create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNA and a guanosine cap nucleotide wherein the cap guanosine contains an N7 methylation and the 5′-terminal nucleotide of the mRNA contains a 2′-O-methyl.
  • Cap1 structure is termed the Cap1 structure.
  • Cap structures include 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')- Attorney Docket No.: 45817-0161WO1 ppp(5')NlmpN2mp (cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).
  • 5′ terminal caps may include endogenous caps or cap analogs.
  • a 5′ terminal cap may include a guanosine analog.
  • Useful guanosine analogs include inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, and 2-azido-guanosine.
  • the nucleic acids described herein may contain a modified 5’-cap.
  • a modification on the 5’-cap may increase the stability of mRNA, increase the half-life of the mRNA, and could increase the mRNA translational efficiency.
  • the modified 5’-cap may include, but is not limited to, one or more of the following modifications: modification at the 2’ and/or 3’ position of a capped guanosine triphosphate (GTP), a replacement of the sugar ring oxygen (that produced the carbocyclic ring) with a methylene moiety (CH 2 ), a modification at the triphosphate bridge moiety of the cap structure, or a modification at the nucleobase (G) moiety.
  • GTP capped guanosine triphosphate
  • CH 2 methylene moiety
  • G nucleobase
  • mRNA: Coding region Provided are nucleic acids that encode antibodies or antigen-binding fragments of the disclosure.
  • protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of this present disclosure.
  • any protein fragment of a reference protein meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical
  • 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or greater than 100 amino acids in length 10.
  • any protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids which are about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein can be utilized in accordance with the present disclosure.
  • a protein sequence to be utilized in accordance Attorney Docket No.: 45817-0161WO1 with the present disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.
  • mRNA Poly-A tail
  • poly(A) tail a long chain of adenosine nucleotides
  • poly(A) polymerase adds a chain of adenosine nucleotides to the RNA.
  • polyadenylation adds a poly-A tail that is between 100 and 250 residues long.
  • Methods for the stabilization of RNA by incorporation of chain-terminating nucleosides at the 3’-terminus include those described in International Patent Publication No. WO2013/103659, incorporated herein in its entirety.
  • Poly(A) tail deadenylation by 3′ exonucleases is a key step in cellular mRNA degradation in eukaryotes. By blocking 3' exonucleases, the functional half-life of mRNA can be increased, resulting in increased protein expression.
  • the poly(A)tail of the mRNA includes a 3’ LA10 or IdT modification.
  • LA10 or IdT modification for example, as described in International Patent Publication No. WO2017/049275, the tail modifications of which are incorporated by reference in their entirety.
  • Annealing an oligonucleotide (e.g., an oligonucleotide conjugate) with a complex secondary structure (e.g., a triple-helix structure or a stem-loop structure) at the 3’end may provide nuclease resistance and increase half-life of mRNA.
  • Unique poly(A) tail lengths may provide certain advantages to the RNAs of the present invention. Generally, the length of a poly(A) tail of the present invention is greater than 30 nucleotides in length. In some embodiments, the poly(A) tail is greater than 35 nucleotides in length. In some embodiments, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides.
  • the length is at least 50 nucleotides. In some embodiments, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 65 nucleotides. In another embodiment, the length is at least 70 nucleotides. In some embodiments, the length is at least 80 nucleotides. In some embodiments, the length is at least 90 nucleotides. In some embodiments, the length is at least 100 nucleotides. In some embodiments, the length is at least 120 nucleotides. In some embodiments, the length is at least 140 nucleotides. In some embodiments, the length is at least 160 nucleotides.
  • the length is at least 180 nucleotides. In some embodiments, the length is at least 200 nucleotides. In some embodiments, the length is at least 250 nucleotides. In some embodiments, the length is at least 300 nucleotides. In some embodiments, the length is at least 350 nucleotides. In some embodiments, the length is at least 400 nucleotides. In some embodiments, the length is at least 450 nucleotides. In some embodiments, the length is at least 500 nucleotides. In some embodiments, the length is at least 600 nucleotides. In some embodiments, the length is at least 700 nucleotides. In some embodiments, the length is at least 800 nucleotides.
  • the length is at least 900 nucleotides. In some embodiments, the length is at least 1000 nucleotides. In some embodiments, the length is at least 1100 nucleotides. In some embodiments, the length is at least 1200 nucleotides. In some embodiments, the length is at least 1300 nucleotides. In some Attorney Docket No.: 45817-0161WO1 embodiments, the length is at least 1400 nucleotides. In some embodiments, the length is at least 1500 nucleotides. In some embodiments, the length is at least 1600 nucleotides. In some embodiments, the length is at least 1700 nucleotides. In some embodiments, the length is at least 1800 nucleotides.
  • the length is at least 1900 nucleotides. In some embodiments, the length is at least 2000 nucleotides. In some embodiments, the length is at least 2500 nucleotides. In some embodiments, the length is at least 3000 nucleotides.
  • the poly(A) tail may be 80 nucleotides, 120 nucleotides, or 160 nucleotides in length. In some embodiments, the poly(A) tail may be 20, 40, 80, 100, 120, 140 or 160 nucleotides in length. In some embodiments, the poly(A) tail is designed relative to the length of the mRNA.
  • poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length than the additional feature.
  • the poly(A) tail may also be designed as a fraction of the mRNA to which it belongs. In this context, the poly(A) tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly(A) tail.
  • engineered binding sites and/or the conjugation of nucleic acids or mRNA for poly(A) binding protein may be used to enhance expression.
  • the engineered binding sites may be sensor sequences which can operate as binding sites for ligands of the local microenvironment of the nucleic acids and/or mRNA.
  • the nucleic acids and/or mRNA may include at least one engineered binding site to alter the binding affinity of poly(A) binding protein (PABP) and analogs thereof.
  • PABP poly(A) binding protein
  • the incorporation of at least one engineered binding site may increase the binding affinity of the PABP and analogs thereof.
  • multiple distinct nucleic acids or mRNA may be linked together to the PABP (poly(A) binding protein) through the 3′-end using nucleotides at the 3′- terminus of the poly(A) tail.
  • Transfection experiments can be conducted in relevant cell lines and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72hr, and day 7 post-transfection.
  • the transfection experiments may be used to evaluate the effect on PABP or analogs thereof binding affinity as a result of the addition of at least one engineered binding site.
  • a poly(A) tail may be used to modulate translation initiation.
  • the poly-A tail recruits PABP which in turn can interact with translation initiation complex and thus may be essential for protein synthesis.
  • a poly(A) tail may also be used in the present invention to protect against 3’-5’ exonuclease digestion.
  • the nucleic acids or mRNA of the present invention are designed to include a poly-A-G quartet.
  • the G-quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail.
  • the resultant nucleic acid or mRNA may be assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the poly-A-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone.
  • the nucleic acids or mRNA of the present invention may include a poly(A) tail and may be stabilized by the addition of a chain terminating nucleoside.
  • the nucleic acids and/or mRNA with a poly(A) tail may further include a 5’cap structure.
  • the nucleic acids or mRNA of the present invention may include a poly-A-G quartet.
  • the nucleic acids and/or mRNA with a poly-A-G quartet may further include a 5’cap structure.
  • the chain terminating nucleoside which may be used to stabilize the nucleic acid or mRNA including a poly(A) tail or poly-A-G quartet may be, but is not limited to, those described in International Patent Publication No. WO2013103659, incorporated herein by reference in its entirety.
  • the chain terminating nucleosides which may be used with the present invention includes, but is not limited to, 3'-deoxyadenosine (cordycepin), 3'- deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'- dideoxynucleosides, such as 2',3'- dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'- dideoxycytosine, 2',3'- dideoxyguanosine, 2',3'-dideoxythymine, a 2'- deoxynucleoside, or a -O- methylnucleoside.
  • 3'-deoxyadenosine cordycepin
  • 3'- deoxyuridine 3'-deoxycytosine
  • 3'-deoxyguanosine 3'-deoxythymine
  • the mRNA which includes a poly(A) tail or a poly-A-G quartet may be stabilized by an alteration to the 3’region of the nucleic acid that can prevent and/or inhibit the addition of oligo(U) (see, e.g., International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety).
  • the mRNA which includes a poly(A) tail or a poly-A-G quartet, may be stabilized by the addition of an oligonucleotide that terminates in a 3’-deoxynucleoside, 2’,3’-dideoxynucleoside 3'-O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and/or described herein.
  • the nucleic acids of the present invention may include a stem-loop such as, but not limited to, a histone stem-loop.
  • the stem-loop may be a nucleotide sequence that is about 25 or about 26 nucleotides in length such as, but not limited to, SEQ ID NOs: 7-17 as described in International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety.
  • the histone stem-loop may be located 3’ relative to the coding region (e.g., at the 3’ terminus of the coding region).
  • the stem-loop may be located at the 3’ end of a nucleic acid described herein. Attorney Docket No.: 45817-0161WO1
  • the stem-loop may be located in the second terminal region.
  • the stem-loop may be located within an untranslated region (e.g., 3’-UTR) in the second terminal region.
  • the nucleic acid such as, but not limited to mRNA, which includes the histone stem-loop may be stabilized by the addition of at least one chain terminating nucleoside.
  • the addition of at least one chain terminating nucleoside may slow the degradation of a nucleic acid and thus can increase the half-life of the nucleic acid.
  • the chain terminating nucleoside may be, but is not limited to, those described in International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety.
  • the chain terminating nucleosides which may be used with the present invention includes, but is not limited to, 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'- deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleosides, such as 2',3'- dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'- dideoxyguanosine, 2',3'-dideoxythymine, a 2'-deoxynucleoside, or a -O- methylnucleoside.
  • 3'-deoxyadenosine cordycepin
  • 3'-deoxyuridine 3'-deoxycytosine
  • 3'- deoxyguanosine 3'-deoxythymine
  • the nucleic acid such as, but not limited to mRNA, which includes the histone stem-loop may be stabilized by an alteration to the 3’region of the nucleic acid that can prevent and/or inhibit the addition of oligo(U) (see, e.g., International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety).
  • the nucleic acid such as, but not limited to, mRNA, which includes the histone stem-loop may be stabilized by the addition of an oligonucleotide that terminates in a 3’-deoxynucleoside, 2’,3’-dideoxynucleoside 3'- O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and/or described herein.
  • an oligonucleotide that terminates in a 3’-deoxynucleoside, 2’,3’-dideoxynucleoside 3'- O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and/or described herein.
  • the nucleic acids of the present invention may include a histone stem-loop, a poly(A) tail sequence, and/or a 5’-cap structure.
  • the histone stem-loop may be before and/or after the poly-A tail sequence.
  • the nucleic acids including the histone stem-loop and a poly(A) tail sequence may include a chain terminating nucleoside described herein.
  • the nucleic acids of the present invention may include a histone stem-loop and a 5’-cap structure.
  • the 5’-cap structure may include, but is not limited to, those described herein and/or known in the art.
  • the nucleic acids described herein may include at least one histone stem-loop and a poly(A) sequence or polyadenylation signal.
  • Non- limiting examples of nucleic acid sequences encoding for at least one histone stem- loop and a poly-A sequence or a polyadenylation signal are described in International Patent Publication Nos. WO2013/120497, WO2013/120629, WO2013/120500, WO2013/120627, WO2013/120498, WO2013/120626, WO2013/120499 and WO2013/120628, the contents of each of which are incorporated herein by reference in their entirety.
  • the nucleic acid encoding for a histone stem- loop and a poly(A) sequence or a polyadenylation signal may code for a pathogen antigen or fragment thereof such as the nucleic acid sequences described in International Patent Publication Nos. WO2013/120499 and WO2013/120628, the contents of both of which are incorporated herein by reference in their entirety.
  • the nucleic acid encoding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal may code for a therapeutic protein such as the nucleic acid sequences described in International Patent Publication Nos.
  • the nucleic acid encoding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal may code for a tumor antigen or fragment thereof such as the nucleic acid sequences described in International Patent Publication Nos. WO2013/120500 and WO2013/120627, the contents of both of which are incorporated herein by reference in their entirety.
  • the nucleic acid encoding for a histone stem- Attorney Docket No.: 45817-0161WO1 loop and a poly(A) sequence or a polyadenylation signal may code for an autoimmune self-antigen such as the nucleic acid sequences described in International Patent Publication Nos. WO2013/120498 and WO2013/120626, the contents of both of which are incorporated herein by reference in their entirety.
