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WO2025024323A1 - Compositions de liaison à la sélectine p et e et leurs procédés d'utilisation - Google Patents

Compositions de liaison à la sélectine p et e et leurs procédés d'utilisation Download PDF

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Publication number
WO2025024323A1
WO2025024323A1 PCT/US2024/038872 US2024038872W WO2025024323A1 WO 2025024323 A1 WO2025024323 A1 WO 2025024323A1 US 2024038872 W US2024038872 W US 2024038872W WO 2025024323 A1 WO2025024323 A1 WO 2025024323A1
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Prior art keywords
seq
amino acid
target molecule
binding polypeptide
sequence
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Inventor
Peyton GREENSIDE
Ryan HENRICI
Hunter ELLIOTT
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Bighat Biosciences Inc
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Bighat Biosciences 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • Cellular adhesion molecules such as selectin molecules, play a fundamental role in a broad range of critical physiological functions, including inflammation, thrombus formation, and wound healing. Dysfunction of cellular adhesion molecules, for example arising from congenital or spontaneous mutation of genes encoding one or more cellular adhesion molecules, can give rise to serious medical conditions.
  • P-selectin which is also known as CD62P, is a transmembrane cellular adhesion molecule expressed by activated endothelial cells and activated platelets. P-selectin is known to play an important role in binding to cells (e.g., circulating immune cells, such as leukocytes). In many cases, interactions between P-selectin and a target cell (e.g., an immune cell) are mediated by interactions between P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) expressed on the target cell’s surface.
  • PSGL-1 P-selectin glycoprotein ligand 1
  • E-selectin also known as CD62E, is a transmembrane cellular adhesion molecule expressed on the surface of endothelial cells following inflammation or injury. E-selectin recognizes sialo-fucosylated Lewis carbohydrates expressed on the surface of the leucocytes, and thus is involved in the recruitment of neutrophils, monocytes, and T cells to inflammatory site.
  • the full-length amino acid sequence of human E-selectin is provided in UniProtKB Accession No. P16581 and NCBI Ref. Sequence NP_000441.2 and the mature form of human E-selectin is provided as amino acid residues 22-610.
  • P-selectin and/or E-selectin expressed on the surface of activated endothelial cells plays a significant role in mediating tethering and initial rolling interactions between circulating immune cells (e g., leukocytes) and the activated endothelial cells on which the P-selectin and/or E- selectin is expressed, for example, as part of a systemic inflammatory response.
  • circulating immune cells e.g., leukocytes
  • P-selectin expressed by activated platelets can mediate interactions between the activated platelet on which it is expressed and circulating immune cells (e.g., leukocytes).
  • a target molecule-binding polypeptide comprising: (a) a first heavy chain complementarity determining region (HCDR1) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 5; (b) a second heavy chain complementarity determining region (HCDR2) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 6; (c) a third heavy chain complementarity determining region (HCDR3) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 7; (d) a first light chain complementarity determining region (LCDR1) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 8-12; (e) a second light chain complementarity determining region (LCDR2) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs:
  • a target molecule-binding polypeptide comprising: (a) a first heavy chain complementarity determining region (HCDR1) comprising a sequence identical to the amino acid sequence set out in SEQ ID NO: 5; (b) a second heavy chain complementarity determining region (HCDR2) comprising a sequence identical to the amino acid sequence set out in SEQ ID NO: 6; (c) a third heavy chain complementarity determining region (HCDR3) comprising a sequence identical to the amino acid sequence set out in SEQ ID NO: 7; (d) a first light chain complementarity determining region (LCDR1) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 8-12; (e) a second light chain complementarity determining region (LCDR2) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 13- 16; and (f) a third light chain complementarity determining
  • a target molecule-binding polypeptide comprising: (a) a heavy chain variable domain (VH) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 24; and (b) a light chain variable domain (VL) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 25.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a target molecule-binding polypeptide comprising: (a) a heavy chain variable domain (VH) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 30; and (b) a light chain variable domain (VL) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 31.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a target molecule-binding polypeptide comprising: (a) a heavy chain variable domain (VH) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 34; and (b) a light chain variable domain (VL) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 35.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a target molecule-binding polypeptide comprising: (a) a heavy chain variable domain (VH) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 34; and (b) a light chain variable domain (VL) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 37.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a target molecule-binding polypeptide comprising: (a) a heavy chain variable domain (VH) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 34; and (b) a light chain variable domain (VL) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 37.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a target molecule-binding polypeptide comprising: (a) a heavy chain variable domain (VH) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 34; and (b) a light chain variable domain (VL) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 40.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a target molecule-binding polypeptide comprising: (a) a heavy chain variable domain (VH) comprising a sequence having at least 80% sequence identity 7 to the amino acid sequence set out in SEQ ID NO: 43; and (b) a light chain variable domain (VL) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 44.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a target molecule-binding polypeptide comprising: (a) a heavy chain variable domain (VH) comprising a sequence having at least 80% sequence identity 7 to the amino acid sequence set out in SEQ ID NO: 46; and (b) a light chain variable domain (VL) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 47.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a target molecule-binding polypeptide comprising: (a) a heavy chain variable domain (VH) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 48; and (b) a light chain variable domain (VL) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 49.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the heavy chain variable domain (VH) is connected to the N-terminus of the light chain variable domain (VL) by a linker.
  • the light chain variable domain (VL) is connected to the N-terminus of the heavy chain variable domain (VH) by a linker.
  • the linker is a polypeptide linker.
  • target molecule-binding polypeptide comprising:
  • a target molecule-binding polypeptide comprising: (a) a heavy (H) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 26; and (b) a light (L) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 27.
  • a target molecule-binding polypeptide comprising: (a) a heavy (H) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 28; and (b) a light (L) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 29.
  • a target molecule-binding polypeptide comprising: (a) a heavy (H) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 32; and (b) a light (L) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 33.
  • a target molecule-binding polypeptide comprising: (a) a heavy (H) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 32; and (b) a light (L) chain comprising a sequence having at least 80% sequence identity’ to the amino acid sequence set out in SEQ ID NO: 36.
  • a target molecule-binding polypeptide comprising: (a) a heavy (H) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 38; and (b) a light (L) chain comprising a sequence having at least 80% sequence identity’ to the amino acid sequence set out in SEQ ID NO: 36.
  • a target molecule-binding polypeptide comprising: (a) a heavy (H) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 32; and (b) a light (L) chain comprising a sequence having at least 80% sequence identity’ to the amino acid sequence set out in SEQ ID NO: 39.
  • a target molecule-binding polypeptide comprising: (a) a heavy (H) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 41; and (b) a light (L) chain comprising a sequence having at least 80% sequence identity’ to the amino acid sequence set out in SEQ ID NO: 42.
  • a target molecule-binding polypeptide comprising: (a) a heavy (H) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 45; and (b) a light (L) chain comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 42.
  • target molecule-binding polypeptide comprising:
  • target molecule-binding polypeptide comprising:
  • the target molecule-binding polypeptide binds one or more amino acid residues within the amino acid sequence set out in any one of SEQ ID NOs: 1-4.
  • the target molecule-binding polypeptide binds a selectin molecule.
  • the selectin molecule is P-selectin, E-selectin, or both.
  • the target molecule-binding comprises a half-life extension mutation.
  • the target molecule-binding polypeptide comprises a scaffold selected from IgGl or IgG4.
  • the target molecule-binding polypeptide is an antibody fragment and the antibody fragment comprises a single-chain variable fragment (scFv), F(ab) fragment, a F(ab’)2 fragment, a monovalent IgG, a diabody, or a minibody.
  • the target molecule-binding polypeptide comprises an scFv.
  • the target moleculebinding polypeptide is a monoclonal antibody.
  • the target moleculebinding polypeptide is human, humanized, or chimeric.
  • the target molecule-binding polypeptide has increased stability compared to stability of a control antibody, as measured by melting temperature (Tm). and wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 and a light chain according to SEQ ID NO: 55.
  • Tm melting temperature
  • the target molecule-binding polypeptide has a Tm of more than 65 °C, 70 °C, 75 °C or 80 °C.
  • the target molecule-binding polypeptide has increased stability under an acidic condition compared to stability under acidic condition of a control antibody, as measured by aggregation temperature (Tagg), and wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 and a light chain according to SEQ ID NO: 55.
  • the target molecule-binding polypeptide has a Tagg of more than 60°C or 65°C.
  • the acidic condition comprises a pH of less than 7.0. a pH of about 2.0 to about 6.0, a pH of about 2.0 to about 5.0, a pH of about 2.0 to about 4.0 or a pH of about 3.0.
  • the target molecule-binding polypeptide has an increased binding affinity to human E-selectin compared to a binding affinity of a control antibody, and wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 and a light chain according to SEQ ID NO: 55.
  • the binding affinity of the target moleculebinding polypeptide to human E-selectin is at least 2-fold, 3-fold, or 4-fold greater than the binding affinity of a control antibody, and wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 and a light chain according to SEQ ID NO: 55.
  • the target molecule-binding polypeptide has a KD for binding to human E-selectin of less than 4.0 X 10-8 M, less than 3.0 X 10-8 M or less than 2.0 X 10-8 M.
  • the target molecule-binding polypeptide has increased humanness compared to humanness of a control antibody, as defined by Observed Antibody Space identity 7 search (OASis) percentile score, and wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 and a light chain according to SEQ ID NO: 55.
  • the target molecule-binding polypeptide has humanness of more than 40%, 50%, 60%, 70%, 80%, or 90% as defined by OASis percentile score.
  • a pharmaceutical composition comprising any of the target molecule-binding polypeptides described herein and a pharmaceutically acceptable carrier or excipient.
  • nucleic acid encoding any of the target moleculebinding polypeptides described herein.