  • mRNA Triple helices
  • nucleic acids of the present invention e.g., the mRNA of the present invention
  • the 3’ end of the nucleic acids of the present invention may include a triple helix alone or in combination with a poly(A) tail.
  • the nucleic acid of the present invention may include at least a first and a second U-rich region, a conserved stem-loop region between the first and second region and an A-rich region.
  • the first and second U-rich region and the A-rich region may associate to form a triple helix on the 3’ end of the nucleic acid.
  • This triple helix may stabilize the nucleic acid, enhance the translational efficiency of the nucleic acid and/or protect the 3’ end from degradation.
  • Triple helices include, but are not limited to, the triple helix sequence of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), MEN- ⁇ and polyadenylated nuclear (PAN) RNA (see, Wilusz et al., Genes & Development 26:2392-2407 (2012); herein incorporated by reference in its entirety).
  • the triple helix may be formed from the cleavage of a MALAT1 sequence prior to the cloverleaf structure.
  • MALAT1 is a long non-coding RNA which, when cleaved, forms a triple helix and a tRNA-like cloverleaf structure.
  • the MALAT1 transcript then localizes to nuclear speckles and the tRNA-like cloverleaf localizes to the cytoplasm (Wilusz et al., Cell.135(5): 919-932 (2008); incorporated herein by reference in its entirety).
  • the terminal end of the nucleic acid of the present invention including the MALAT1 sequence can then form a triple helix structure, after RNaseP cleavage from the cloverleaf structure, which stabilizes the nucleic acid Attorney Docket No.: 45817-0161WO1 (Peart et al., WIREs RNA.4(5):491-506 (2013); incorporated herein by reference in its entirety).
  • the nucleic acids or mRNA described herein include a MALAT1 sequence.
  • the nucleic acids or mRNA may be polyadenylated.
  • the nucleic acids or mRNA is not polyadenylated but has an increased resistance to degradation compared to unaltered nucleic acids or mRNA.
  • the nucleic acids of the present invention may include a MALAT1 sequence in the second flanking region (e.g., the 3’-UTR).
  • the MALAT1 sequence may be human or mouse.
  • TEEs Translation Enhancer Elements
  • TEEs translation enhancer element
  • TEEs translation enhancer element
  • TEEs are conserved elements in the UTR which can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation. The conservation of these sequences has been previously shown by Pánek et al., Nucleic Acids Research. 41(16): 7625-7634 (2013); incorporated herein by reference in its entirety) across 14 species including humans.
  • the 5’-UTR of the mRNA includes at least one TEE.
  • the TEE may be located between the transcription promoter and the start codon.
  • the mRNA with at least one TEE in the 5’-UTR may include a cap at the 5’-UTR. Further, at least one TEE may be located in the 5’-UTR of mRNA undergoing cap- dependent or cap-independent translation.
  • the TEEs known may be in the 5′-leader of the Gtx homeodomain protein (Chappell et al., Proc. Natl. Acad. Sci. USA 101:9590-9594 (2004), incorporated herein by reference in their entirety).
  • Attorney Docket No.: 45817-0161WO1 In another non-limiting example, TEEs are disclosed as SEQ ID NOs: 1-35 in US Patent Publication No.
  • the TEE may be an internal ribosome entry site (IRES), HCV-IRES or an IRES element such as, but not limited to, those described in US Patent No.
  • the IRES elements may include, but are not limited to, the Gtx sequences (e.g., Gtx9-nt, Gtx8-nt, Gtx7-nt) described by Chappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594 (2004)) and Zhou et al. (PNAS 102:6273-6278 (2005)) and in US Patent Publication Nos. US20070048776 and US20110124100 and International Patent Publication No.
  • Gtx sequences e.g., Gtx9-nt, Gtx8-nt, Gtx7-nt
  • Chappell et al. Proc. Natl. Acad. Sci. USA 101:9590-9594 (2004)
  • Zhou et al. PNAS 102:6273-6278 (2005)
  • WO2007025008 each of which is incorporated herein by reference in its entirety. Additional exemplary TEEs are disclosed in US Patent Nos. US6310197, US6849405, US7456273, US7183395; US Patent Publication Nos. US20090226470, US20070048776, US20110124100, US20090093049, US20130177581; International Patent Publication Nos. WO2009075886, WO2007025008, WO2012009644, WO2001055371 WO1999024595; and European Patent Publications Nos. EP2610341A1 and EP2610340A1; each of which is incorporated herein by reference in its entirety.
  • the polynucleotides, primary constructs, alternative nucleic acids and/or mRNA may include at least one TEE that is described in International Patent Publication Nos. WO1999024595, WO2012009644, WO2009075886, WO2007025008, WO1999024595, European Patent Publication Attorney Docket No.: 45817-0161WO1 Nos. EP2610341A1 and EP2610340A1, US Patent Nos. US6310197, US6849405, US7456273, US7183395, US Patent Publication No. US20090226470, US20110124100, US20070048776, US20090093049, and US20130177581 each of which is incorporated herein by reference in its entirety.
  • the TEE may be located in the 5’-UTR of the mRNA.
  • the polynucleotides, primary constructs, alternative nucleic acids and/or mmRNA may include at least one TEE that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity with the TEEs described in US Patent Publication Nos. US20090226470, US20070048776, US20130177581 and US20110124100, International Patent Publication Nos. WO1999024595, WO2012009644, WO2009075886 and WO2007025008, European Patent Publication No.
  • EP2610341A1 and EP2610340A1 and US Patent Nos. US6310197, US6849405, US7456273, and US7183395, each of which is incorporated herein by reference in its entirety.
  • Multiple copies of a specific TEE can be present in mRNA.
  • the TEEs in the translational enhancer polynucleotides can be organized in one or more sequence segments.
  • a sequence segment can harbor one or more of the specific TEEs exemplified herein, with each TEE being present in one or more copies.
  • When multiple sequence segments are present in a translational enhancer polynucleotide they can be homogenous or heterogeneous.
  • the multiple sequence segments in a translational enhancer polynucleotide can harbor identical or different types of the specific TEEs exemplified herein, identical or different number of copies of each of the specific TEEs, and/or identical or different organization of the TEEs within each sequence segment.
  • the 5’-UTR of the mRNA may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least Attorney Docket No.: 45817-0161WO1 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences.
  • the TEE sequences in the 5’-UTR of mRNA of the present invention may be the same or different TEE sequences.
  • the TEE sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C represent a different TEE sequence at the nucleotide level.
  • the 5’-UTR may include a spacer to separate two TEE sequences. As a non-limiting example, the spacer may be a 15 nucleotide spacer and/or other spacers known in the art.
  • the 5’-UTR may include a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times and at least 9 times, or more than 9 times in the 5’-UTR.
  • the TEE in the 5’-UTR of the mRNA of the present invention may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more than 99% of the TEE sequences disclosed in US Patent Publication Nos. US20090226470, US20070048776, US20130177581 and US20110124100, International Patent Publication Nos.
  • the TEE in the 5’-UTR of the mRNA of the present invention may include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotide fragment of the TEE sequences disclosed in US Patent Publication Nos.
  • the TEE in the 5’-UTR of the mRNA of the present invention may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more than 99% of the TEE sequences disclosed in Chappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594 (2004)) and Zhou et al.
  • the TEE in the 5’-UTR of the polynucleotides, primary constructs, alternative nucleic acids and/or mmRNA of the present invention may include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotide fragment of the TEE sequences disclosed in Chappell et al. (Proc. Natl.
  • the TEE used in the 5’-UTR of the mRNA of the present invention is an IRES sequence such as, but not limited to, those described in US Patent No. US7468275 and International Patent Publication No. WO2001055369, each of which is incorporated herein by reference in its entirety.
  • the TEEs used in the 5’-UTR of the mRNA of the present invention may be identified by the methods described in US Patent Publication Nos. US20070048776 and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2012009644, each of which is incorporated herein by reference in its entirety. Attorney Docket No.: 45817-0161WO1
  • the TEEs used in the 5’-UTR of the mRNA of the present invention may be a transcription regulatory element described in US Patent Nos. US7456273 and US7183395, US Patent Publication No. US20090093049, and International Publication No.
  • WO2001055371 each of which is incorporated herein by reference in its entirety.
  • the transcription regulatory elements may be identified by methods known in the art, such as, but not limited to, the methods described in US Patent Nos. US7456273 and US7183395, US Patent Publication No. US20090093049, and International Publication No. WO2001055371, each of which is incorporated herein by reference in its entirety.
  • the TEE used in the 5’-UTR of the mRNA of the present invention is an oligonucleotide or portion thereof as described in US Patent No. US7456273 and US7183395, US Patent Publication No. US20090093049, and International Publication No.
  • WO2001055371 each of which is incorporated herein by reference in its entirety.
  • the 5’-UTR including at least one TEE described herein may be incorporated in a monocistronic sequence such as, but not limited to, a vector system or a nucleic acid vector.
  • the vector systems and nucleic acid vectors may include those described in US Patent Nos.7456273 and US7183395, US Patent Publication Nos. US20070048776, US20090093049, and US20110124100 and International Patent Publication Nos. WO2007025008 and WO2001055371, each of which is incorporated herein by reference in its entirety.
  • the TEEs described herein may be located in the 5’- UTR and/or the 3’-UTR of the mRNA.
  • the TEEs located in the 3’-UTR may be the same and/or different than the TEEs located in and/or described for incorporation in the 5’-UTR.
  • the 3’-UTR of the mRNA may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least Attorney Docket No.: 45817-0161WO1 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences.
  • the TEE sequences in the 3’-UTR of the polynucleotides, primary constructs, alternative nucleic acids and/or mmRNA of the present invention may be the same or different TEE sequences.
  • the TEE sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C represent a different TEE sequence at the nucleotide level.
  • the 3’-UTR may include a spacer to separate two TEE sequences. As a non-limiting example, the spacer may be a 15-nucleotide spacer and/or other spacers known in the art.
  • the 3’-UTR may include a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times and at least 9 times or more than 9 times in the 3’-UTR.
  • mRNA Heterologous 5’-UTRs 5’-UTRs of an mRNA of the invention may be homologous or heterologous to the coding region found in the mRNA. Multiple 5′ UTRs may be included in mRNA and may be the same or of different sequences.
  • any portion of the mRNA, including none, may be codon optimized and any may independently contain one or more different structural or chemical alterations, before and/or after codon optimization.
  • each 5’-UTR (5’-UTR-005 to 5’-UTR 68511) is identified by its start and stop site relative to its native or wild type (homologous) transcript (ENST; the identifier used in the ENSEMBL database).
  • 5’-UTRs which are heterologous to the coding region of the mRNA are engineered into the mRNA.
  • the mRNA e.g., an mRNA in a composition described herein
  • the mRNA is administered to cells, tissue, or organisms, and outcomes such as protein level, localization, and/or half-life are measured to evaluate the beneficial effects the heterologous 5’-UTR may have on mRNA.
  • Variants of the 5’ UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.5’-UTRs may also be codon-optimized or altered in any manner described herein.
  • mRNA RNA motifs for RNA binding proteins
  • RBPs RNA binding proteins
  • RNA-binding domains such as, but not limited to, RNA recognition motif (RR) and hnRNP K-homology (KH) domains, typically regulate the sequence association between RBPs and their RNA targets (Ray et al., Nature.499:172-177 (2013); incorporated herein by reference in its entirety).
  • the canonical RBDs can bind short RNA sequences.
  • the canonical RBDs can recognize structure RNAs.
  • an mRNA encoding HuR is co-transfected or co-injected along with the mRNA of interest into the cells or into the tissue.
  • These proteins can also be tethered to the mRNA of interest in vitro and then administered to the cells together.
  • Poly A tail binding protein, PABP interacts with eukaryotic translation initiation factor eIF4G to stimulate translational initiation.
  • Co-administration of mRNAs encoding these RBPs along with the mRNA drug and/or tethering these proteins to the mRNA drug in vitro and administering the protein-bound mRNA into the cells can increase the translational efficiency of the mRNA.
  • the same concept can be extended to co- administration of mRNA along with mRNAs encoding various translation factors and facilitators as well as with the proteins themselves to influence RNA stability and/or translational efficiency.
  • the nucleic acids and/or mRNA may include at least one RNA-binding motif such as, but not limited to an RNA-binding domain (RBD).
  • RBD RNA-binding domain
  • the RBD may be any of the RBDs, fragments or variants thereof descried by Ray et al., (Nature.499:172-177 (2013); incorporated herein by reference in its entirety).