  • an expression comprising a nucleic acid encoding any of the target molecule-binding polypeptides described herein.
  • a host cell comprising any nucleic acid or expression vector described herein.
  • a method of treating sickle cell disease in a subject comprising administering to the subject any of the target molecule-binding polypeptides described herein, any of the pharmaceutical compositions described herein, any of the nucleic acids described herein, any of the expression vectors described herein, or any of the host cells described herein.
  • the subject has experienced or is experiencing a vasoocclusive crisis.
  • the administering is intravenous, subcutaneous, or intramuscular.
  • the subject has a blood hemoglobin level of from about 6 g/dL to about 11 g/dL.
  • the subject has a mutation in the hemoglobin beta gene (HBB).
  • a method of treating lupus in a subject comprising administering to the subject any of the target molecule-binding polypeptides described herein, any of the pharmaceutical compositions described herein, any of the nucleic acids described herein, any of the expression vectors described herein, or any of the host cells described herein.
  • the lupus is selected from the group consisting of systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, and drug-induced lupus erythematosus.
  • the administering is intravenous, subcutaneous, or intramuscular.
  • the subject has a blood hemoglobin level of from about 6 g/dL to about 11 g/dL. In some embodiments, the subject has a mutation in the hemoglobin beta gene (HBB).
  • HBB hemoglobin beta gene
  • the administering increases blood oxygen saturation or decreases frequency of chest pain, severity of chest pain, frequency of limb pain, severity' of limb pain, a risk of stroke, or a risk of heart attack, in the subject.
  • kits comprising any of the target molecule-binding polypeptides described herein, any of the pharmaceutical compositions described herein, any of the nucleic acids described herein, any of the expression vectors described herein, or any of the host cells described herein, and instructions for use.
  • the cardiovascular condition is clinically associated with sickle cell disease, systemic lupus erythematosus, or primary myelofibrosis.
  • target molecule-binding polypeptides also known as antigen-binding polypeptides
  • the target molecule also referred to as the target antigen
  • the target antigen is selectin, for example, P-selectin. E-selectin, or both.
  • a target moleculebinding polypeptide disclosed herein has superior functional inhibition of cell adhesion.
  • a target molecule-binding polypeptide disclosed herein targeting both P-selectin and E-selectin has superior functional inhibition of cell adhesion.
  • a target molecule-binding polypeptide disclosed herein targeting both P-selectin and E-selectin has superior functional inhibition of cell adhesion in blood compared to a target molecule-binding polypeptide targeting P-selectin.
  • a target molecule-binding polypeptide disclosed herein targeting both P-selectin and E-selectin has superior functional inhibition of cell adhesion in blood compared to a target molecule targeting E-selectin.
  • a target molecule-binding polypeptide disclosed herein binds to P-selectin and/or E-selectin, but not L-selectin.
  • target molecule-binding polypeptides comprising: (a) a first light chain complementarity determining region (LCDR1) comprising a sequence having a sequence identical to the amino acid sequence set out in any one of SEQ ID NOs: 8-12; (b) a second light chain complementarity determining region (LCDR2) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 13-16; and (c) a third light chain complementarity determining region (LCDR3) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 17- 20.
  • LCDR1 first light chain complementarity determining region
  • LCDR2 second light chain complementarity determining region
  • LCDR3 a third light chain complementarity determining region
  • target molecule-binding polypeptides comprising: (a) a first light chain complementarity determining region (LCDR1) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 8-12; (b) a second light chain complementarity determining region (LCDR2) comprising a sequence having a sequence identical to the amino acid sequence set out in any one of SEQ ID NOs: 13-16; and (c) a third light chain complementarity determining region (LCDR3) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 17-20.
  • LCDR1 first light chain complementarity determining region
  • LCDR2 second light chain complementarity determining region
  • LCDR3 a third light chain complementarity determining region
  • target molecule-binding polypeptides comprising: (a) a first light chain complementarity determining region (LCDR1) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 8-12; (b) a second light chain complementarity determining region (LCDR2) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 13- 16; and (c) a third light chain complementarity determining region (LCDR3) comprising a sequence having a sequence identical to the amino acid sequence set out in any one of SEQ ID NOs: 17-20.
  • LCDR1 first light chain complementarity determining region
  • LCDR2 second light chain complementarity determining region
  • LCDR3 a third light chain complementarity determining region
  • the LCDR1 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 8.
  • the LCDR2 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 13.
  • the LCDR3 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 18.
  • the target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 25.
  • the target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 25. In some embodiments, the target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 23. In some embodiments, the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 23.
  • the LCDR1 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 9.
  • the LCDR2 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 13.
  • the LCDR3 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 17.
  • the target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 31.
  • the target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 31. In some embodiments, the target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 29. In some embodiments, the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 29.
  • the LCDR1 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 8.
  • the LCDR2 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 13.
  • the LCDR3 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 19.
  • the target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 35.
  • the target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 35. In some embodiments, the target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 33. In some embodiments, the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 33. [0057] In some embodiments, the LCDR1 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 8.
  • the LCDR2 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 14.
  • the LCDR3 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 17.
  • the target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 37.
  • the target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 37.
  • the target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 36. In some embodiments, the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 36.
  • the LCDR1 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 10.
  • the LCDR2 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 15.
  • the LCDR3 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 17.
  • the target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 40.
  • the target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 40. In some embodiments, the target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 39. In some embodiments, the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 39.
  • the LCDR1 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 11.
  • the LCDR2 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 13.
  • the LCDR3 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 17.
  • the target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 44.
  • the target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 44. In some embodiments, the target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 42. In some embodiments, the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 42.
  • the LCDR1 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 8.
  • the LCDR2 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 16.
  • the LCDR3 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 20.
  • the target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 47.
  • the target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 47.
  • the LCDR1 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 12.
  • the LCDR2 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 16.
  • the LCDR3 of a target molecule-binding polypeptide disclosed herein comprises a sequence identical to SEQ ID NO: 17.
  • the target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 49.
  • the target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 49.
  • a target molecule-binding polypeptide disclosed herein comprises: (a) a first heavy chain complementarity determining region (HCDR1) comprising a sequence having at least 80% homology relative to SEQ ID NO: 5; (b) a second heavy chain complementarity determining region (HCDR2) comprising a sequence having at least 80% homologj' relative to SEQ ID NO: 6; (c) a third heavy chain complementarity determining region (HCDR3) comprising a sequence having at least 80% homology relative to SEQ ID NO: 7; or (d) a combination thereof.
  • HCDR1 first heavy chain complementarity determining region
  • HCDR2 comprising a sequence having at least 80% homologj' relative to SEQ ID NO: 6
  • HCDR3 a third heavy chain complementarity determining region
  • a target molecule-binding polypeptide disclosed herein comprises: (a) a HCDR1 comprising a sequence identical to the amino acid sequence set out in SEQ ID NO: 5; (b) a HCDR2 comprising a sequence identical to the amino acid sequence set out in SEQ ID NO: 6; (c) a HCDR3 comprising a sequence identical to the amino acid sequence set out in SEQ ID NO: 7; or (d) a combination thereof.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 24. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 24.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 22. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 22.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 30. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 30.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 28. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 28.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 34. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 34.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 32. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 32. [0070] In some embodiments, a target molecule-binding polypeptide disclosed herein comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 38. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 38.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 43. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 43.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 41. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 41.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 46. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 46.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 48. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 48.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 26. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 26.
  • a target molecule-binding polypeptide disclosed herein comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 45. In some embodiments, the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 45.
  • a target molecule-binding polypeptide disclosed herein comprises a half-life extension mutation.
  • the target molecule-binding polypeptide comprises a scaffold selected from IgGl or IgG4.
  • a target molecule-binding polypeptide disclosed herein is an antibody fragment and the antibody fragment comprises a single-chain variable fragment (scFv), F(ab) fragment, a F(ab’)2 fragment, a monovalent IgG, a diabody, or a minibody.
  • the target molecule-binding polypeptide comprises an scFv.
  • a target molecule-binding polypeptide disclosed herein is a monoclonal antibody. In some embodiments, the target molecule-binding polypeptide is human, humanized, or chimeric.
  • a target molecule-binding polypeptide disclosed herein binds to one or more amino acid residue within the amino acid sequence set forth in SEQ ID NO: 1 . In some embodiments, the target molecule-binding polypeptide binds to one or more amino acid residue within the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the target molecule-binding polypeptide binds to one or more amino acid residue within the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the target molecule-binding polypeptide binds to one or more amino acid residue within the amino acid sequence set forth in SEQ ID NO: 4.
  • the target molecule is a selectin molecule.
  • the selectin molecule is P-selectin. E-selectin. or both.
  • composition comprising the target molecule-binding polypeptide disclosed herein and a pharmaceutically acceptable carrier or excipient.
  • nucleic acid sequence encoding the target molecule-binding polypeptide disclosed herein.
  • an expression vector comprising a nucleic acid sequence encoding target molecule-binding polypeptide disclosed herein.
  • a host cell comprising the nucleic acid disclosed herein or the expression vector disclosed herein.
  • the target molecule-binding polypeptide disclosed herein, the pharmaceutical composition disclosed herein, the nucleic acid sequence disclosed herein, the expression vector disclosed herein, or the host cell disclosed herein for use in therapy.
  • a method of treating a subject in need thereof comprising, administering to the subject the target molecule-binding polypeptide disclosed herein, the pharmaceutical composition disclosed herein, the nucleic acid sequence disclosed herein, the expression vector disclosed herein, or the host cell disclosed herein.
  • the administering is subcutaneous or intramuscular.
  • the administering increases blood oxygen saturation or decreases frequency of chest pain, severity of chest pain, frequency of limb pain, severity of limb pain, a risk of stroke, or a risk of heart attack, in the subject.