  • the nucleic acids or mRNA of the present invention may include a sequence for at least one RNA-binding domain (RBDs).
  • the RBDs do not need to be from the same species or even the same structural class.
  • at least one flanking region e.g., the 5’-UTR and/or the 3’-UTR
  • the first flanking region and the second flanking region may both include at least one RBD.
  • the RBD may be the same or each of the RBDs may have at least 60% (e.g., at least 70%, 80%, or 90%) sequence identity to the other RBD.
  • At least on RBD may be located before, after and/or within the 3’-UTR of the nucleic acid or mRNA of the present invention.
  • at least one RBD may be located before or within the first 300 nucleosides of the 3’-UTR.
  • the nucleic acids and/or mRNA of the present invention may include at least one RBD in the first region of linked nucleosides.
  • the RBD may be located before, after, or within a coding region (e.g., the ORF).
  • the first region of linked nucleosides and/or at least one flanking region may include at least on RBD.
  • the first region of linked nucleosides may include a RBD related to splicing factors and at least one flanking region may include a RBD for stability and/or translation factors.
  • the nucleic acids and/or mRNA of the present invention may include at least one RBD located in a coding and/or non-coding region of the nucleic acids and/or mRNA.
  • Attorney Docket No.: 45817-0161WO1 at least one RBD may be incorporated into at least one flanking region to increase the stability of the nucleic acid and/or mRNA of the present invention.
  • an antisense locked nucleic acid (LNA) oligonucleotides and exon-junction complexes (EJCs) may be used in the RNA binding protein motif.
  • the LNA and EJCs may be used around a start codon (-4 to +37 where the A of the AUG codons is +1) in order to decrease the accessibility to the first start codon (AUG).
  • Nucleic acids as agents for delivering anti-HSA antibodies or binding proteins The compositions of the disclosure can be administered not only as antibodies or antigen-binding fragments, but also in the form of nucleic acids.
  • the exemplary nucleic acids described herein may be used to deliver antibodies or antigen-binding fragments to a subject.
  • nucleic acids may be used as therapeutic agents to express antibodies or antigen-binding fragments of the disclosure as a therapy to treat a target disease.
  • Pharmaceutical Compositions Pharmaceutical compositions containing an anti-HSA antibody, antigen- binding fragment, binding protein, or nucleic acid encoding the same, described herein can be prepared using methods known in the art. Pharmaceutical compositions described herein may contain an anti-HSA antibody, antigen-binding fragment, binding protein, or a nucleic acid encoding the same, described herein in combination with one or more pharmaceutically acceptable excipients.
  • compositions described herein can be prepared using physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (19th ed., 1995), incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.
  • the compositions can also be prepared so as to contain the active agent (e.g., an anti-HSA antibody, antigen-binding fragment, binding protein, or a nucleic acid encoding the same) at a desired Attorney Docket No.: 45817-0161WO1 concentration.
  • a pharmaceutical composition described herein may contain at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) active agent by weight (w/w).
  • an active agent that can be incorporated into a pharmaceutical formulation can itself have a desired level of purity.
  • a polypeptide or nucleic acid described herein may be characterized by a certain degree of purity after isolating the antibody or binding protein from cell culture media or after chemical synthesis.
  • An antibody, antigen-biding fragment, binding protein, or nucleic acid described herein may be at least 10% pure prior to incorporating the antibody, antigen-biding fragment, binding protein or nucleic acid into a pharmaceutical composition (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% pure).
  • compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the active agent having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art, e.g., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives.
  • optional pharmaceutically acceptable carriers e.g., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives.
  • Such additives must be nontoxic to the recipients at the dosages and concentrations employed. Buffering agents Buffering agents help to maintain the pH in the range which approximates physiological conditions.
  • Suitable buffering agents for use with the pharmaceutical compositions of the disclosure include both organic and inorganic acids and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid- monosodium succinate mixture, succinic acid- sodium hydroxide mixture, succinic acid- disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate Attorney Docket No.: 45817-0161WO1 mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid- disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.
  • phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can be used.
  • Preservatives can be added to a composition described herein, for example, to inhibit microbial growth. Suitable preservatives for use with the pharmaceutical compositions of the disclosure include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.
  • benzalconium halides e.g., chloride, bromide, and iodide
  • alkyl parabens such as methyl or propyl paraben,
  • Isotonifiers also known as “stabilizers,” can be added to ensure isotonicity of liquid compositions described herein and include polhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, arabitol, xylitol, sorbitol, and mannitol.
  • Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall.
  • Typical stabilizers can be polyhydric sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L- leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as Attorney Docket No.: 45817-0161WO1 urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a- monothioglycerol and sodium thio
  • non-ionic surfactants or detergents are added to the pharmaceutical composition, for example, to help solubilize the therapeutic agent as well as to protect the therapeutic agent against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein.
  • Suitable non-ionic surfactants include, for example and without limitation, polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.).
  • Alternative pharmaceutically acceptable carriers that can be incorporated into a pharmaceutical composition described herein may include dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to.
  • a pharmaceutical composition described herein may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative.
  • LNP Lipid Nanoparticle
  • the present disclosure provides LNP compositions with advantageous properties.
  • the lipid nanoparticle compositions described herein may be used for the delivery of therapeutic and/or prophylactic agents, e.g., mRNAs, to mammalian cells or organs.
  • the lipid nanoparticles described herein have little or no immunogenicity.
  • the lipid compounds disclosed herein have a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA).
  • a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent, e.g., mRNA has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.
  • the present application provides pharmaceutical compositions comprising: (a) a delivery agent comprising a lipid nanoparticle; and (b) a polynucleotide encoding an antibody or antigen-binding fragment of the disclosure.
  • a delivery agent comprising a lipid nanoparticle
  • a polynucleotide encoding an antibody or antigen-binding fragment of the disclosure are included in a lipid nanoparticle (LNP).
  • Lipid nanoparticles according to the present disclosure may comprise: (i) an ionizable lipid (e.g., an ionizable amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-modified lipid.
  • lipid nanoparticles according to the present disclosure further comprise one or more polynucleotides of the present disclosure (e.g., mRNA).
  • the lipid nanoparticles according to the present disclosure can be generated using components, compositions, and methods as are generally known in the art, see, for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117; Attorney Docket No.: 45817-0161WO1 PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety.
  • the lipid nanoparticle comprises an ionizable cationic lipid (e.g., an ionizable amino lipid) at a content of 20-60 mol.%, 25-60 mol.%, 30-60 mol.%, 35-60 mol.%, 40-60 mol.%, 45-60 mol.%, 20-55 mol.%, 25-55 mol.%, 30-55 mol.%, 35-55 mol.%, 40-55 mol.%, 45-55 mol.%, 20-50 mol.%, 25-50 mol.%, 30-50 mol.%, 35-50 mol.%, or 40-50 mol.%.
  • an ionizable cationic lipid e.g., an ionizable amino lipid
  • the lipid nanoparticle may comprise an ionizable cationic lipid (e.g., an ionizable amino lipid) at a content of 40- 50 mol.%, 45-50 mol.%, 45-46 mol.%, 46-47 mol.%, 47-48 mol.%, 48-49 mol.%, or 49-50 mol.%, for example about 45 mol.%, about 45.5 mol.%, about 46 mol.%, about 46.5 mol.%, about 47 mol.%, about 47.5 mol.%, about 48 mol.%, about 48.5 mol.%, about 49 mol.%, or about 49.5 mol.% ionizable cationic lipid (e.g., an ionizable amino lipid).
  • an ionizable cationic lipid e.g., an ionizable amino lipid
  • the lipid nanoparticle comprises a non-cationic helper lipid or phospholipid at a content of 5-25 mol.%.
  • the lipid nanoparticle may comprise a non-cationic helper lipid or phospholipid at a content of molar ratio of 5-25 mol.%, 5-20 mol.%, 5-15 mol.%, 10-25 mol.%, 10-20 mol.%, 10-15 mol.%, 5-6 mol.%, 6-7 mol.%, 7-8 mol.%, 8-9 mol.%, 9-10 mol.%, 10-11 mol.%, 11-12 mol.%, 12-13 mol.%, 13-14 mol.%, 14-15 mol.%, 10-14 mol.%, 10-13 mol.%, 10-12 mol.%, 10-11 mol.%, 9-15 mol.%, 9-14 mol.%, 9-13 mol.%, 9-12 mol.%, or 9-11 mol.% non-cationic lipid.
  • the lipid nanoparticle comprises a sterol or other structural lipid at a content molar ratio of 25-55 mol.%, 25-50 mol.%, 25-45 mol.%, 25-40 mol.%, 25-35 mol.%, 30-55 mol.%, 30-50 mol.%, 30-45 mol.%, 30-40 mol.%, 30-35 mol.%, 35-55 mol.%, 35-50 mol.%, 35-45 mol.%, 35-40 mol.%, 25-30 mol.%, 30-35 mol.%, 25-28 mol.%, 28-30 mol.%, 30-33 mol.%, 35-38 mol.%, 38-40 mol.%, 36-40 mol.%, 37-40 mol.%, 38-40 mol.%, 38-39 mol.%, 36-40 mol.%, 37-40 mol.%, 36-39 mol.%, 36-39 mol.%, or 37-39 mol.%.
  • the lipid nanoparticle may comprise a Attorney Docket No.: 45817-0161WO1 sterol or other structural lipid at a content of about 30 mol.%, about 30.5 mol.%, about 31.0 mol.%, about 31.5 mol.%, about 32.0 mol.%, about 32.5 mol.%, about 33.0 mol.%, about 33.5 mol.%, about 34.0 mol.%, about 34.5 mol.%, about 35.0 mol.%, about 35.5 mol.%, about 36.0 mol.%, about 36.5 mol.%, about 37.0 mol.%, about 37.5 mol.%, about 38.0 mol.%, about 38.5 mol.%, about 39.0 mol.%, about 39.5 mol.%, about 40.0 mol.%, about 40.5 mol.%, about 41.0 mol.%, about 41.5 mol.%, about 42.0 mol.%, about 42.5 mol.%, about 43.0 mol.%
  • the lipid nanoparticle comprises a PEG-modified lipid at a content of 0.5-15 mol.%, 1.0-15 mol.%, 1.5-15 mol.%, 2.0-15 mol.%, 2.5-15 mol.%, 3.0-15 mol.%, 3.5-15 mol.%, 4.0-15 mol.%, 4.5-15 mol.%, 5.0-15 mol.%, 10- 15 mol.%, 0.5-10 mol.%, 0.5-5 mol.%, 0.5-4.5 mol.%, 0.5-4.0 mol.%, 0.5-3.5 mol.%, 0.5-3.0 mol.%, 0.5-2.5 mol.%, 0.5-2.0 mol.%, 0.5-1.5 mol.%, 0.5-1.0 mol.%, 1.0-10 mol.%, 1.0-5 mol.%, 1.0-4.5 mol.%, 1.0-4.0 mol.%, 1.0-3.5 mol.%, 1.0-3.0 mol.%, 1.0-
  • the lipid nanoparticle may comprise a PEG-modified lipid at a content of a about 0.5 mol.%, about 1.0 mol.%, about 1.5 mol.%, about 2.0 mol.%, about 2.5 mol.%, about 3.0 mol.%, about 3.5 mol.%, about 4.0 mol.%, about 4.5 mol.%, about 5.0 mol.%, about 6.0 mol.%, about 7.0 mol.%, about 8.0 mol.%, about 9.0 mol.%, about 10.0 mol.%, or about 15.0 mol.%.
  • the lipid nanoparticle comprises: (i) 20 to 60 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 25 to 55 mol.% sterol or other structural lipid, (iii) 5 to 25 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 0.5 to 15 mol.% PEG-modified lipid.
  • ionizable cationic lipid e.g., ionizable amino lipid
  • sterol or other structural lipid e.g., sterol or other structural lipid
  • non-cationic lipid e.g., phospholipid
  • iv 0.5 to 15 mol.% PEG-modified lipid.
  • the lipid nanoparticle comprises: (i) 40 to 50 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 30 to 45 mol.% sterol or other Attorney Docket No.: 45817-0161WO1 structural lipid, (iii) 5 to 15 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 1 to 5 mol.% PEG-modified lipid.
  • ionizable cationic lipid e.g., ionizable amino lipid
  • sterol or other Attorney Docket No.: 45817-0161WO1 structural lipid e.g., 5 to 15 mol.% non-cationic lipid (e.g., phospholipid)
  • iv 1 to 5 mol.% PEG-modified lipid.