  • the subject has a blood hemoglobin level of from about 6 g/dL to about 1 1 g/dL.
  • the subject has sickle cell anemia, lupus, or primary myelofibrosis.
  • the subject has a mutation in the hemoglobin beta gene (HBB).
  • HBB hemoglobin beta gene
  • the target molecule-binding polypeptide disclosed herein is a use of the target molecule-binding polypeptide disclosed herein, the pharmaceutical composition disclosed herein, the nucleic acid sequence disclosed herein, the expression vector disclosed herein, or the host cell disclosed herein, in the manufacture of a medicament for a blood disease, an autoimmune condition, a cardiovascular condition or a cancer.
  • the cardiovascular condition is sickle cell disease, systemic lupus erythematosus, or primary myelofibrosis.
  • kits comprising the target molecule-binding polypeptide disclosed herein, the pharmaceutical composition disclosed herein, the nucleic acid sequence disclosed herein, the expression vector disclosed herein, or the host cell disclosed herein and instructions for use.
  • the target molecule-binding polypeptide has increased humanness compared to humanness of a control antibody, as defined by Observed Antibody Space identity search (OASis) percentile score, optionally wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 or a light chain according to SEQ ID NO: 55.
  • the target molecule-binding polypeptide has humanness of more than 40%, 50%, 60%, 70%, 80%, or 90% as defined by OASis percentile score.
  • the target molecule-binding polypeptide has increased stability compared to stability of a control antibody, as measured by melting temperature (T m ), optionally wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 or a light chain according to SEQ ID NO: 55.
  • T m melting temperature
  • the target molecule-binding polypeptide has a Tm of more than 65 °C, 70 °C, 75 °C or 80 °C.
  • the target molecule-binding polypeptide has increased stability under an acidic condition compared to stability under acidic condition of a control antibody, as measured by aggregation temperature (T agg ), optionally wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 or a light chain according to SEQ ID NO: 55.
  • the target molecule-binding polypeptide has a T agg of more than 60°C or 65 °C.
  • the acidic condition comprises a pH of less than 7.0, a pH of about 2.0 to about 6.0, a pH of about 2.0 to about 5.0, a pH of about 2.0 to about 4.0 or a pH of about 3.0.
  • the target molecule-binding polypeptide has an increased binding affinity to human E-selectin compared to a binding affinity of a control antibody, optionally wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 or a light chain according to SEQ ID NO: 55.
  • the binding affinity of the target molecule-binding polypeptide to human E-selectin is at least 2-fold, 3-fold, or 4-fold greater than the binding affinity of a control antibody, optionally wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 or a light chain according to SEQ ID NO: 55.
  • the target molecule-binding polypeptide has a KD for binding to human E-selectin of less than 4.0 X 10' 8 M, less than 3.0 X 10' 8 M or less than 2.0 X 10' 8 M.
  • the control antibody comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 54 and a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 55.
  • the target molecule-binding polypeptide is the target molecule-binding polypeptide disclosed herein.
  • FIG. 1 shows antibody affinity of PDL huEP5C7 antibodies to Human P-Selectin-His.
  • FIG. 2 shows antibody affinity of PDL huEP5C7 antibodies to Rhesus P-Selectin-His.
  • FIG. 3 shows antibody affinity of PDL huEP5C7 antibodies to Rat P-Selectin-His.
  • FIG. 4 shows antibody affinity of PDL huEP5C7 antibodies to Human E-Selectin-His.
  • FIG. 5 shows antibody affinity of PDL huEP5C7 antibodies to Cyno E-Selectin-
  • FIG. 6 shows antibody affinity of PDL huEP5C7 antibodies to Rat E-Selectin-His.
  • FIG. 7 shows time-dependent aggregation of PDL huEP5C7 antibodies after incubation under thermal stress conditions.
  • FIG. 8 shows binding of PDL huEP5C7 antibodies to Human E-Selectin after incubation under thermal stress conditions.
  • FIG. 9 shows binding of PDL huEP5C7 antibodies to Human P-Selectin after incubation under thermal stress conditions.
  • FIG. 10 shows time-dependent aggregation of PDL huEP5C7 antibodies after incubation under thermal stress conditions.
  • FIG. 11 shows binding of PDL huEP5C7 antibodies to Human E-Selectin after incubation under thermal stress conditions.
  • FIG. 12 shows binding of PDL huEP5C7 antibodies to Human P-Selectin after incubation under thermal stress conditions.
  • FIG. 13 shows time-dependent aggregation of PDL huEP5C7 antibodies after forced isomerization/acidic stress.
  • FIG. 14 shows time-dependent aggregation of PDL huEP5C7 antibodies after forced isomerization/acidic stress.
  • FIG. 15 shows time-dependent aggregation of PDL huEP5C7 antibodies after forced deamidation/basic stress.
  • FIG. 16 shows time-dependent aggregation of PDL huEP5C7 antibodies after forced deamidation/basic stress.
  • FIG. 17 shows binding of PDL huEP5C7 antibodies to Human E-Selectin after forced deamidation/basic stress.
  • FIG. 18 shows binding of PDL huEP5C7 antibodies to Human P-Selectin after forced deamidation/basic stress.
  • FIG. 19 shows antibody inhibition of endothelial cell-red blood cell (RBC) aggregates under venous flow conditions simulating acute hemolysis.
  • FIG. 20 shows antibody inhibition of endothelial cell-RBC aggregates under venous flow conditions simulating chronic hemolysis.
  • FIG. 21 shows a schematic of an endothelial cell monolayer on a microfluidic chip stimulated with heme.
  • FIG. 22 shows antibody affinity of Ab_0296 antibodies to Human P-Selectin-His.
  • FIG. 23 shows antibody affinity of Ab_0349 antibodies to Human P-Selectin-His.
  • FIG. 24 shows antibody affinity' of Ab_0350 antibodies to Human P-Selectin-His.
  • FIG. 25 shows antibody affinity of Ab_0352 antibodies to Human P-Selectin-His.
  • FIG. 26 shows antibody affinity of Ab_0354 antibodies to Human P-Selectin-His.
  • FIG. 27 shows antibody affinity of Ab_0357 antibodies to Human P-Selectin-His.
  • FIG. 28 shows antibody affinity' of Ab_0296 antibodies to Human E-Selectin-His.
  • FIG. 29 shows antibody affinity of Ab_0349 antibodies to Human E-Selectin-His.
  • FIG. 30 shows antibody affinity of Ab 0350 antibodies to Human E-Selectin-His.
  • FIG. 31 shows antibody affinity of Ab_0352 antibodies to Human E-Selectin-His.
  • FIG. 32 shows antibody affinity' of Ab_0354 antibodies to Human E-Selectin-His.
  • FIG. 33 shows antibody affinity' of Ab_0357 antibodies to Human E-Selectin-His.
  • FIG. 34 shows time-dependent aggregation of Ab_0296 antibodies after incubation under thermal stress conditions.
  • FIG. 35 shows aggregation of Ab_0296 antibodies after incubation under thermal stress conditions for four weeks.
  • FIG. 36 shows binding of Ab_0296 antibodies to Human E-Selectin after incubation under thermal stress conditions.
  • FIG. 37 shows binding of Ab_0296 antibodies to Human P-Selectin after incubation under thermal stress conditions.
  • FIG. 38 shows binding of PDL huEP5C7_IgG2 antibodies to Human E-Selectin after incubation under thermal stress conditions.
  • FIG. 39 shows binding of PDL huEP5C7_IgG2 antibodies to Human P-Selectin after incubation under thermal stress conditions.
  • FIG. 40 shows time-dependent aggregation of Ab_0352 antibodies after incubation under thermal stress conditions.
  • FIG. 41 shows aggregation of Ab_0352 antibodies after incubation under thermal stress conditions for four weeks.
  • FIG. 42 shows binding of Ab_0352 antibodies to Human E-Selectin-His after incubation under thermal stress conditions.
  • FIG. 43 shows binding of Ab_0352 antibodies to Human P-Selectin-His after incubation under thermal stress conditions.
  • FIG. 44 shows binding of PDL huEP5C7_IgG2 antibodies to Human E-Selectin after incubation under thermal stress conditions.
  • FIG. 45 shows binding of PDL huEP5C7_IgG2 antibodies to Human P-Selectin after incubation under thermal stress conditions.
  • FIG. 46 shows time-dependent aggregation of Ab_0357 antibodies after incubation under thermal stress conditions.
  • FIG. 47 shows aggregation of Ab_0357 antibodies after incubation under thermal stress conditions for four weeks.
  • FIG. 48 shows binding of Ab 0357 antibodies to Human E-Selectin-His after incubation under thermal stress conditions.
  • FIG. 49 shows binding of Ab_0357 antibodies to Human P-Selectin-His after incubation under thermal stress conditions.
  • FIG. 50 shows binding of PDL huEP5C7_IgG2 antibodies to Human E-Selectin after incubation under thermal stress conditions.
  • FIG. 51 shows binding of PDL huEP5C7_IgG2 antibodies to Human P-Selectin after incubation under thermal stress conditions.
  • FIG. 52 shows off-target binding of Ab_0296, Ab_0352, Ab_0357, and PDL huEP5C7 after incubation under thermal stress conditions.
  • FIG. 53 shows time-dependent aggregation of Ab_0296 antibodies before forced isomerization/acidic stress.
  • FIG. 54 shows time-dependent aggregation of Ab_0296 antibodies after forced isomerization/acidic stress.
  • FIG. 55 shows aggregation of Ab_0296 and PDL huEP5C7 antibodies after forced isomerization/acidic stress.
  • FIG. 56 shows time-dependent aggregation of Ab_0349 antibodies before forced isomerization/acidic stress.
  • FIG. 57 shows time-dependent aggregation of Ab_0349 antibodies after forced isomerization/acidic stress.