  • the lipid nanoparticle comprises: (i) 45 to 50 mol.% ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 35 to 45 mol.% sterol or other structural lipid, (iii) 8 to 12 mol.% non-cationic lipid (e.g., phospholipid), and (iv) 1.5 to 3.5 mol.% PEG-modified lipid.
  • “Compounds” numbered with an “I-” prefix e.g., “Compound I-1,” “Compound I-2,” “Compound I-3,” “Compound I-VI,” etc., indicate specific ionizable lipid compounds.
  • the lipid nanoparticle of the present disclosure comprises an ionizable cationic lipid (e.g., an ionizable amino lipid) that is a compound of Formula (I): or its N-oxide, or a salt or isomer thereof, R’ branched denotes a point of attachment; wherein selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl; Attorney Docket No.: 45817-0161WO1 R 4 is selected from the group consisting of -(CH2)nOH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein denotes a point R
  • R’ a is R’ branched ; R’ branched is a point of attachment; R a ⁇ , R a ⁇ , R a ⁇ , and R a ⁇ are each R 4 is -(CH ) OH; n is 2; each R 5 is H 6 2 n ; each R is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; l is 5; and m is 7.
  • R’ a is R’ branched ;
  • R’ branched is denotes a point of attachment;
  • R a ⁇ , R a ⁇ , R a ⁇ , and R a ⁇ are C1-14 alkyl;
  • R 4 is -(CH2)nOH; n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M’ are each -C(O)O-;
  • R’ is a C 1-12 alkyl; l is 3; and m is 7.
  • R’ a is R’ branched ; R’ branched ; denotes a point of attachment; R a ⁇ is C 2-12 alkyl; R a ⁇ , R a ⁇ , alkyl; R 4 is ; R 10 is NH(C1-6 alkyl); n2 is 2; R 5 is H; each R 6 is H; M and M’ is a C 1-12 alkyl; l is 5; and m is 7.
  • R’ a is R’ branched ; R’ branched ; denotes a point of attachment; R a ⁇ , R a ⁇ , nd R a ⁇ a are each 12 14 alkyl; R 4 is -(CH 2 ) n OH; n is 2; each R 5 is H; each R 6 is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; l is 5; and m is 7.
  • the compound of Formula (I) is selected from: (Compound I-1), , (Compound I-2), and (Compound I-3).
  • the compound of Formula (I) is: (Compound I-1). In (I) is: (Compound I-2). In (I) is: (Compound I-3).
  • the disclosure relates to a compound of Formula (Ia): or its N-oxide, or a salt or isomer thereof, denotes a point of attachment; selected from the group consisting of H, C 2-12 alkyl, and C 2-12 alkenyl; Attorney Docket No.: 45817-0161WO1 R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein denotes a point R 10 is N(R) 2 ; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H;
  • the disclosure relates to a compound of Formula (Ib): or its N-oxide, or a salt or isomer thereof, R’ branched is: ; wherein denotes a point of attachment; Attorney Docket No.: 45817-0161WO1 wherein R a ⁇ , R a ⁇ , R a ⁇ , and R a ⁇ are each independently selected from the group consisting of H, C 2-12 alkyl, and C 2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; R 4 is -(CH2)nOH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; each R 5 is independently selected from the group consisting of C1-3 alkyl, C 2-3 alkenyl, and H; each R 6 is independently selected from the group consisting of C1-3 alkyl, C 2-3 alkenyl, and H; M and M’ are each independently selected from the group consisting of -
  • R’ a is R’ branched ; R’ branched is ; denotes a point of attachment; R a ⁇ , R a ⁇ , and R a ⁇ are each H; R 2 C 1-14 alkyl; R 4 is -(CH 2 ) n OH; n is 2; each R 5 is H; each R 6 is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; l is 5; and m is 7.
  • R’ a is R’ branched ;
  • R’ branched is denotes a point of attachment;
  • R a ⁇ and R a ⁇ are each H;
  • R a ⁇ is R 3 are each C 1-14 alkyl;
  • R 4 is -(CH 2 ) n OH; n is 2;
  • each R 5 is H;
  • each R 6 is H;
  • M and M’ are each -C(O)O-;
  • R’ is a C1-12 alkyl; l is 5; and m is 7.
  • the disclosure relates to a compound of Formula (Ic): Attorney Docket No.: 45817-0161WO1 or its N-oxide, or a salt or isomer thereof, R’ branched denotes a point of attachment; wherein selected from the group consisting of H, C 2-12 alkyl, and C 2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; , a point of attachment; wherein R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R 5 is independently selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H; each R 6 is independently selected from the group consisting of C 1-3 alkyl, C2-3 alkenyl, and H; M and M’ are each independently selected from the group consisting
  • R a ⁇ and R a ⁇ are each independently selected from the group consisting of H, C 1-12 alkyl, and C 2-12 alkenyl, wherein at least one of R a ⁇ and R a ⁇ is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;
  • R b ⁇ and R b ⁇ are each independently selected from the group consisting of H, C1-12 alkyl, and C2-12 alkenyl, wherein at least one of R b ⁇ and R b ⁇ is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;
  • R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl;
  • R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from
  • the disclosure relates to a compound of Formula (II-a): (II-a) or its N-oxide, or a salt or isomer thereof, Attorney Docket No.: 45817-0161WO1 wherein R’ a is R’ branched or R’ cyclic ; wherein R’ branched ; wherein R a ⁇ and each independently selected from the group consisting of H, C 1-12 alkyl, and C 2-12 alkenyl, wherein at least one of R a ⁇ and R a ⁇ is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; R b ⁇ and R b ⁇ are each independently selected from the group consisting of H, C1-12 alkyl, and C2-12 alkenyl, wherein at least one of R b ⁇ and R b ⁇ is selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl
  • the disclosure relates to a compound of Formula (II-b): Attorney Docket No.: 45817-0161WO1 ; R a ⁇ and each independently selected from the group consisting of C1-12 alkyl and C 2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; R 4 is selected from the group consisting of -(CH 2 ) n OH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein denotes a point R 10 is N each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a C 1-12 alkyl or C 2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the disclosure relates to a compound of Formula (II-c): Attorney Docket No.: 45817-0161WO1 wherein selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl; R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein denotes a point R 10 is N(R)2; each R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R’ is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the disclosure relates to a compound of Formula (II-d): Attorney Docket No.: 45817-0161WO1 wherein R a ⁇ and R b ⁇ are each independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl; R 4 is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, , wherein denotes a point R 10 is N(R)2; each R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a C 1-12 alkyl or C 2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the disclosure relates to a compound of Formula (II-e): (II-e) or its N-oxide, or a salt or isomer thereof, wherein R’ a is R’ branched or R’ cyclic ; wherein Attorney Docket No.: 45817-0161WO1 ; wherein selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; R 2 and R 3 are each independently selected from the group consisting of C1-14 alkyl and C 2-14 alkenyl; R 4 is -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; R’ is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • m and l are each independently selected from 4, 5, and 6. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each 5. In some embodiments of the compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), each R’ independently is a C1-12 alkyl.
  • each R’ independently is a C2-5 alkyl.
  • R’ b and R 2 and R 3 are each independently a C1-14 alkyl.
  • R’ b is: and R 2 and R 3 are each independently a C 6-10 alkyl.
  • R’ b is: and R 2 and R 3 are each a C8 alkyl.
  • R’ branched is: , R a ⁇ is a C1-12 alkyl and R 2 and R 3 are of the compound of Formula (II), , , , , or , R 3 are each Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R’ branched is: , R a ⁇ is a C2-6 alkyl, and R 2 and R 3 are each a C8 alkyl.
  • R’ branched R b ⁇ are each a C1-12 alkyl.
  • a), (II-b), (II-c), (II-d), or (II-e), R’ branched is: are each a C2-6 alkyl.
  • some compound of Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), m and l are each independently selected from 4, 5, and 6 and each R’ independently is a C 1-12 alkyl.
  • m and l are each 5 and each R’ independently is a C 2-5 alkyl.
  • R’ branched l are each independently and R a ⁇ and R b ⁇ are each a 1 2 Formula (II), (II-a), (II-b), (II-c), (II-d), or (II-e), R’ branched is: , m and l are each 5, each R’ independently is a C 2-5 alkyl, and R a ⁇ and R b ⁇ are each a C2-6 alkyl.
  • R’ branched l are each independently selected 2 and R and R 3 are each independently a C6-10 alkyl.
  • R’ branched and R’ b m and l are each 5, R’ is a C2-5 alkyl, R a ⁇ is a C2-6 alkyl, and R 2 and R 3 are alkyl.
  • R 10 is NH(C1-6 alkyl) and n2 is 2.
  • R 4 is , wherein R 10 is NH(CH 3 ) and n2 is 2.
  • R’ branched l are each independently R a ⁇ and R b ⁇ are each a C1-12 alkyl, and R 4 , wherein R 10 is NH(C 1-6 alkyl), and n2 is 2.
  • R 10 is NH(CH3) and n2 is 2.
  • R’ branched l are each independently selected each independently a C6-10 alkyl, R a ⁇ is a C1-12 alkyl, and R 4 , wherein R 10 is NH(C 1-6 alkyl) and n2 is 2.
  • R’ branched R’ b is: , m and l are each 5, R’ is a C2-5 alkyl, R a ⁇ is a C2-6 alkyl, R 2 and R 3 Attorney Docket No.: 45817-0161WO1 is (II-d), or (II-e), R 4 is -(CH2)nOH and n is 2, 3, or 4.
  • R 4 is -(CH 2 ) n OH and n is 2.
  • R branched l are each independently R a ⁇ and R b ⁇ are each a C1-12 alkyl, R 4 is -(CH2)nOH, and n is 2, 3, or 4.
  • R’ n is 2.
  • the disclosure relates to a compound of Formula (II-f): R’ branched is: and R’ b is: ; Attorney Docket No.: 45817-0161WO1 wherein denotes a point of attachment; R a ⁇ is a alkyl; 2 R and are each independently a C1-14 alkyl; R 4 is -(CH 2 ) n OH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; R’ is a C 1-12 alkyl; m is selected from 4, 5, and 6; and l is selected from 4, 5, and 6.
  • m and l are each 5, and n is 2, 3, or 4.
  • R’ is a C2-5 alkyl, Ra ⁇ is a C2-6 alkyl, and R2 and R3 are each a C6-10 alkyl.
  • m and l are each 5, n is 2, 3, or 4, R’ is a C2-5 alkyl, Ra ⁇ is a C2-6 alkyl, and R2 and R3 are each a C6-10 alkyl.
  • the disclosure relates to a compound of Formula (II-g): , wherein R’ is a C2-5 alkyl; and R 4 is selected from the group consisting of -(CH 2 ) n OH wherein n is selected from the group consisting of 3, 4, and 5, , wherein denotes a point of attachment, R 10 is NH(C 1-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3.
  • the disclosure relates to a compound of Formula (II-h): 5
  • R 4 is selected from the group consisting of -(CH 2 ) n OH wherein n is selected from the group consisting of 3, 4, and 5, , wherein denotes a point of , and n2 is selected from consisting of 1, 2, and 3.
  • R 4 is , wherein n2 is 2.
  • R 4 is - (CH 2 ) 2 OH.
  • the disclosure relates to a compound having the Formula (III): , or a salt or Attorney Docket No.: 45817-0161WO1 R1, R2, R3, R4, and R5 are independently selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, -R”MR’, -R*YR”, -YR”, and -R*OR”; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, an aryl group, and a heteroaryl group; X 1 , X 2 , and X 3
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each C 5-20 alkyl; X 1 is -CH 2 -; and X 2 and X 3 are each -C(O)-.
  • the compound of Formula (III) is: (Compound I-VI), or a salt or isomer thereof.
  • Attorney Docket No.: 45817-0161WO1 Phospholipids The lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Particular phospholipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid- containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • elements e.g., a therapeutic agent
  • a lipid- containing composition e.g., LNPs
  • Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a Attorney Docket No.: 45817-0161WO1 nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).
  • a targeting or imaging moiety e.g., a dye
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.
  • a phospholipid of the present disclosure comprises 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine
  • a phospholipid useful or potentially useful in the present disclosure is an analog or variant of DSPC.