  • FIG. 58 shows aggregation of Ab_0349 and PDL huEP5C7 antibodies after forced isomerization/acidic stress.
  • FIG. 59 shows time-dependent aggregation of Ab_0350 antibodies before forced isomerization/acidic stress.
  • FIG. 60 shows time-dependent aggregation of Ab_0350 antibodies after forced isomerization/acidic stress.
  • FIG. 61 shows aggregation of Ab_0350 and PDL huEP5C7 antibodies after forced isomerization/acidic stress.
  • FIG. 62 shows time-dependent aggregation of Ab_0352 antibodies before forced isomerization/acidic stress.
  • FIG. 63 shows time-dependent aggregation of Ab_0352 antibodies after forced isomerization/acidic stress.
  • FIG. 64 shows aggregation of Ab 0352 and PDL huEP5C7 antibodies after forced isomerization/acidic stress.
  • FIG. 65 shows time-dependent aggregation of Ab_0354 antibodies before forced isomerization/acidic stress.
  • FIG. 66 shows time-dependent aggregation of Ab_0354 antibodies after forced isomerization/acidic stress.
  • FIG. 67 shows aggregation of Ab_0354 and PDL huEP5C7 antibodies after forced isomerization/acidic stress.
  • FIG. 68 shows time-dependent aggregation of Ab_0357 antibodies before forced isomerization/acidic stress.
  • FIG. 69 shows time-dependent aggregation of Ab_0357 antibodies after forced isomerization/acidic stress.
  • FIG. 70 shows aggregation of Ab_0357 and PDL huEP5C7 antibodies after forced isomerization/acidic stress.
  • FIG. 71 shows inhibition by Ab_0357 of platelet-leukocyte aggregates under static conditions in citrate-anti coagulated whole blood from a healthy donor.
  • FIG. 72 shows inhibition by Ab_0357 of platelet-leukocyte aggregates under static conditions in citrate-anticoagulated whole blood from a healthy donor.
  • FIG. 73 shows inhibition by Ab_0357 of platelet-leukocyte aggregates under static conditions in citrate-anticoagulated whole blood from a healthy donor.
  • FIG. 74 shows inhibition by Crizanlizumab of platelet-leukocyte aggregates under static conditions in citrate-anti coagulated whole blood from a healthy donor.
  • FIG. 75 shows inhibition by Crizanlizumab of platelet-leukocyte aggregates under static conditions in citrate-anticoagulated whole blood from a healthy donor.
  • FIG. 76 shows inhibition by Crizanlizumab of platelet-leukocyte aggregates under static conditions in citrate-anticoagulated whole blood from a healthy donor.
  • FIG. 77 shows basal levels of red blood cell adhesion from samples from healthy donors on heme activated HUVECs under physiologic flow conditions compared to nonactivated HUVECs using a HUVEC coated biochip microfluidic platform.
  • FIG. 78 shows red blood cell adhesion data from samples from SCD donors treated with a test compound (crizanlizumab, BHB00070, and PF-07209326) on long term (chronic) activated HUVECs under physiologic flow conditions using a HUVEC coated biochip microfluidic platform.
  • a test compound crizanlizumab, BHB00070, and PF-07209326
  • FIG. 79 shows red blood cell adhesion data from samples from SCD donors treated with a test compound (crizanlizumab, BHB00070, and PF-07209326) on short term (acute) activated HUVECs under physiologic flow conditions using a HUVEC coated biochip microfluidic platform.
  • a test compound crizanlizumab, BHB00070, and PF-07209326
  • a and “an” refers to one or more (i.e., at least one) of the grammatical object of the article.
  • a polypeptide encompasses one or more polypeptides.
  • exogenous refers to any material introduced from or originating from outside a cell, a tissue or an organism that is not produced by or does not originate from the same cell, tissue, or organism in which it is being introduced.
  • transduced refers to a process by which exogenous nucleic acid is introduced or transferred into a cell.
  • a “transduced,” “transfected,” or “transformed” cell e.g., a mammalian cell, a hepatocyte
  • exogenous nucleic acid e.g. an expression vector
  • nucleic acid refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either a single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some embodiments of any of the nucleic acids described herein, the nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA.
  • Modifications can be introduced into a nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis.
  • Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., arginine, lysine and histidine
  • acidic side chains e.g., aspartic acid and glutamic acid
  • uncharged polar side chains e.g., asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, and tryptophan
  • nonpolar side chains e.g..
  • alanine isoleucine, leucine, methionine, phenylalanine, proline, and valine
  • beta-branched side chains e.g., isoleucine, threonine, and valine
  • aromatic side chains e.g., histidine, phenylalanine, try ptophan, and ty rosine
  • aromatic side chains e.g., histidine, phenylalanine, tryptophan, and tyrosine
  • amino acid sequence refers to the arrangement of amino acids in protein. In some instances, the amino acid sequence is one that occurs in nature. In other instances, the amino acid sequence is engineered by humans.
  • the amino acids may be the L- optical isomer or the D-optical isomer.
  • a polypeptide can be a chain of at least three amino acids, peptide-mimetics, a protein, a recombinant protein, an antibody (monoclonal or polyclonal), an antibody fragment, a single-chain variable fragment (scFv), an antigen, an epitope, an enzy me, a receptor, a vitamin, or a structure analogue or combinations thereof.
  • a polypeptide chain may vary in length.
  • L-enantiomeric and D-enantiomeric amino acids that form a polypeptide are as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G. Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F. Phe); proline (P, Pro); serine (S. Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Vai).
  • X or Xaa can indicate any amino acid.
  • an antigen-binding domain is used to refer to one or more antibodyvariable domain(s) (e.g.. formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides) that is capable of specifically binding to one or more different antigen(s).
  • an antigen-binding domain can bind to an antigen or epitope with specificity and affinity- similar to that of naturally-occurring antibodies.
  • the antigen-binding domain can be an antibody or a fragment thereof.
  • an antigen-binding domain can include an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are know n in the art.
  • nucleotide sequence encoding a protein includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence.
  • N-terminally positioned when referring to a position of a first domain or sequence relative to a second domain or sequence in a poly peptide primary- amino acid sequence means that the first domain is located closer to the N-terminus of the polypeptide primary' amino acid sequence. In some embodiments, there may be additional sequences and/or domains between the first domain or sequence and the second domain or sequence.
  • C-terminally positioned when referring to a position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence means that the first domain is located closer to the C-terminus of the polypeptide primary' amino acid sequence. In some embodiments, there may be additional sequences and/or domains betw een the first domain or sequence and the second domain or sequence.
  • '‘antibody” refers to a protein with an immunoglobulin fold that specifically binds to an antigen via its variable region or regions.
  • antibody is used herein in the broadest sense and encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller et al., J. Immunol. 170:4854-4861, 2003). Antibodies may be murine, human, humanized, chimeric, or derived from other species. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immunol. Biology, 5th Ed., Garland Publishing. New York).
  • a target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs (complementarity determining regions) on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.
  • An antibody can, e.g., include a full- length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds an antigen of a target of interest or part thereof.
  • the immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • the immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin.
  • Antibodies bound to various types of molecules such as polyethylene glycols (PEGs) may be used as modified antibodies.
  • PEGs polyethylene glycols
  • antibody fragments refers to a portion of a full-length antibody or a polypeptide that includes a portion of a full-length antibody, that retains antigen-binding activity’ via its variable region or regions.
  • antibody fragments include Fab, Fab 1 , F(ab')2, and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al., Protein Eng. Design & Sei. 17(4):315-323, 2004). fragments produced by a Fab expression library, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • CDR complementarity determining region
  • HVRs hypervariable regions
  • the three CDRs in the antibody heavy chain and the antibody light chain interrupt four framework regions in the heavy chain variable domain and the light chain variable domain.
  • the CDRs of each chain are ty pically referred to as CDR1 , CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
  • framework regions of different light immunoglobulin chains and different heavy immunoglobulin chains are relatively conserved within different antibodies produced by a mammal.
  • the framework regions of light and heavy immunoglobulin chains serve to position and align the CDRs in three-dimensional space.
  • Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBASE2” germline variable gene sequence database for human and mouse sequences.
  • “Monospecific” antigen-binding protein refers to the ability 7 of the antigen-binding protein, such as an antibody, to bind only one epitope.
  • “Bispecific” antigen-binding protein refers to the ability of the antigen-binding protein to bind two different epitopes.
  • “Multispecific” antigen-binding protein refers to the ability of the antigen-binding protein to bind more than one epitope.
  • a multispecific antigen-binding protein such as a multispecific antibody, encompasses a bispecific antigen-binding protein or a bispecific antibody.
  • the epitopes can be on the same antigen, or each epitope can be on a different antigen. Therefore, in certain embodiments, a multispecific antigen-binding protein provided herein, such as a bispecific antibody, binds to two different antigens. In certain embodiments, the multispecific antigen-binding protein, such as a bispecific antibody, binds to different epitopes on one antigen.
  • a multispecific antigen-binding protein binds to each epitope with a dissociation constant (Kd) of about ⁇ 1 M, about ⁇ 100 nM, about ⁇ 10 nM, about ⁇ 1 nM, about ⁇ 0.1 nM, about ⁇ 0.01 nM, or about ⁇ 0.001 nM (e.g., about 10' 8 M or less, e.g., from about 10' 8 M to about 10’ 1? M, e.g., from about 10' 9 M to about 10' 10 M).
  • Kd dissociation constant
  • Fully human antibodies can be obtained using a variety of methods, for example using yeast-based libraries or transgenic animals (e.g., mice) that can produce repertoires of human antibodies.
  • yeast-based libraries or transgenic animals e.g., mice
  • Yeast presenting human antibodies on their surface that bind to an antigen of interest can be selected using FACS (Fluorescence-Activated Cell Sorting) based methods or bycapture on beads using labelled antigens.