  • a phospholipid useful or potentially useful in the present disclosure is a compound of Formula (IV): Attorney Docket No.: 45817-0161WO1 or a salt thereof, wherein: each R 1 is independently optionally substituted alkyl; or optionally two R 1 are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R 1 are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is of the ; each instance of substituted C1-6 alkylene, wherein one methylene unit of the optionally substituted C 1-6 alkylene is optionally replaced with O, N(R N)
  • the phospholipids may be one or more of the phospholipids described in U.S. Application No.62/520,530.
  • Phospholipid Head Modifications In certain embodiments, a phospholipid useful or potentially useful in the present disclosure comprises a modified phospholipid head (e.g., a modified choline group). In certain embodiments, a phospholipid with a modified head is DSPC, or analog thereof, with a modified quaternary amine.
  • at least one of R 1 is not methyl. In certain embodiments, at least one of R 1 is not hydrogen or methyl.
  • the compound of Formula (IV) is of one of the following Formulae: , each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Attorney Docket No.: 45817-0161WO1 each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each v is independently 1, 2, or 3.
  • a compound of Formula (IV) is of Formula (IV-a): or a salt thereof.
  • a phospholipid useful or potentially useful in the present disclosure comprises a cyclic moiety in place of the glyceride moiety.
  • a phospholipid useful in the present disclosure is DSPC, or analog thereof, with a cyclic moiety in place of the glyceride moiety.
  • a phospholipid useful or potentially useful in the present disclosure comprises a modified tail.
  • a phospholipid useful or potentially useful in the present disclosure is DSPC, or analog thereof, with a modified tail.
  • a “modified tail” may be a tail with shorter or longer aliphatic chains, aliphatic chains with branching introduced, aliphatic chains with substituents introduced, aliphatic chains wherein one or more methylenes are replaced by cyclic or heteroatom groups, or any combination thereof.
  • the compound of (IV) is of Formula (IV-a), or a salt thereof, wherein at least one instance of R 2 is each instance of R 2 is optionally substituted C 1- Attorney Docket No.: 45817-0161WO1 30 alkyl, wherein one or more methylene units of R 2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, - C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, - OC N , NR N C O, C S, SC , C , C N , NR N C , c): (IV-c), or a salt thereof, wherein: each x is independently an integer between 0-30, inclusive; and each instance is G is independently
  • a phospholipid useful or potentially useful in the present disclosure comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid useful or potentially useful Attorney Docket No.: 45817-0161WO1 in the present disclosure is a compound of Formula (IV), wherein n is 1, 3, 4, 5, 6, 7, 8, 9, or 10.
  • a compound of Formula (IV) is of one of the following Formulae: , or a salt Alternative Lipids
  • a phospholipid useful or potentially useful in the present disclosure comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid useful.
  • an alternative lipid is used in place of a phospholipid of the present disclosure.
  • an alternative lipid of the present disclosure is oleic acid.
  • the alternative lipid is one of the following: , , Attorney Docket No.: 45817-0161WO1 ,
  • the lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids.
  • structural lipid refers to sterols and also to lipids containing sterol moieties. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle.
  • Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha- Attorney Docket No.: 45817-0161WO1 tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
  • the structural lipid is a sterol.
  • sterols are a subgroup of steroids consisting of steroid alcohols.
  • the structural lipid is a steroid.
  • the structural lipid is cholesterol.
  • the structural lipid is an analog of cholesterol.
  • the structural lipid is alpha-tocopherol. In some embodiments, the structural lipids may be one or more of the structural lipids described in U.S. Application No.62/520,530.
  • Polyethylene Glycol (PEG)-Lipids The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more a polyethylene glycol (PEG) lipid. As used herein, the term “PEG-lipid” refers to polyethylene glycol (PEG)- modified lipids.
  • Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3-amines.
  • PEGylated lipids are also referred to as PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-lipid includes, but not limited to 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG- disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG- DPPE), or PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).
  • PEG-DMG 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol
  • PEG-DSPE 1,2-distearoyl-sn- g
  • the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the lipid moiety of the PEG-lipids includes those having lengths of from about C 14 to about C 22 , preferably from about C 14 to about C 16 .
  • a PEG moiety for example an mPEG-NH2 has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.
  • the PEG- lipid is PEG2k-DMG.
  • the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG.
  • Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.
  • PEG-lipids are known in the art, such as those described in U.S. Patent No. 8,158,601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety.
  • lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids.
  • a PEG lipid is a lipid modified with polyethylene glycol.
  • a PEG lipid may be selected from the non- limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG- modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.
  • a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-modified lipids are a modified form of PEG DMG.
  • PEG-DMG has the following structure: Attorney Docket No.: 45817-0161WO1
  • any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain.
  • the PEG lipid is a PEG-OH lipid.
  • a “PEG-OH lipid” (also referred to herein as “hydroxy- PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (–OH) groups on the lipid.
  • the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain.
  • a PEG-OH or hydroxy-PEGylated lipid comprises an —OH group at the terminus of the PEG chain.
  • a PEG lipid useful in the present disclosure is a compound of Formula (V).
  • R 3 is –OR O ;
  • R O is hydrogen, optionally substituted alkyl, or an oxygen protecting group;
  • r is an integer between 1 and 100, inclusive;
  • L 1 is optionally substituted C1-10 alkylene, wherein at least one methylene of the optionally substituted C 1-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R N ), S, C(O), - C(O)N(R N ), NR N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), NR N C(O)O, or - physiological conditions;
  • Attorney Docket No.: 45817-0161WO1 m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • A is of the ; each instance of L 2 substitute
  • the compound of Fomula (V) is a PEG-OH lipid (i.e., R 3 is –OR O , and R O is hydrogen).
  • the compound of Formula (V) is of Formula (V-OH): (V-OH), or a salt thereof.
  • Attorney Docket No.: 45817-0161WO1 a PEG lipid useful in the present disclosure is a PEGylated fatty acid.
  • a PEG lipid useful in the present disclosure is a compound of Formula (VI).
  • R 3 is–OR O ;
  • R O is hydrogen, optionally substituted alkyl or an oxygen protecting group;
  • r is an integer between 1 and 100, inclusive;
  • R 5 is optionally substituted C10-40 alkyl, optionally substituted C10-40 alkenyl, or optionally substituted C 10-40 alkynyl; and optionally one or more methylene groups of R 5 are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), - , - , - or a nitrogen protecting group.
  • the compound of Formula (VI) is of Formula (VI- OH): (VI-OH), or a salt thereof.
  • the compound of Formula (VI) is: Attorney Docket No.: 45817-0161WO1 . or a compositions disclosed herein does not comprise a PEG-lipid.
  • the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No.62/520,530.
  • a PEG lipid of the present disclosure comprises a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
  • the PEG- modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG- DSG and/or PEG-DPG.
  • a LNP of the present disclosure comprises an ionizable cationic lipid of any of Formula I, II or III, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG. In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of any of Formula I, II or III, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the present disclosure comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid comprising a compound having Formula IV, a structural lipid, and the PEG lipid comprising a compound having Formula V or VI.
  • Attorney Docket No.: 45817-0161WO1 In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid comprising a compound having Formula IV, a structural lipid, and the PEG lipid comprising a compound having Formula V or VI.
  • a LNP of the present disclosure comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid having Formula IV, a structural lipid, and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the present disclosure comprises an ionizable cationic lipid of , and a In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of , In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of , Attorney Docket No.: 45817-0161WO1 an alternative lipid comprising oleic acid, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI. In some embodiments, a LNP of the present disclosure comprises an ionizable cationic lipid of and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the present disclosure comprises an ionizable cationic lipid of a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI.
  • a LNP of the present disclosure comprises an N:P ratio of from about 2:1 to about 30:1.
  • a LNP of the present disclosure comprises an N:P ratio of about 6:1.
  • a LNP of the present disclosure comprises an N:P ratio of about 3:1.
  • a LNP of the present disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of from about 10:1 to about 100:1. In some embodiments, a LNP of the present disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 20:1. In some embodiments, a LNP of the present disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 10:1. In some embodiments, a LNP of the present disclosure has a mean diameter from about 50nm to about 150nm.
  • a LNP of the present disclosure has a mean diameter from about 70nm to about 120nm.
  • Other Lipid Composition Components The lipid composition of a pharmaceutical composition disclosed herein can include one or more components in addition to those described above.
  • the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants), or other components.
  • a permeability enhancer molecule can be a molecule described by U.S. Patent Application Publication No.2005/0222064.
  • Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).
  • a polymer can be included in and/or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form).
  • a polymer can be biodegradable and/or biocompatible.
  • a polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, Attorney Docket No.: 45817-0161WO1 polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • the ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt/wt). In some embodiments, the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt).
  • the wt/wt ratio of the lipid composition to the polynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.
  • the pharmaceutical composition disclosed herein can contain more than one polypeptides.
  • a pharmaceutical composition disclosed herein can contain two or more polynucleotides (e.g., RNA, e.g., mRNA).
  • the lipid nanoparticles described herein can comprise polynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, but not limited to, 5:1 to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about 20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, from about 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 to about 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 to about 25
  • the lipid nanoparticles described herein can comprise the polynucleotide in a concentration from approximately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.
  • Nanoparticle Compositions are Formulated as lipid nanoparticles (LNP). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as compound as described herein, and (ii) a polynucleotide encoding a polypeptide. In such nanoparticle composition, the lipid composition disclosed herein can encapsulate the polynucleotide encoding a polypeptide. Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer.
  • Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes.
  • a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.
  • Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes.
  • nanoparticle compositions are vesicles including one or more lipid bilayers.
  • a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and/or crosslinked to one another.
  • Lipid bilayers can include one or more ligands, proteins, or channels.
  • a lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and mRNA.
  • the LNP Attorney Docket No.: 45817-0161WO1 comprises an ionizable amino lipid, a PEG-modified lipid, a sterol and a structural lipid.
  • the LNP has a molar ratio of about 40-50% ionizable amino lipid; about 5-15% structural lipid; about 30-45% sterol; and about 1-5% PEG- modified lipid.
  • the lipid nanoparticle comprises 47-49 mol.% ionizable cationic lipid (e.g. ionizable amino lipid, e.g., Compound I-1, Compound I-2, or Compound I-3), 10-12 mol.% non-cationic lipid (e.g., phospholipid, e.g., DSPC), 38- 40 mol.% sterol (e.g., cholesterol) or other structural lipid, and 1-3 mol.% PEG- modified lipid (e.g., PEG-DMG or Compound P-I).
  • ionizable cationic lipid e.g. ionizable amino lipid, e.g., Compound I-1, Compound I-2, or Compound I-3
  • 10-12 mol.% non-cationic lipid e.g., phospholipid, e.g., DSPC
  • 38- 40 mol.% sterol e.g., cholesterol
  • PEG- modified lipid
  • the lipid nanoparticle (“LNP-1”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-1 (ii) 35-45 mol.% sterol (e.g., cholesterol); (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and (iv) 1.5-3.5 mol.% PEG-lipid (e.g., Compound P-I or PEG-DMG).
  • the lipid nanoparticle (“LNP-1A”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-1 (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-DMG.
  • the lipid nanoparticle (“LNP-1B”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-1 (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% Compound P-I.
  • the lipid nanoparticle (“LNP-2”) may comprise the following: (i) 45-50 mol.% Compound I-2; (ii) 35-45 mol.% sterol (e.g., Cholesterol); (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and (iv) 1.5-3.5 mol.% PEG-lipid (e.g., Compound P-I or PEG-DMG).
  • the lipid nanoparticle (“LNP-2A”) may comprise the following: (i) 45-50 mol.% Compound I-2; (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-DMG.
  • the lipid nanoparticle (“LNP-2B”) may comprise the following components at the following molar ratios: (i) 45-50 mol.% Compound I-2; (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% Compound P-I.
  • the lipid nanoparticle (“LNP-3”) may comprise the following: (i) 45-50 mol.% Compound I-3; (ii) 35-45 mol.% sterol (e.g., Cholesterol); (iii) 8-12 mol.% phospholipid (e.g., DSPC or DOPE); and (iv) 1.5-3.5 mol.% PEG-lipid (e.g., Compound P-I or PEG-DMG).
  • sterol e.g., Cholesterol
  • 8-12 mol.% phospholipid e.g., DSPC or DOPE
  • PEG-lipid e.g., Compound P-I or PEG-DMG
  • the lipid nanoparticle (“LNP-3A”) may comprise the following: (i) 45-50 mol.% Compound I-3; Attorney Docket No.: 45817-0161WO1 (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% PEG-DMG.