  • Transgenic animals that have been modified to express human immunoglobulin genes can be immunized with an antigen of interest and antigen-specific human antibodies isolated using B-cell sorting techniques. Human antibodies produced using these techniques can then be characterized for desired properties such as affinity, developability and selectivity.
  • alternative antibody formats can be used.
  • Alternative antibody formats include alternative scaffolds in which the one or more CDRs of an antibody disclosed herein can be arranged onto a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301) or an EGF domain.
  • a “humanized antibody” refers to a type of engineered antibody having its CDRs derived from anon-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one or more human immunoglobulin(s).
  • framework support residues may be altered to preserve binding affinity (see, e.g., Queen et al. Proc. Natl Acad Sci USA, 86: 10029-10032 (1989), Hodgson et al. Bio/Technology, 9:421 (1991)).
  • a suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABAT database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody.
  • a human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs.
  • a suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.
  • the prior art describes several ways of producing such humanized antibodies - see, for example, EP-A- 0239400 and EP-A-054951.
  • single variable domain refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains such as VH, VHH and VL and modified antibody variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.
  • a single variable domain can bind an antigen or epitope independently of a different variable region or domain.
  • a "domain antibody” or “DAB” may be considered the same as a "single variable domain”.
  • a single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent (for example, as disclosed in WO 00/29004 Al), nurse shark and Camelid VHH DABs.
  • Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains.
  • Such VHH domains may be humanized according to standard techniques available in the art, and such domains are considered to be "single variable domains".
  • VH includes camelid VHH domains.
  • VH and/or VL domains disclosed herein may be incorporated, e.g., in the form of a scFv. into CAR-T therapeutics.
  • the term '‘promoter” means a DNA sequence recognized by enzymes/proteins in a cell (e.g., a mammalian cell, a hepatocyte) required to initiate the transcription of an operably linked coding sequence (e.g., a nucleic acid encoding a polypeptide (e.g., any of the exemplary polypeptides described herein).
  • a promoter typically refers, to e.g., a nucleotide sequence to which an RNA polymerase and/or any associated factor binds and at which transcription is initiated.
  • the promoter can be constitutive, inducible, or tissue-specific (e.g., a liver-specific promoter).
  • the term “enhancer” refers to a nucleotide sequence that can increase the transcription of an operably linked nucleic acid (e.g., a nucleic acid encoding a polypeptide (e.g., any of the exemplary polypeptides described herein).
  • An enhancer can increase the level of transcription by providing additional binding sites for transcription-associated proteins (e.g., transcription factors). Unlike promoters, enhancers can act at distances further away from the transcription start site (e.g., as compared to a promoter).
  • '‘homology” or “similarity” between two proteins is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one protein sequence to the second protein sequence. Similarity may be determined by procedures which are well-known in the art, for example, a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information).
  • nucleic or percent “identity ,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
  • sequence comparison of polypeptides typically one amino acid sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g., visual alignment or using publicly available software using known algorithms to achieve maximal alignment. Such programs include the BLAST programs, ALIGN, ALIGN-2 or Megalign. The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.
  • affinity refers to the strength of the sum of all non-covalent interactions between an antigen-binding site and its antigen. Unless otherwise indicated, “affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between an antigenbinding domain and an antigen. Affinity can be measured, e.g., using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).
  • SPR surface plasmon resonance
  • BIACORE® BIACORE®
  • biolayer interferometry e.g., FORTEBIO®
  • single-chain polypeptide means a polypeptide comprising a single polypeptide chain.
  • multi-chain polypeptide means a complex of two or more (e.g., 2. 3, 4, 5, 6, 7, or 8) polypeptide chains (e.g., the same or different polypeptide chains) that covalently and/or non-covalently associate with each other.
  • polypeptide chains of a multi-chain polypeptide can associate through the use of tw o domains that associate with each other (e.g., tw o Fc domains or IL-15 and the sushi domain of IL-15 receptor alpha).
  • variable domain residue numbering as in Kabat or “amino acid position numbering as in Kabat”, and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain few er or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "‘standard’’ Kabat numbered sequence. It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full-length antibody sequences. There are also alternative numbering conventions for CDR sequences, for example those set out in Chothia et al. (1989) Nature 342:877-883.
  • CDR sequences available to a skilled person include “AbM” (University' of Bath) and “contact’’ (university' College London) methods.
  • the minimum overlapping region using at least two of the Kabat, Chothia, AbM and contact methods can be determined to provide the ‘minimum binding unit’.
  • the minimum binding unit may be a sub-portion of a CDR.
  • Table I below represents one definition using each numbering convention for each CDR or binding unit.
  • the Kabat numbering scheme is used in Table 1 to number variable domain amino acid sequence.
  • some of the CDR definition may vary depending on the individual publication used.
  • the target molecule-binding polypeptides include an antigen-binding domain specific for E-selectin, P- selectin, or both E-selectin and P- selectin.
  • the target molecule-binding polypeptides described herein may have at least one or more amino acid modifications in one or more regions of an antibody, including but not limited to, a heavy chain, light chain, variable heavy chain, variable light chain, heavy chain constant domain, light chain constant domain, Fc region. Fab region, hinge region, VH domain, VL domain, CFI1 domain, CH2 domain, CH3 domain, and paratrope.
  • the target molecule-binding polypeptides may be modified by at least one or more amino acid substitution, deletion, addition, or a combination thereof at one or more position while the variant target molecule-binding polypeptide substantially retains the biological characteristics of the unmodified protein, such as binding to P-selectin, E-selectin, or both P-selectin and E-selectin.
  • the amino acid sequence of the target molecule-binding polypeptides may have 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, or at least 15 amino acid insertions, deletions, substitutions, or a combination thereof
  • one or more amino acid modifications described herein can be in the antigen-binding (Fab) fragments of an antibody or antibody binding fragment thereof.
  • the modification can be within the Fab region of a heavy chain polypeptide (H) and a first light chain polypeptide (L).
  • the modification can be within a heavy chain variable domain (VH), a heavy chain constant domain (CHI), a light chain variable domain (VL), a light chain constant domain (CL), or a combination thereof.
  • one or more amino acid modification can be in the Fab VH and VL with the N- terminal region. The VH, VL.
  • HC or LC sequence disclosed herein may be a variant sequence with up to 15 amino acid substitutions, insertion, or deletions.
  • the variant sequence may have up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s), addition(s). or deletion(s).
  • the sequence variation may exclude one or more or all of the CDRs, for example, the CDRs are the same as the VH, VL.
  • HC or LC sequence and the variation is in the remaining portion of the VH or VL, HC, or LC sequence, so that the CDR sequences are fixed and intact.
  • the target molecule-binding polypeptides described herein may have 60, 70, 80, 90 ,95, 100% identity to at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 90, 100, consecutive amino acid of a HCDR, LCDR, light chain variable region, heavy chain vanable region, light chain, heavy chain, constant region, or a framework region disclosed herein.
  • Each of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 may be modified alone or in combination with any other CDR, in any permutation or combination.
  • the modification is a substitution, particularly a conserved substitution, for example in Table 2 below.
  • the target molecule-binding polypeptide may comprise CDRs: HCDR1 comprising SEQ ID NO: 5, HCDR2 comprising SEQ ID NO: 6, HCDR3 comprising SEQ ID NO: 7, LCDR1 selected from the group consisting of SEQ ID NOs: 8-12, LCDR2 of SEQ ID NOs: 13-16, and LCDR3 selected from the group consisting of SEQ ID NO: 17-20, for the same, functional fragments thereof, functional variants thereof, and combination thereof.
  • any of the target molecule-binding polypeptides described herein bind a sequence set forth in SEQ ID NOs: 1-4 and 62-63, or a portion thereof.
  • the target molecule-binding polypeptide may comprise CDRs: HCDR1 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or 100% homolog ⁇ ’ to SEQ ID NO: 5, HCDR2 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%.
  • any of the target molecule-binding polypeptides described herein bind a sequence set forth in SEQ ID NOs: 11-4 and 62-63, or a portion or thereof.
  • the target molecule-binding polypeptide may comprise CDRs: an HCDR1 having an amino acid sequence set forth in SEQ ID NO: 5, an HCDR2 having an amino acid set forth in SEQ ID NO: 6, an HCDR3 having an amino acid set forth in SEQ ID NO: 7, an LCDR1 having an amino acid set forth in any one of SEQ ID NOs: 8-12, an LCDR2 having an amino acid set forth in any one of SEQ ID NOs: 13-16, and an LCDR3 having an amino acid set forth in any one of SEQ ID NOs: 17-20.
  • the target molecule-binding polypeptide may comprise CDRs: an HCDR1 having an amino acid sequence set forth in SEQ ID NO: 5 containing 1 amino acid substitution, an HCDR2 having an amino acid set forth in SEQ ID NO: 6 containing 1 , 2 or 3 amino acid substitutions, an HCDR3 having an amino acid set forth in SEQ ID NO: 7 containing 1 amino acid substitution, an LCDR1 having an amino acid set forth in any one of SEQ ID NOs: 8-12 containing 1 or 2 amino acid substitutions, an LCDR2 having an amino acid set forth in any one of SEQ ID NOs: 13-16 containing 1 amino acid substitution, and an LCDR3 having an amino acid set forth in any one of SEQ ID NOs: 17- 20 containing 1 amino acid substitution.
  • the amino acid substitution is a conserved substitution.
  • the target molecule-binding polypeptide may comprise an HCDR1 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 5.
  • the target molecule-binding polypeptide may comprise an HCDR2 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%.
  • the target molecule-binding polypeptide may comprise an HCDR3 having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 7.
  • the target molecule-binding polypeptide may comprise an LCDR1 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NOs: 8-12.
  • the target molecule-binding polypeptide may comprise LCDR2 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%.
  • the target molecule-binding polypeptide may comprise an LCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NO: 17- 20.
  • the target molecule-binding polypeptide may comprise an HCDR1, HCDR2 and HCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NOS: 5. 6, and 7 respectively and an LCDR1, LCDR2 and LCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%.
  • the target molecule-binding polypeptide may comprise an HCDR1, HCDR2 and HCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NOS: 5, 6, and 7 respectively and an LCDR1, LCDR2 and LCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NOS: 9, 13, and 17, respectively.
  • the target molecule-binding polypeptide may comprise an HCDR1, HCDR2 and HCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to SEQ ID NOS: 5, 6, and 7 respectively and an LCDR1, LCDR2 and LCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%.
  • the target molecule-binding polypeptide may comprise an HCDR1, HCDR2 and HCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99%. or 100% homology to SEQ ID NOS: 5, 6.
  • an LCDR1, LCDR2 and LCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to SEQ ID NOS: 8, 14, and 17, respectively.
  • the target molecule-binding polypeptide may comprise an HCDR1, HCDR2 and HCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to SEQ ID NOS: 5, 6, and 7 respectively and an LCDR1, LCDR2 and LCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NOS: 10, 15, and 17, respectively.
  • the target molecule-binding polypeptide may comprise an HCDR1.
  • HCDR2 and HCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to SEQ ID NOS: 5, 6, and 7 respectively and an LCDR1, LCDR2 and LCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%.
  • the target molecule-binding polypeptide may comprise an HCDR1, HCDR2 and HCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NOS: 5, 6, and 7 respectively and an LCDR1.
  • LCDR2 and LCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to SEQ ID NOS: 8, 16, and 20, respectively.
  • the target molecule-binding polypeptide may comprise an HCDR1, HCDR2 and HCDR3 having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NOS: 5, 6, and 7 respectively and an LCDR1, LCDR2 and LCDR3 having at least 80%, 81%, 82%, 83%, 84%, 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%.
  • any of the target molecule-binding polypeptides described herein bind a sequence set forth in SEQ ID NOs: 1-4 and 62-63, or a portion thereof.
  • the target molecule-binding polypeptide may comprise an HCDR1.
  • HCDR2 and HCDR3 having amino acid sequences set forth in SEQ ID NOS: 5, 6, and 7, respectively and having at least 1, 2, or 3 amino acid substitutions, and an LCDR1, LCDR2 and LCDR3 amino acid sequences set forth in SEQ ID NOS: 8, 13, and 18, respectively and having at least 1, 2, or 3 amino acid substitutions.
  • the target molecule-binding polypeptide may comprise an HCDR1, HCDR2 and HCDR3 having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NOS: 5. 6, and 7 respectively and an LCDR1, LCDR2 and LCDR3 having at least 80%, 81%, 82%, 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%.
  • the target molecule-binding polypeptide may comprise an HCDR1, HCDR2 and HCDR3 having an amino acid sequence set forth in SEQ ID NOS: 5, 6, and 7, respectively and an LCDR1, LCDR2 and LCDR3 having an amino acid sequence set forth in SEQ ID NOS: 12, 16, and 17, respectively, SEQ ID NOs: 8, 13, and 17, respectively, SEQ ID NOs: 8, 14, and 17, respectively, SEQ ID NOs: 8, 15, and 17, respectively, SEQ ID NOs: 8, 16, and 17.
  • the target molecule-binding polypeptide may comprise an HCDR1, HCDR2 and HCDR3 having a variant amino acid sequence with 1 , 2, or 3 amino acid substitutions in the amino acid sequence set forth in SEQ ID NOS: 5, 6, and 7, respectively, and an LCDR1, LCDR2 and LCDR3 having a variant amino acid sequence with 1 or 2 amino acid substitutions in the amino acid sequence set forth in SEQ ID NOS: 12, 16, and 17, respectively, SEQ ID NOs: 8, 13, and 17, respectively, SEQ ID NOs: 8, 14, and 17, respectively, SEQ ID NOs: 8, 15, and 17, respectively, SEQ ID NOs: 8, 16, and 17, respectively, SEQ ID NOs: 9, 13, and 17, respectively, SEQ ID NOs: 9, 14, and 17, respectively, SEQ ID NOs: 9, 15, and 17, respectively, SEQ ID NOs: 9, 16, and 17, respectively, SEQ ID NOs: 10, 13, and 17.
  • any of the target molecule-binding polypeptides described herein bind a sequence set forth in SEQ ID NOs: 1-4 and 62-63, or a portion thereof.
  • the target molecule-binding polypeptide may comprise VH having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NOs: 24, 30, 34, 43, 46, or 48.
  • the target molecule-binding polypeptide may comprise VL having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99%. or 100% homology to any one of SEQ ID NOs: 25, 31, 35, 37, 40, 44, 47, or 49.
  • the target molecule-binding polypeptide may comprise a VH having an amino acid sequence set forth in any one of SEQ ID NOs: 24, 30, 34, 43, 46, or 48, and a VL having an amino acid sequence set forth in any one of SEQ ID NOs: 25, 31, 35, 37, 40. 44, 47, or 49.
  • the target molecule-binding polypeptide may comprise a VH having a variant amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acid substitutions in an amino acid sequence set forth in any one of SEQ ID NOs: 24, 30, 34, 43.
  • any of the target molecule-binding polypeptides described herein bind a sequence set forth in SEQ ID NOs: 1-4 and 62-63, or a portion thereof.
  • the target molecule-binding polypeptide may comprise heavy chain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NOs: 22, 26, 28, 32, 38, 41 , or 45.
  • the target molecule-binding polypeptide may comprise light chain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NOs: 23, 27, 29, 33, 36, 39, or 42.
  • the target molecule-binding polypeptide may comprise a heavy chain having an amino acid sequence set forth in any one of SEQ ID NOs: 22, 26, 28, 32, 38, 41, or 45, and a light chain having an amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 29, 33, 36, 39, or 42.
  • the target molecule-binding polypeptide may comprise a heavy chain having a variant amino acid sequence containing from 1 to 88 ammo acid substitutions in an amino acid sequence set forth in any one of SEQ ID NOs: 22, 26, 28, 32, 38, 41, or 45, and a light chain having a variant amino acid sequence containing from 1 to 42 amino acid substitutions in an amino acid sequence set forth in any one of SEQ ID NOs: 23. 27, 29, 33, 36, 39, or 42.
  • the target molecule-binding polypeptide may comprise a heavy chain having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in an amino acid sequence set forth in any one of SEQ ID NOs: 22, 26, 28, 32, 38, 41, or 45, and a light chain having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in an amino acid sequence set forth in any one of SEQ ID NOs: 23. 27. 29. 33. 36, 39, or 42.
  • the target molecule-binding polypeptide may comprise a heavy chain having a variant amino acid sequence containing from 1 to 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) amino acid substitutions in an amino acid sequence set forth in any one of SEQ ID NOs: 22, 26, 28, 32, 38, 41, or 45, and a light chain having a variant amino acid sequence containing from 1 to 11 (e.g., 1. 2, 3, 4, 5. 6, 7, 8. 9, 10. or 11) amino acid substitutions in an amino acid sequence set forth in any one of SEQ ID NOs: 23, 27, 29, 33, 36, 39, or 42.
  • any of the target molecule-binding polypeptides described herein bind a sequence set forth in SEQ ID NOs: 1-4 and 62-63, or a portion thereof.
  • the target molecule-binding polypeptide can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NOs: 5- 49 as shown in Table 3.
  • the target molecule-binding polypeptide can comprise a sequence having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NOs: 5-49.
  • the target-molecule-binding polypeptide comprises a sequence having at least 90%, 95%, 97%, 99%, or 100% homology to any one of SEQ ID NOs: 5-49. In some embodiments, the target molecule-binding polypeptide comprises a sequence that is identical to any one of SEQ ID NOs: 5-49. In various embodiments, any of the target molecule-binding polypeptides described herein bind a sequence set forth in SEQ ID NOs: 1-4 and 62-63, or a portion thereof.
  • the target molecule-binding polypeptide comprises a heavy chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO:22 and comprises a light chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%. 99%. or 100% homolog ⁇ ’ to SEQ ID NO: 23.
  • the target molecule-binding polypeptide comprises a heavy chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homolog ⁇ ’ to SEQ ID NO: 26 and comprises a light chain sequence having at least 80%, 81%, 82%, 83%, 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology to SEQ ID NO:27.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 22, and a light chain sequence having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 23.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 22.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence set forth in SEQ ID NO: 22, and a light chain sequence set forth in SEQ ID NO: 23.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 26, and a light chain sequence having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 27.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 26, and a light chain sequence having a variant amino acid sequence containing from 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 27.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence set forth in SEQ ID NO: 26, and a light chain sequence set forth in SEQ ID NO: 27.
  • the target molecule-binding polypeptide comprises a heavy chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 28 and comprises a light chain sequence having at least 80%, 81%, 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%, 99%, or 100% homology' to SEQ ID NO: 29.
  • the target moleculebinding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 28, and a light chain sequence having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 29.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 22 (e.g., 1, 2, 3, 4, 5, 6, 7.
  • the target moleculebinding polypeptide may comprise a heavy chain sequence set forth in SEQ ID NO: 28 and a light chain sequence set forth in SEQ ID NO: 29.
  • the target moleculebinding polypeptide comprises a heavy chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 32 and comprises a light chain sequence having at least 80%, 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%, 98%, 99%, or 100% homology to SEQ ID NO: 33.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 32, and a light chain sequence having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 33.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or 22) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 32, and a light chain sequence having a variant amino acid sequence containing from 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 33.