  • the lipid nanoparticle (“LNP-3B”) may comprise the following: (i) 45-50 mol.% Compound I-3; (ii) 35-45 mol.% Cholesterol; (iii) 8-12 mol.% DSPC; and (iv) 1.5-3.5 mol.% Compound P-I.
  • the LNP has a polydispersity value of less than 0.4. In some embodiments, the LNP has a net neutral charge at a neutral pH. In some embodiments, the LNP has a mean diameter of 50-150 nm. In some embodiments, the LNP has a mean diameter of 80-100 nm.
  • the term “lipid” refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic.
  • lipids examples include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids.
  • the amphiphilic properties of some lipids lead them to form liposomes, vesicles, or membranes in aqueous media.
  • a lipid nanoparticle may comprise an ionizable amino lipid.
  • the term “ionizable amino lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties.
  • an ionizable amino lipid may be positively charged or negatively charged.
  • An ionizable amino lipid may be positively charged, in which case it can be referred to as “cationic lipid”.
  • an ionizable amino lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipid.
  • a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or - Attorney Docket No.: 45817-0161WO1 3), etc.
  • the charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged).
  • positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups.
  • the charged moieties comprise amine groups.
  • negatively- charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like.
  • the charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired. It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge” or “partial positive charge” on a molecule. The terms “partial negative charge” and “partial positive charge” are given their ordinary meaning in the art. A “partial negative charge” may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom.
  • the ionizable amino lipid is sometimes referred to in the art as an “ionizable cationic lipid”.
  • the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure.
  • an ionizable amino lipid may also be a lipid including a cyclic amine group.
  • the ionizable amino lipid may be selected from, but not limited to, an ionizable amino lipid described in International Publication Nos.
  • the ionizable amino lipid may be selected from, but not limited to, Formula CLI-CLXXXII of US Patent No.7,404,969; each of which is herein incorporated by reference in their entirety.
  • the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety.
  • the lipid may be synthesized by methods known in the art and/or as described in International Publication Nos.
  • Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light scattering can also be utilized to determine particle sizes.
  • microscopy e.g., transmission electron microscopy or scanning electron microscopy
  • Dynamic light scattering or potentiometry e.g., potentiometric titrations
  • Dynamic light scattering can also be utilized to determine particle sizes.
  • Nanoparticle compositions such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.
  • the size of the nanoparticles can help counter biological reactions such as, but not limited to, inflammation, or can increase the biological effect of the polynucleotide.
  • size or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.
  • the polynucleotide encoding a polypeptide are formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 Attorney Docket No.: 45817-0161WO1 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm,
  • the nanoparticles have a diameter from about 10 to 500 nm. In some embodiments, the nanoparticle has a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.
  • the largest dimension of a nanoparticle composition is 1 ⁇ m or shorter (e.g., 1 ⁇ m, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter).
  • a nanoparticle composition can be relatively homogenous.
  • a polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition.
  • a small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution.
  • a nanoparticle composition can have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25.
  • the polydispersity index of a nanoparticle composition disclosed herein can be from about 0.10 to about 0.20.
  • the zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition.
  • the zeta potential can describe the surface charge of a nanoparticle composition.
  • Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species can interact undesirably with cells, tissues, and other elements in the body.
  • the zeta potential of a nanoparticle composition disclosed herein can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about 10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about 0 mV to about +20
  • the zeta potential of the lipid nanoparticles can be from about 0 mV to about 100 mV, from about 0 mV to about 90 mV, from about 0 mV to about 80 mV, from about 0 mV to about 70 mV, from about 0 mV to about 60 mV, from about 0 mV to about 50 mV, from about 0 mV to about 40 mV, from about 0 mV to about 30 mV, from about 0 mV to about 20 mV, from about 0 mV to about 10 mV, from about 10 mV to about 100 mV, from about 10 mV to about 90 mV, from about 10 mV to about 80 mV, from about 10 mV to about 70 mV, from about 10 mV to about 60 mV, from about 10 mV to about 50 mV, from about 10 mV to about 40 mV, from about 10
  • the zeta potential of the lipid nanoparticles can be from about 10 mV to about 50 mV, from about 15 mV to about 45 mV, from about 20 mV to about 40 mV, and from about 25 mV to about 35 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be about 10 mV, about 20 mV, about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about 80 mV, about 90 mV, and about 100 mV.
  • encapsulation efficiency of a polynucleotide describes the amount of the polynucleotide that is encapsulated by or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided.
  • encapsulation can refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement. Encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of the polynucleotide in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free polynucleotide in a solution.
  • the encapsulation efficiency of a polynucleotide can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.
  • the amount of a polynucleotide present in a pharmaceutical composition disclosed herein can depend on multiple factors such as the size of the polynucleotide, Attorney Docket No.: 45817-0161WO1 desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the polynucleotide.
  • the amount of an mRNA useful in a nanoparticle composition can depend on the size (expressed as length, or molecular mass), sequence, and other characteristics of the mRNA.
  • the relative amounts of a polynucleotide in a nanoparticle composition can also vary.
  • the relative amounts of the lipid composition and the polynucleotide present in a lipid nanoparticle composition of the present disclosure can be optimized according to considerations of efficacy and tolerability.
  • the N:P ratio can serve as a useful metric.
  • the N:P ratio of a nanoparticle composition controls both expression and tolerability, nanoparticle compositions with low N:P ratios and strong expression are desirable.
  • N:P ratios vary according to the ratio of lipids to RNA in a nanoparticle composition. In general, a lower N:P ratio is preferred.
  • the one or more RNA, lipids, and amounts thereof can be selected to provide an N:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1.
  • the N:P ratio can be from about 2:1 to about 8:1.
  • the N:P ratio is from about 5:1 to about 8:1.
  • the N:P ratio is between 5:1 and 6:1.
  • the N:P ratio is about is about 5.67:1.
  • the present disclosure also provides methods of producing lipid nanoparticles comprising encapsulating a polynucleotide.
  • Such method comprises using any of the pharmaceutical compositions disclosed herein and producing lipid nanoparticles in accordance with methods of production of lipid nanoparticles known in the art. See, e.g., Wang et al., Adv. Drug Deliv. Rev.87:68-80 (2015); Silva et al., Curr. Pharm. Technol.16: 940-954 (2015); Attorney Docket No.: 45817-0161WO1 Naseri et al. Adv. Pharm. Bull.5:305-13 (2015); Silva et al. Curr. Pharm. Biotechnol.
  • the LNP formulations described herein can additionally comprise a permeability enhancer molecule.
  • Non-limiting permeability enhancer molecules are described in U.S. Pub. No. US20050222064, herein incorporated by reference in its entirety.
  • the LNP formulations can further contain a phosphate conjugate.
  • the phosphate conjugate can increase in vivo circulation times and/or increase the targeted delivery of the nanoparticle.
  • Phosphate conjugates can be made by the methods described in, e.g., Intl. Pub. No. WO2013033438 or U.S. Pub. No. US20130196948.
  • the LNP formulation can also contain a polymer conjugate (e.g., a water-soluble conjugate) as described in, e.g., U.S. Pub. Nos. US20130059360, US20130196948, and US20130072709.
  • a polymer conjugate e.g., a water-soluble conjugate
  • the LNP formulations can comprise a conjugate to enhance the delivery of nanoparticles of the present disclosure in a subject. Further, the conjugate can inhibit phagocytic clearance of the nanoparticles in a subject.
  • the conjugate can be a "self" peptide designed from the human membrane protein CD47 (e.g., the "self” particles described by Rodriguez et al, Science.339:971-975 (2013), herein incorporated by reference in its entirety).
  • the self peptides delayed macrophage-mediated clearance of nanoparticles which enhanced delivery of the nanoparticles.
  • the LNP formulations can comprise a carbohydrate carrier.
  • the carbohydrate carrier can include, but is not limited to, an anhydride- modified phytoglycogen or glycogen-type material, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin (e.g., Intl. Pub. No. WO2012109121, herein incorporated by reference in its entirety).
  • anhydride-modified phytoglycogen beta-dextrin e.g., Intl. Pub. No. WO2012109121, herein incorporated by reference in its entirety.
  • the LNP formulations can be coated with a surfactant or polymer to improve the delivery of the particle.
  • the LNP can be coated with a hydrophilic coating such as, but not limited to, PEG coatings and/or coatings that have a neutral surface charge as described in U.S. Pub. No. US20130183244, herein incorporated by reference in its entirety.
  • the LNP formulations can be engineered to alter the surface properties of particles so that the lipid nanoparticles can penetrate the mucosal barrier as described in U.S. Pat. No.8,241,670 or Intl. Pub. No. WO2013110028, each of which is herein incorporated by reference in its entirety.
  • the LNP engineered to penetrate mucus can comprise a polymeric material (i.e., a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co- polymer.
  • the polymeric material can include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • LNP engineered to penetrate mucus can also include surface altering agents such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin ⁇ 4 do
  • the mucus penetrating LNP can be a hypotonic formulation comprising a mucosal penetration enhancing coating.
  • the formulation can be hypotonic for the epithelium to which it is being delivered.
  • Non-limiting Attorney Docket No.: 45817-0161WO1 examples of hypotonic formulations can be found in, e.g., Intl. Pub. No. WO2013110028, herein incorporated by reference in its entirety.
  • the polynucleotide described herein is Formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids (Aleku et al., Cancer Res.68:9788-9798 (2008); Strumberg et al., Int. J. Clin. Pharmacol.
  • a lipoplex such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids (Aleku
  • the polynucleotides described herein are Formulated as a solid lipid nanoparticle (SLN), which can be spherical with an average diameter between 10 to 1000 nm.
  • SSN solid lipid nanoparticle
  • SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactants and/or emulsifiers.
  • Exemplary SLN can be those as described in Intl. Pub. No. WO2013105101, herein incorporated by reference in its entirety.
  • the polynucleotides described herein can be Formulated for controlled release and/or targeted delivery.
  • controlled release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
  • the polynucleotides can be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery.
  • encapsulate means to enclose, surround or encase. As it relates to the formulation of the compounds of the present disclosure, encapsulation can be substantial, complete or partial. The term “substantially encapsulated” means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or greater than 99% of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or encased within the delivery agent.
  • Partial encapsulation or “partially encapsulate” means that less than 10, 10, 20, 30, 4050 or less of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or encased within the delivery agent.
  • encapsulation can be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the present disclosure using fluorescence and/or electron micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, or greater than 99% of the pharmaceutical composition or compound of the present disclosure are encapsulated in the delivery agent.
  • the polynucleotides described herein can be encapsulated in a therapeutic nanoparticle, referred to herein as "therapeutic nanoparticle polynucleotides.”
  • Therapeutic nanoparticles can be Formulated by methods described in, e.g., Intl. Pub. Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, and WO2012054923; and U.S. Pub. Nos.
  • the therapeutic nanoparticle polynucleotide can be Formulated for sustained release.
  • sustained release refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time.
  • the period of time can include, but is not limited to, hours, Attorney Docket No.: 45817-0161WO1 days, weeks, months and years.
  • the sustained release nanoparticle of the polynucleotides described herein can be Formulated as disclosed in Intl. Pub. No. WO2010075072 and U.S. Pub. Nos. US20100216804, US20110217377, US20120201859 and US20130150295, each of which is herein incorporated by reference in their entirety.
  • the therapeutic nanoparticle polynucleotide can be Formulated to be target specific, such as those described in Intl. Pub. Nos.
  • LNPs can be prepared using microfluidic mixers or micromixers.
  • Exemplary microfluidic mixers can include, but are not limited to, a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggered herringbone micromixer (SHM) (see, Zhigaltsev et al., Langmuir.28:3633-40 (2012); Belliveau et al., Molecular Therapy-Nucleic Acids.1:e37 (2012); Chen et al., J. Am. Chem. Soc. 134(16):6948-51 (2012); each of which is herein incorporated by reference in its entirety).
  • SHM herringbone micromixer
  • Exemplary micromixers include Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM,) from the Institut für Mikrotechnik Mainz GmbH, Mainz Germany.
  • methods of making LNP using SHM further comprise mixing at least two input streams wherein mixing occurs by microstructure- induced chaotic advection (MICA).
  • MICA microstructure- induced chaotic advection
  • fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other.
  • This method can also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling.
  • Methods of generating LNPs using SHM include those disclosed in U.S. Pub.