  • 1 to 22 e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or 22
  • 1 to 11 e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11
  • the target molecule-binding polypeptide may comprise a heavy chain sequence set forth in SEQ ID NO: 32 and a light chain sequence set forth in SEQ ID NO: 33.
  • the target molecule-binding polypeptide comprises a heavy chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 32, and a light chain sequence having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 36.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 22 (e.g., 1, 2, 3. 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 32, and a light chain sequence having a variant amino acid sequence containing from 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 36.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence set forth in SEQ ID NO: 32 and a light chain sequence set forth in SEQ ID NO: 36.
  • the target molecule-binding polypeptide comprises a heavy chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99%. or 100% homology to SEQ ID NO: 38 and comprises a light chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology- to SEQ ID NO: 36.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 38, and a light chain sequence having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 36.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 22 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence set forth in SEQ ID NO: 38 and a light chain sequence set forth in SEQ ID NO: 36.
  • the target molecule-binding polypeptide comprises a heavy chain sequence having at least 80%, 81%, 82%, 83%. 84%. 85%. 86%. 87%. 88%. 89%.
  • SEQ ID NO: 39 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%, or 100% homology to SEQ ID NO: 32 and comprises a light chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 39.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 32, and a light chain sequence having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 39.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 32, and a light chain sequence having a variant amino acid sequence containing from 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, or 11) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 39.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence set forth in SEQ ID NO: 32 and a light chain sequence set forth in SEQ ID NO: 39.
  • the target molecule-binding polypeptide comprises a heavy chain sequence having at least 80%, 81%, 82%, 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%, or 100% homologj’ to SEQ ID NO: 41 and comprises a light chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to SEQ ID NO: 42.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 41, and a light chain sequence having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 42.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 41, and a light chain sequence having a variant amino acid sequence containing from 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 42.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence set forth in SEQ ID NO: 41 and a light chain sequence set forth in SEQ ID NO: 42.
  • the target molecule-binding polypeptide comprises a heavy chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology 7 to SEQ ID NO: 45 and comprises a light chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology 7 to SEQ ID NO: 42.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 45, and a light chain sequence having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 42.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 45, and a light chain sequence having a variant amino acid sequence containing from 1 to 11 (e.g., 1, 2. 3, 4, 5. 6. 7, 8, 9. 10. or 11) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 42.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence set forth in SEQ ID NO: 45 and a light chain sequence set forth in SEQ ID NO: 42.
  • the target molecule-binding polypeptide comprises a heavy chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology 7 to SEQ ID NO: 32 and comprises a light chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology 7 to SEQ ID NO: 60.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 32, and a light chain sequence having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 60.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 22 (e.g.. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. 12. 13.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence set forth in SEQ ID NO: 32 and a light chain sequence set forth in SEQ ID NO: 60.
  • the target molecule-binding polypeptide comprises a heavy chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 32 and comprises a light chain sequence having at least 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 61 .
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 44 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 32, and a light chain sequence having a variant amino acid sequence containing from 1 to 21 amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 61.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence having a variant amino acid sequence containing from 1 to 22 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 32.
  • the target molecule-binding polypeptide may comprise a heavy chain sequence set forth in SEQ ID NO: 32 and a light chain sequence set forth in SEQ ID NO: 61.
  • any of the target molecule-binding polypeptides described herein bind a sequence set forth in SEQ ID NOs: 1-4 and 62-63, or a portion thereof.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 24 and comprises a light chain variable domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 25.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 30 and comprises a light chain variable domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 31.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology to SEQ ID NO: 34 and comprises a light chain variable domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to SEQ ID NO: 35.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80%, 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%, 98%, 99%, or 100% homology to SEQ ID NO: 34 and comprises a light chain variable domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to SEQ ID NO: 37.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 25.
  • the target moleculebinding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing about 1-1 1 (e g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable domain having a variant amino acid sequence containing about 1-10 (e g., 1, 2.
  • the target molecule-binding polypeptide such as a scFv comprises a heavy chain variable domain set forth in SEQ ID NO: 24 and a light chain variable domain set forth in SEQ ID NO: 25.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 30 and a light chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 31.
  • the target molecule- binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing about 1-11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 30 and a light chain variable domain having a variant amino acid sequence containing about 1-10 (e.g., 1, 2.
  • the target molecule-binding polypeptide such as a scFv comprises a heavy chain variable domain set forth in SEQ ID NO: 30 and a light chain variable domain set forth in SEQ ID NO: 31.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a vanant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 34 and a light chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 35.
  • the target moleculebinding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing about 1 -1 1 (e g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 34 and a light chain variable domain having a variant amino acid sequence containing about 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9. or 10) amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 35.
  • the target molecule-binding polypeptide such as a scFv comprises a heavy chain variable domain set forth in SEQ ID NO: 34 and a light chain variable domain set forth in SEQ ID NO: 35.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 34 and a light chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 37.
  • the target moleculebinding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing about 1-11 (e.g., 1, 2, 3, 4, 5. 6, 7, 8.
  • the target molecule-binding polypeptide such as a scFv comprises a heavy chain variable domain set forth in SEQ ID NO: 34 and a light chain variable domain set forth in SEQ ID NO: 37.
  • any of the target molecule-binding polypeptides described herein bind a sequence set forth in SEQ ID NOs: 1-4 and 62-63, or a portion thereof.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 34 and comprises a light chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 40.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 34 and a light chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 40.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing about 1-11 (e g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 34 and a light chain variable domain having a variant amino acid sequence containing about 1 -10 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 40.
  • the target molecule-binding polypeptide such as a scFv comprises a heavy chain variable domain set forth in SEQ ID NO: 34 and a light chain variable domain set forth in SEQ ID NO: 40.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 43 and comprises a light chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology to SEQ ID NO: 44.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 43 and a light chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 44.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing about 1-11 (e g., 1, 2.
  • the target molecule-binding polypeptide such as a scFv comprises a heavy chain variable domain set forth in SEQ ID NO: 43 and a light chain variable domain set forth in SEQ ID NO: 44.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homolog ⁇ ’ to SEQ ID NO: 46 and comprises a light chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 47.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 46 and a light chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 47.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing about 1-11 (e.g., 1. 2, 3, 4, 5. 6, 7, 8.
  • the target molecule-binding polypeptide such as a scFv comprises a heavy chain variable domain set forth in SEQ ID NO: 46 and a light chain variable domain set forth in SEQ ID NO: 47.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%. or 100% homology to SEQ ID NO: 48 and comprises a light chain sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 49.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 48 and a light chain variable domain having a variant amino acid sequence containing at least 1 amino acid substitution in the amino acid sequence set forth in SEQ ID NO: 49.
  • the target molecule-binding polypeptide comprises a heavy chain variable domain having a variant amino acid sequence containing about 1-11 (e.g., 1, 2, 3, 4, 5.
  • the target molecule-binding polypeptide such as a scFv comprises a heavy chain variable domain set forth in SEQ ID NO: 48 and a light chain variable domain set forth in SEQ ID NO: 49.
  • any of the target molecule-binding polypeptides described herein bind a sequence set forth in SEQ ID NOs: 1-4 and 62-63, or a portion thereof.
  • a heavy chain variable domain and/or a light chain variable domain described herein may be modified by at least one amino acid substitution, deletion, or insertion, wherein the variant target molecule binding protein substantially retains the biological characteristics of the unmodified protein, such as binding to P-selectin, E-selectin, or both P- selectin and E-selectin.
  • a heavy chain variable domain and/or a light chain variable domain may be modified alone or in combination with any other CDR, in any permutation or combination.
  • the heavy chain variable domain and/or light chain variable domain may be modified by the substitution, deletion, or insertion of 1 amino acid to 10 amino acids.
  • the heavy chain variable domain and/or light chain variable domain may have a 1 amino acid to 2 amino acids, 1 amino acid to 3 amino acids, 1 amino acid to 4 amino acids, 1 amino acid to 5 amino acids, 1 amino acid to 6 amino acids, 1 amino acid to 7 amino acids, 1 amino acid to 8 amino acids, 1 amino acid to 9 amino acids, 1 amino acid to 10 amino acids, 2 amino acids to 3 amino acids, 2 amino acids to 4 amino acids. 2 amino acids to 5 amino acids, 2 amino acids to 6 amino acids, 2 amino acids to 7 amino acids, 2 amino acids to 8 amino acids, 2 amino acids to 9 amino acids, 2 amino acids to 10 amino acids, 3 amino acids to 4 amino acids, 3 amino to 5 amino acids, 3 amino acids to 6 amino acids, 3 amino acids to 7 amino acids.
  • amino acids to 8 amino acids 3 amino acids to 9 amino acids, 3 amino acids to 10 amino acids, 4 amino acids to 5 amino acids, 4 amino acids to 6 amino acids.
  • 4 amino acids to 7 amino acids 4 amino acids to 8 amino acids, 4 amino acids to 9 amino acids, 4 amino acids to 10 amino acids, 5 amino acids to 6 amino acids, 5 amino acids to 7 amino acids, 5 amino acids to 8 amino acids, 5 amino acids to 9 amino acids, 5 amino acids to 10 amino acids, 6 amino acids to 7 amino acids.
  • the LCDR e.g., LCDR1, LCDR2, or LCDR3
  • HCDR e.g., HCDR1, HCDR2. or HCDR3
  • a modification e.g., a substitution, insertion or deletion
  • the heavy chain variable domain and/or light chain variable domain may have a modification (e g., a substitution, insertion or deletion) of at least 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids or 10 amino acids. In some embodiments, the heavy chain variable domain and/or light chain variable domain may have a modification (e.g.. a substitution, insertion or deletion) of at most 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids.