  • the polynucleotides described herein can be Formulated in lipid nanoparticles using microfluidic technology (see, Whitesides, George M., Nature 442: 368-373 (2006); and Abraham et al., Science 295: 647-651 (2002); each of which is herein incorporated by reference in its entirety).
  • the polynucleotides can be Formulated in lipid nanoparticles using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK).
  • a micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.
  • the polynucleotides described herein can be Formulated in lipid nanoparticles having a diameter from about 1 nm to about 100 nm such as, but not limited to, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70
  • the lipid nanoparticles can have a diameter from about 10 to 500 nm. In some embodiments, the lipid nanoparticle can have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.
  • the polynucleotides can be delivered using smaller LNPs.
  • Such particles can comprise a diameter from below 0.1 ⁇ m up to 100 nm such as, but not limited to, less than 0.1 ⁇ m, less than 1.0 ⁇ m, less than 5 ⁇ m, less than 10 ⁇ m, less than 15 um, less than 20 um, less than 25 um, less than 30 um, less than 35 um, less than 40 um, less than 50 um, less than 55 um, less than 60 um, less than 65 um, less than 70 um, less than 75 um, less than 80 um, less than 85 um, less than 90 um, less than 95 um, less than 100 um, less than 125 um, less than 150 um, less than 175 um, less than 200 um, less than 225 um, less than 250 um, less than 275 um, less than 300 um, less than 325 um, less than 350 um, less than 375 um, less than 400 um, less than 425 um, less than 450 um, less than 475 um, less than 500 um, less than 0.1
  • the nanoparticles and microparticles described herein can be geometrically engineered to modulate macrophage and/or the immune response.
  • the geometrically engineered particles can have varied shapes, sizes and/or surface charges to incorporate the polynucleotides described herein for targeted delivery such as, but not limited to, pulmonary delivery (see, e.g., Intl. Pub. No. WO2013082111, herein incorporated by reference in its entirety).
  • Other physical features the geometrically Attorney Docket No.: 45817-0161WO1 engineering particles can include, but are not limited to, fenestrations, angled arms, asymmetry and surface roughness, charge that can alter the interactions with cells and tissues.
  • the nanoparticles described herein are stealth nanoparticles or target-specific stealth nanoparticles such as, but not limited to, those described in U.S. Pub. No. US20130172406, herein incorporated by reference in its entirety.
  • the stealth or target-specific stealth nanoparticles can comprise a polymeric matrix, which can comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polyesters, polyanhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates, or combinations thereof.
  • polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyester
  • antibodies, antigen-binding fragments, and/or binding proteins of the present disclosure are administered to a subject in need thereof.
  • the subject in need thereof is a subject with a disease, disorder, and/or condition that may be treated with technologies described herein.
  • the subject in need thereof is a subject with, e.g., a cancer, an infection, an inflammatory condition, or an autoimmune condition, depending on the binding specificity and effector function of any additional binding moieties present in a construct comprising a VHH of the of the present disclosure.
  • a mammal includes, for example and without limitation, a household Attorney Docket No.: 45817-0161WO1 pet (e.g., a dog, a cat, a rabbit, a ferret, a hamster), a livestock or farm animal (e.g., a cow, a pig, a sheep, a goat, a pig, a chicken or another poultry), a horse, a monkey, a laboratory animal (e.g., a mouse, a rat, a rabbit) and a human.
  • a household Attorney Docket No.: 45817-0161WO1 pet e.g., a dog, a cat, a rabbit, a ferret, a hamster
  • a livestock or farm animal e.g., a cow, a pig, a sheep, a goat, a pig, a chicken or another poultry
  • a horse e.g., a monkey, a laboratory animal (e.g.,
  • the subject in need thereof is a human. Technologies of the present disclosure can be practiced in any subject in need thereof that is likely to benefit from administration of technologies of the present disclosure (e.g., a subject with cancer).
  • a subject in need thereof is a human.
  • the human is male.
  • the human is female.
  • the human is an adult (e.g., 18 or more years of age).
  • the adult is greater than 18 years old, greater than 25 years old, greater than 30 years old, greater than 40 years old, greater than 50 years old, greater than 55 years old, greater than 60 years old, greater than 65 years old, greater than 70 years old, greater than 75 years old, greater than 80 years old, greater than 85 years old, greater than 90 years old, greater than 95 years old, greater than 100 years old, or greater than 105 years old in age.
  • the human is a child.
  • the child is greater than 2 years old, greater than 3 years old, greater than 4 years old, greater than 5 years old, greater than 6 years old, greater than 7 years old, greater than 8 years old, greater than 9 years old, greater than 10 years old, greater than 11 years old, greater than 12 years old, greater than 13 years old, greater than 14 years old, greater than 15 years old, or greater than 16 years old in age.
  • a subject in need thereof is administered an antibody or binding protein of the present disclosure.
  • Anti-HSA antibodies or antigen-binding fragments of the disclosure, and nucleic acids encoding the same can be administered to a subject (e.g., a mammalian subject, such as a human) by a variety of routes.
  • the antibody or nucleic acid is administered to the subject intravenously, subcutaneously, Attorney Docket No.: 45817-0161WO1 intramuscularly, parenterally, intrathecally, intracerebroventricularly, transdermally, or orally.
  • the most suitable route for administration in any given case will depend on the particular therapeutic agent administered, the patient, pharmaceutical formulation methods, and various patient-specific parameters, such as the patient's age, body weight, sex, severity of the diseases being treated, the patient’s diet, and the patient’s excretion rate.
  • An appropriate dosage of anti-HSA antibodies, antigen-binding fragments, or binding proteins, or nucleic acids encoding the same of the present disclosure will vary with the particular condition, disease and/or disease being treated, various subject-specific parameters (e.g., age, weight, physical condition of the subject), severity of the particular condition, disease, and/or disorder being treated, the nature of current or combination therapy (if any), the specific route of administration and other factors within the knowledge and expertise of a health practitioner.
  • a maximally tolerated dose of technologies described herein is to be used, e.g., the highest safe dose according to sound medical judgement.
  • technologies described herein are administered in an effective amount, e.g., a dose sufficient to provide one or more medically desirable results.
  • a therapeutic regiment for use in accordance with technologies described herein may include administration of such technologies or compositions comprising such technologies once a day, once every two days, once every three days, twice a week, once a week, once every two weeks, once every three weeks, once every month or four weeks, once every six weeks, once every two months or eight weeks, once every three months or twelve weeks.
  • a subject receives a single dose of a technology described herein.
  • a subject receives a plurality of doses of a technology described herein (e.g., at least two, at least three, at least four, at least five, at least six, at least eight, at least ten, or more doses).
  • technologies described herein are administered over a period of time, such as one week, two weeks, three weeks, four weeks, six weeks, two Attorney Docket No.: 45817-0161WO1 months, three months, four months, five months, six months, one year or more.
  • Appropriate therapeutic regimens are readily understood by medical practitioners and such regimens may be designed by a medical practitioner for a particular patient (e.g., a patient-specific regimen).
  • kits that contain anti-HSA antibodies, antigen- binding fragments, binding proteins, and/or nucleic acids encoding the same.
  • the kits provided herein contain one or more cells engineered to express and secrete an anti-HSA antibody, antigen-binding fragment, of binding protein of the disclosure, such as a cell containing a nucleic acid molecule of the disclosure.
  • a kit described herein may include reagents that can be used to produce a pharmaceutical composition of the invention.
  • kits described herein may include reagents that can induce the expression of anti-HSA antibodies, antigen- binding fragments, or relate proteins of the present disclosure within cells (e.g., mammalian cells).
  • kits described herein may include tools for engineering a prokaryotic or eukaryotic cell (e.g., a CHO cell or a BL21(DE3) E. Coli cell or an immune cell) so as to express and secrete an anti-HSA antibody or binding protein described herein.
  • a kit may contain CHO cells stored in an appropriate media and optionally frozen according to methods known in the art.
  • the kit may also contain a nucleic acid encoding the desired antibody or binding protein as well as reagents for expressing the antibody or binding protein in the cell.
  • a kit described herein may also provide an anti-HSA antibody, antigen- binding fragment or binding protein of the disclosure, or a nucleic acid encoding the same in combination with a package insert describing how the antibody, antigen- binding fragment, binding protein, or nucleic acid may be administered to a subject, for example, for the treatment of a disease, disorder and/or condition (e.g., cancer).
  • a disease, disorder and/or condition e.g., cancer
  • a disease, disorder and/or condition e.g., cancer
  • a disease, disorder and/or condition e.g., cancer
  • 45817-0161WO1 EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods claimed herein can be performed, made, and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of the present disclosure. Methods: The following materials and methods were used in the examples.
  • a Llama was immunized with a primary dose immunization of 200 ⁇ g each of HSA and HSA DII in complete Freund’s Adjuvant (CFA), given subcutaneously. Following the primary immunization, incomplete Freund’s Adjuvant (IFA) was used for all the following boosts.
  • CFA complete Freund’s Adjuvant
  • IFA incomplete Freund’s Adjuvant
  • CSA was alternated with HSA DII in a total of 4 injections. HSA was included in all injections.
  • a test bleed was taken on day 52 post immunization, to check for antibody titers against the immunized targets in the serum. The second bleed was performed on day 70, for a production bleed of 600 ml and was used to isolate and cryopreserve PBMCs.
  • FACS sorting of B-cells Antibody discovery was performed by Fluorescence Activated Cell Sorting (FACS) and culture of sorted Llama B cells, based on methods available on mammalian B cell culture (see, e.g., Kwakkenbos et al., Nat. Med. 16(1):123-128 (2010); WO2013076139 A1; Carbonetti et al., JIM 448: 66-73 (2017), herein incorporated by reference in their entirety). To ensure the isolation of CSA- cross reactive anti-HSA single domain antibodies, Llama PBMCs were stained and sorted for CSA-binding VHH+ B cells.
  • B-cell culture B-cells were seeded at 2-3 cells/well and co-cultured with irradiated CD40L expressing feeder cells and appropriate cytokines in 96 well plates. The culture plates were incubated for 10-12 days in a CO2 incubator at 37 o C and 5% CO 2 . Proliferating B cell cultures were processed to harvest culture supernatants for screening the secreted antibodies against the target antigens, and the cell pellets were frozen for isolation of RNA and antibody sequencing.
  • MSD assays were performed using 384-well MSD plates (MSD #L25XA) that were coated with 15 ⁇ l/well of 5 ⁇ g/ml of one of the antigens, HSA, HSA DII, or CSA, in 1x PBS overnight at 4 oC. Supernatants were diluted five-fold for use in the assays. Antigen-binding total IgG wells were detected with 1 ⁇ g/ml of sulfo-tag labelled polyclonal anti-Llama IgG (Abcam, ab112784).
  • MSD assays for identification of VHH+ clones were performed similarly by coating wells of 384-well MSD plates with 5 ⁇ g/ml of polyclonal anti-Llama IgG (Abcam, ab112784).
  • the captured VHH antibodies present in the B cell culture supes were detected with 1 ⁇ g/ml of rabbit anti-camelid VHH (Genscript, A01860) followed by probing the secondary rabbit antibody with sulfo-tag labeled anti-rabbit IgG.
  • Next Generation Sequencing of Antibodies from B-cells mRNA was isolated from B cell culture lysates using the Dynabeads mRNAdirect purification kit (Invitrogen, 61012) according to manufacturer’s instructions.
  • cDNA synthesis was performed using Superscript IV first strand synthesis system (Invitrogen, 18091050). Two step PCR was performed according to standard procedures, to amplify the variable domain of the heavy chain with historical primers used in Llama antibody discovery (see, e.g., Conrath et al., Antimicro.-Agents Chemother. v45(10):2807-2812 (2001); Els Pardon, Nat. Protoc.9(3):674-93 (2014), herein incorporated by reference in their entirety).
  • the primary PCR involved using CALL001 forward primer and CALL002 reverse primer that anneal to the leader sequence and 5 ⁇ end of CH2 domain of the heavy chain respectively.
  • the PCR product was used in a nested PCR with forward and reverse primers listed in the above references linked with NGS adaptors, to amplify a 400bp VH or VHH amplicon.
  • the nested PCR amplicon was used in a final PCR reaction using barcoded primers that bind to the adaptors introduced in the nested PCR.
  • VHH expression Recombinant antibody expression was performed in 2 ml cultures. Small scale transfections of the engineered plasmid constructs in expi293 cells were set up in 24 deep well plates.
  • the purified proteins were analyzed by SDS- PAGE under non-reducing conditions to check for purity and validation of molecular weight.
  • V5 or FLAG-tagged sdAb VHH clones exhibited abundant protein expression. An average of 4-5 mg protein was purified from 30 ml transfected culture supernatants.
  • BLI interferometry assays The expressed his-tagged sdAbs present in the transfection supernatants were captured on anti-penta his biosensors using an Octet Red instrument, by dipping the biosensors in 1:100 diluted culture supernatants at 1000 rpm for 180 s. The biosensors were subsequently dipped in the running buffer (1x PBS, pH7.4) for 60 s to wash off the unbound antibody.
  • biosensors with the captured sdAbs were then interacted with 1 ⁇ M solution of the antigen (HSA/HSA DII/CSA) for a period of 180 s at 1000 rpm, followed by dipping the biosensors with the antibody-antigen complex in running buffer for a period of 300 s to measure the dissociation rate.
  • Small scale purification and size exclusion chromatography SEC-MALS: Small scale purification of transiently expressed sdAbs with a c-terminal 6xHis tag (SEQ ID NO:208) was performed with His-mag sepharose excel beads (Cytiva; cat# 17371222) from 1 ml transfection supernatants.
  • V5 and Flag tagged VHH ELISAs For the quantitation of V5-tagged sdAbs, ELISA plates were coated with 50 ⁇ l/well of 2.5 ⁇ g/ml of anti-V5 antibody (Abcam, ab9137).
  • V5-tagged sdAb proteins were used as standards and diluted in a 3x dilution series over 12 dilutions starting from 2.5 ⁇ g/ml, including a buffer blank. Sera were diluted 750x and 1500x for assaying the V5-tagged sdAbs.
  • the wells of the ELISA plates were coated with 50 ⁇ l of 2.5 ⁇ g/ml of anti-FLAG-tag antibody (Sigma, F3165).2.5 ⁇ g/ml of the 6 different FLAG-tagged sdAb proteins were diluted in a 2x dilution series over 12 dilutions and used as standards in the assay.400x and 800x dilutions of the sera were used to assay the FLAG-tagged sdAbs.
  • the plate bound V5-tagged/FLAG-tagged sdAbs from the samples were detected with 1:10,000 dilution of anti-his HRP (Abcam, ab1187).
  • the captured ligands were interacted with different dilutions of the antigen (CSA, HSA or HSA DII) that were diluted in a 2-fold dilution series from 1000 nM (CSA, HSA) or 250 nM (HSA DII) in HBS-EP+ buffer.
  • the samples were injected at 100 ⁇ l/min in all the four flow cells for a contact time of 120 s. Dissociation of the samples from the captured sdAbs was monitored over 300 s. This was followed by regeneration of the capture ligand surface on the chip with a 30 s pulse of 10 mM glycine, pH 1.5, at 30 ⁇ l/min.
  • Antibody humanization was done using the general methods of CDR grafting (Hanf et al., Methods.65(1):68-76 (2014)). The method involves grafting the mature llama CDR sequences onto a germline human acceptor framework with high homology to the parent sequence. Structural defects due to sequence mismatches at the graft interface were fixed by mutating some framework residues to llama, or by mutating some CDR residues.
  • Llama Immunization to generate anti-HSA VHH domains One Llama glama was immunized with sub-cutaneous injection (sc) of a cocktail of different target antigens, which included human serum albumin (HSA, domain II of human serum albumin (HSA DII), and cynomolgus serum albumin (CSA).
  • HSA human serum albumin
  • HSA DII domain II of human serum albumin
  • CSA cynomolgus serum albumin
  • Sorted wells that showed a binding signal 50x more than the background/negative wells were considered antigen positive.
  • B cell sorting was performed by gating CSA+ sdAb+ B cells, the gate was not stringent for VHH+ B cells.
  • 165 wells showed CSA+HSA+HSA DII+ binding signal, which is 2.8% of the total B cells sorted.
  • 74 wells were VHH+ and 91 wells were IgG1+. Hence, 45% of the total antigen positive wells were identified as VHH+, while the remaining were IgG+.
  • Wells that were identified as having a positive signal in in vitro MSD binding assays for CSA, HSA, and HSA DII antigens and VHH were selected as candidates for antibody sequencing. Using this selection criteria 137 wells containing proliferated B cells were selected for antibody sequencing. Wells with an MSD signal 50x above the background were considered antigen positive. The selected 137 wells also included those that did not show high MSD signal for the antigens. Frozen proliferated B cell culture pellets from these wells were processed for mRNA isolation, cDNA synthesis, PCR and NGS for sequencing the variable heavy chain domain of the antibodies.
  • NGS Next Generation Sequencing
  • Analysis and in vitro characterization of candidate antibody sequence 137 sequences obtained from MSD supernatant screening were cloned into pCDNA3.4 expression vectors for protein expression in mammalian cells. These sequences were ranked based on (1) the MSD assay signal of their respective B cell culture well supernatants for antigen binding and (2) sequence diversity.
  • Binding of the sdAbs to serum albumin (HSA, CSA, or HSA DII) at acidic pH is very important since the extended half-life of serum albumins is attributed to their ability to bind to FcRn at low pH in the acidic environment of the endosomes of the vascular epithelial cells following pinocytosis (Sand et al., (2014).
  • Neonatal Fc receptor (FcRn) binds to domains I and III of HSA.
  • domains I and III of albumin is required for optimal pH-dependent binding to the neonatal Fc receptor (FcRn). J. Biol. Chem.2014; 289:34583-94, herein incorporated by reference in its entirety).
  • binding of anti-HSA antibodies to domain II of HSA is preferred to prevent any obstruction of binding of HSA to huFcRn.
  • 53 VHH sequences were selected for expression, that included clones with varied antigen binding signal intensity in the MSD assays as shown in Table 6 below. Of the 53 clones, 17 clones showed high binding to all three antigens, 8 bound only CSA and HSA, 2 clones bound CSA only and 1 clone bound only HSA.19 clones showed different specificities and binding signal levels to each of these antigens as listed in Table 6. Six clones showed no binding to any of the 3 antigens.
  • Each of the 53 sdAb sequences selected was expressed with a C-terminal 6xhis-tag (SEQ ID NO:208) to facilitate purification using immobilized affinity chromatography (IMAC).5-days post expi293 transfection culture supernatants of the sdAb-6xhis molecules were harvested. The supernatants were screened for binding to HSA, CSA, and HSA DII at pH 7.4 in assay by bi-layer interferometry (BLI) (FIGs.4 and 5). Samples were judged to have positive binding to the target based on a binding of ⁇ 0.2 units (nM) during the binding phase and an off rate that did not return to baseline during first 200 sec of the monitored off-rate time.
  • IMAC immobilized affinity chromatography
  • VHH-his tagged antibodies Following fermentation of the VHH-his tagged antibodies, as described in the methods, the level of aggregated protein was assessed by size exclusion chromatography (SEC). No significant fraction of aggregated molecules was observed by SEC (FIG.5) for the tested molecules. 12 VHH-his antibodies showed high affinity to the three antigens (CSA, HSA and HSA-DII) in the BLI antigen binding screen (FIG 4). These 11 clones VHH antibodies were next evaluated in an in vivo PK study to determine their half-life.
  • SEC size exclusion chromatography
  • VHH2 VHH7, VHH1, VHH4, VHH6, VHH10, VHH9, VHH5, VHH3, VHH11, and VHH17.
  • VHH13 One clone, VHH13, that did not bind to HSA DII was included as a presumptive negative control for comparative purposes.
  • EXAMPLE 5 Preparation and characterization of V5- and FLAG-tagged sdAb clones for in vivo PK study The selected sdAb VHH clones were evaluated in GenoWay double knockin mice expressing both human serum albumin and neonatal Fc receptor (HSA/huFcRn transgenic mice).
  • mice express physiological levels of HSA and maintain autologous receptor-ligand interaction, mimic physiological drug clearance in humans Attorney Docket No.: 45817-0161WO1 and thus form an accurate in vivo model to evaluate the PK profile of anti-huSA antibodies.
  • These models have been validated to generate data and have shown to be predictive of similar results in humans (see, Viuff et al., Journal of Controlled Release.223:22–30 (2016), herein incorporated by reference in its entirety).
  • the constructs that were designed for in vitro expression in the previous screen were reformatted with either a C-terminal V5-(His)6 or FLAG-(His)6 tag as shown in FIG.6.
  • the antibodies were expressed in expi293 cells purified and characterized first by non-reducing SDS-PAGE of sdAb which revealed a single protein band of 15- 17kD (FIG.7) as expected.
  • the antibodies were also analyzed by SEC-MALS as described in the methods.
  • SEC-MALS analysis of the expressed sdAb VHH clones revealed a single homogenous peak of >95% purity, eluted between 9.5-10.5 min (FIG.8).
  • the double knock-in HSA/huFcRn mice were used to evaluate the 12 selected antibodies in vivo, each animal containing two different sdAbs with the respective V5 or FLAG tag dosed at 6 mg/kg using a total of 18 groups of 3 mice each. VHH17 was eliminated due to its rapid dissociation rate as shown in Table 12.
  • mice were bled at seven time points – 6 h, 24 h, 48 h, 72 h, 96 h, 192 h, and 216 h.
  • Each article ID was dosed in 3 groups so that each mouse group was bled only twice per week over the time course of the study.
  • the detailed study design is shown in FIG.9 and Table 9 below.
  • Table 10 Summary of in vivo PK profiles of V5-tagged clones determined by ELISA Sample VHH9 VHH1 VHH5 VHH7 VHH3 VHH11 Dilution d d by 3 Attorney Docket No.: 45817-0161WO1 400x Half 64.4 64.44 63.68 51.01 42.86 22.92 Life h EXAMPLE 6.
  • Affinity measurements of the sdAbs Affinity measurements on each of the sdAbs that was tested in the animal model for each of the three different antigens was performed by surface plasmon resonance (SPR) assays. Assays were repeated three times to derive the average kinetic constants.
  • Table 12 lists results for clones in decreasing order of affinity to HSA.
  • Table 13 lists results for clones in decreasing order of affinity to CSA.
  • Table 14 lists results for clones in decreasing order of affinity to HSA DII.
  • Table 12 affinity measurements of sdAbs by surface plasmon resonance (SPR) assay in decreasing order of affinity to HSA anti-HSA sdAb ka (1/Ms) kd (1/s) KD (M) VHH2 1.70E+05 5.53E-04 3.26
  • Table 13 — affinity measurements of sdAbs by surface plasmon resonance (SPR) assay in decreasing order of affinity to CSA anti-HSA sdAb ka (1/Ms) kd (1/s) KD (M) Attorney Docket No.: 45817-0161WO1 VHH2 1.41E+05 4.79E-04 3.4 VHH1 1.06E+05 5.93E-04 5.6
  • Table 14 affinity measurements of sdAbs by surface plasmon resonance (SPR) assay in decreasing order of affinity to HSA DII.
  • the summary data of the 5 selected clones from the sequential selection rounds is shown in Table 16 containing the MSD data for the initial binding.
  • FIG.12 showing the sequence diversity of the chosen clones.
  • the selection of the lead molecules came from a single clade.
  • FIG.13 and Table 17 are a comparison of the various PK profiles of each five final molecules with the half-life varying between 57.3 and 69.9 h.
  • FIG.14 and Table 18 are the SPR binding data for each of the final molecules on HSA, CSA and HSA DII and the calculated binding constants respectively.
  • the selected clones consistently showed similar affinity from the initial round of selection.
  • Table 16 MSD signals from the initial B cell culture supernatants for 5 clones selected for humanization Clone ID MSD signal from the initial B cell culture supernatants Attorney Docket No.: 45817-0161WO1 VHH5 128788 445545 443428 162239 Table 17 – In vivo PK data from humanized mice of the 5 selected clones sdAB Half-life (h) VHH1 69.94 Table 18 – In vitro bin ding affinity measurements measured using Biacore at pH 7.4 Anti-HSA Affinity (KD) M sdAB HSA CSA HSA DII EXAMPLE 8.
  • KD Anti-HSA Affinity

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Abstract

L'invention concerne des anticorps anti-albumine sérique humaine (HSA), des fragments de liaison à l'antigène de ceux-ci, et des protéines de liaison comprenant de tels domaines de liaison, ainsi que des acides nucléiques codant pour ceux-ci. L'invention concerne également des procédés d'utilisation de tels anticorps, fragments de liaison à l'antigène et des protéines de liaison pour prolonger la demi-vie de composés thérapeutiques.
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