  • the target molecule-binding polypeptide comprises one or more variable regions, for example, wherein one or more of the variable regions comprises a sequence having 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%.
  • a variable region of a target molecule-binding polypeptide can be a heavy chain variable region (VH).
  • a heavy chain variable region of the target molecule-binding polypeptide can comprise a sequencing having 80%. 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to any one of SEQ ID NOs: 24, 30, 34, 43, 46, or 48.
  • a variable region of a target molecule-binding polypeptide can be a light chain variable region (VL).
  • a light chain variable region of the target molecule-binding polypeptide can comprise a sequencing having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to any one of SEQ ID NOs: 25, 31,
  • the target molecule-binding polypeptide comprises one or more variable regions containing an amino acid sequence set forth in any one of SEQ ID NOs: 24. 30. 34, 43, 46, 48, 25, 31, 35, 37, 40, 44, 47, or 49.
  • the target moleculebinding polypeptide comprises one or more variable regions containing a variant amino acid sequence set forth in any one of SEQ ID NOs: 24. 30, 34, 43, 46, 48, 25, 31, 35, 37, 40, 44, 47, or 49 and having at least 1, 2. 3, 4, 5. 6, 7, 8, 9. 10 amino acid modifications.
  • each domain of three complementarity-determining region LCDR: LCDR1, LCDR2, and LCDR3 for VL, and HCDR1, HCDR2, and HCDR3 for VH may be modified alone or in combination with any other CDR. in any permutation or combination.
  • the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 may be modified by substitution, deletion, or insertion of 1 amino acid to 10 amino acids.
  • the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 may have 1 amino acid to 2 amino acids, 1 amino acid to 3 amino acids, 1 amino acid to 4 amino acids, 1 amino acid to 5 amino acids, 1 amino acid to 6 amino acids, 1 amino acid to 7 amino acids, 1 amino acid to 8 amino acids, 1 amino acid to 9 amino acids, 1 amino acid to 10 amino acids, 2 amino acids to 3 amino acids, 2 amino acids to 4 amino acids, 2 amino acids to 5 amino acids, 2 amino acids to 6 amino acids.
  • the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 may have 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids modifications.
  • the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 may have at least 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, or 9 amino acids modifications. In some embodiments, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 may have at most 2 amino acids. 3 amino acids, 4 amino acids. 5 amino acids. 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids modifications.
  • the target molecule-binding polypeptide comprises one or more complementarity determining regions (CDRs), for example, wherein one or more of the CDRs has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NOs: 5-20.
  • a CDR of a target molecule-binding polypeptide can be a heavy chain CDR (HCDR).
  • one or more of the HCDRs of the target molecule-binding polypeptide can have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology to any one of SEQ ID NOs: 5-7.
  • a target molecule-binding polypeptide can have three HCDRs (e.g., HCDR1, HCDR2, and HCDR3).
  • an HCDR1 of the target molecule-binding polypeptide can have 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 5.
  • an HCDR1 can have 1, 2, 3, 4, 5, 6, or 7 different nucleic acid residues (e.g., inserted, substituted, or deleted amino acid residues) compared to the sequence of SEQ ID NO: 5.
  • an HCDR2 of the target molecule-binding polypeptide can have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologj' to any one of SEQ ID NO: 6.
  • an HCDR2 can have 1, 2, 3, 4, 5, or 6 different nucleic acid residues (e.g., inserted, substituted, or deleted amino acid residues) compared to the sequence of SEQ ID NO: 6.
  • an HCDR3 of the target moleculebinding polypeptide can have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homolog)' to SEQ ID NO: 7.
  • an HCDR3 can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 different nucleic acid residues (e.g.. inserted, substituted, or deleted amino acid residues) compared to the sequence of SEQ ID NO: 7.
  • a CDR of a target molecule-binding polypeptide can be a light chain CDR (LCDR).
  • one or more of the LCDRs of the target molecule-binding polypeptide can have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%. 95%, 96%, 97%, 98%, 99%, or 100% homolog)' to any one of SEQ ID NOs: 8-20.
  • a target molecule-binding polypeptide can have three LCDRs (e.g., LCDR1, LCDR2, and LCDR3).
  • an LCDR1 of the target molecule-binding polypeptide can have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homolog)' to any one of SEQ ID NO: 8-12.
  • an LCDR1 can have 1, 2, 3, 4, 5, 6, 7, 8, or 9 different nucleic acid residues (e.g., inserted, substituted, or deleted amino acid residues) compared to the sequence of SEQ ID NO: 8-12.
  • an LCDR2 of the target molecule-binding polypeptide can have 80%, 81%, 82%, 83%, 84%, 85%, 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology to any one of SEQ ID NO: 13-16.
  • an LCDR2 can have 1, 2, 3, 4, or 5 different nucleic acid residues (e.g., inserted, substituted, or deleted amino acid residues) compared to the sequence of SEQ ID NO: 13-16.
  • an LCDR3 of the target molecule-binding polypeptide can have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NOs: 17-20.
  • an LCDR3 can have 1, 2, 3, 4, 5, 6, or 7 different nucleic acid residues (e.g., inserted, substituted, or deleted amino acid residues) compared to the sequence of SEQ ID NO: 17-20.
  • a target molecule-binding polypeptide can comprise a HCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%.
  • a HCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 6, and a HCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%.
  • the HCDR1 sequence is identical to SEQ ID NO: 5.
  • the HCDR2 sequence is identical to SEQ ID NO: 6.
  • the HCDR3 sequence is identical to SEQ ID NO: 7.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 24.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence identical to SEQ ID NO: 24. In some cases, the target molecule-binding polypeptide comprises a heavy chain region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 22. In some cases, the target molecule-binding polypeptide comprises a heavy chain region sequence identical to SEQ ID NO: 22.
  • the target molecule-binding polypeptide comprises a heavy chain region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 26.
  • the target molecule-binding polypeptide comprises a heavy’ chain region sequence identical to SEQ ID NO: 26.
  • a target molecule-binding polypeptide can comprise a LCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to SEQ ID NO: 8, a LCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%. 96%. 97%. 98%. 99%.
  • the LCDR1 sequence is identical to SEQ ID NO: 8.
  • the LCDR2 sequence is identical to SEQ ID NO: 13.
  • the LCDR3 sequence is identical to SEQ ID NO: 18.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology’ to SEQ ID NO: 25.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence identical to SEQ ID NO: 25.
  • the target molecule-binding polypeptide comprises a light chain region sequence having 80%, 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology to SEQ ID NO: 23.
  • the target molecule-binding polypeptide comprises a light chain region sequence identical to SEQ ID NO: 23.
  • the target molecule-binding polypeptide comprises a light chain region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology’ to SEQ ID NO: 27.
  • the target molecule-binding polypeptide comprises a light chain region sequence identical to SEQ ID NO: 27.
  • a target molecule-binding polypeptide can comprise a HCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%. 89%. 90%.
  • a HCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology' to SEQ ID NO: 6, and a HCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99%. or 100% homology’ to SEQ ID NO: 30. In some cases, the target molecule-binding polypeptide comprises a heavy chain variable region sequence identical to SEQ ID NO: 30.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99%. or 100% homology’ to SEQ ID NO: 30. In some cases, the target molecule-binding polypeptide comprises a heavy chain variable region sequence identical to SEQ ID NO: 30.
  • a target molecule-binding polypeptide can comprise a LCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 9, a LCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 13, and a LCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID
  • the LCDR1 sequence is identical to SEQ ID NO: 9.
  • the LCDR2 sequence is identical to SEQ ID NO: 13.
  • the LCDR3 sequence is identical to SEQ ID NO: 17.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 31.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence identical to SEQ ID NO: 31.
  • the target molecule-binding polypeptide comprises a light chain region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 29. In some cases, the target molecule-binding polypeptide comprises a light chain region sequence identical to SEQ ID NO: 29.
  • a target molecule-binding polypeptide can comprise a HCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
  • a HCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 6
  • a HCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NO: 7.
  • the HCDR1 sequence is identical to SEQ ID NO: 5.
  • the HCDR2 sequence is identical to SEQ ID NO: 6.
  • the HCDR3 sequence is identical to SEQ ID NO: 7.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homolog ⁇ ’ to SEQ ID NO: 34.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence identical to SEQ ID NO: 34.
  • the target molecule-binding polypeptide comprises a heavy chain region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology 7 to SEQ ID NO: 32.
  • the target molecule-binding polypeptide comprises a heavy chain region sequence identical to SEQ ID NO: 32.
  • a target molecule-binding polypeptide can comprise a LCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 8, a LCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%.
  • the LCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NO: 19.
  • the LCDR1 sequence is identical to SEQ ID NO: 8.
  • the LCDR2 sequence is identical to SEQ ID NO: 13.
  • the LCDR3 sequence is identical to SEQ ID NO: 19.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence having 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homolog ⁇ 7 to SEQ ID NO: 35.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence identical to SEQ ID NO: 35.
  • the target molecule-binding polypeptide comprises a light chain region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homolog ⁇ ' to SEQ ID NO: 33.
  • the target molecule-binding polypeptide comprises a light chain region sequence identical to SEQ ID NO: 33.
  • a target molecule-binding polypeptide can comprise a LCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homolog ⁇ 7 to SEQ ID NO: 8, a LCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%.
  • the LCDR1 sequence is identical to SEQ ID NO: 8.
  • the LCDR2 sequence is identical to SEQ ID NO: 14.
  • the LCDR3 sequence is identical to SEQ ID NO: 17.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homolog ⁇ 7 to SEQ ID NO: 37.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence identical to SEQ ID NO: 37.
  • the target molecule-binding polypeptide comprises a light chain region sequence having 80%, 81%. 82%. 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology to SEQ ID NO: 36.
  • the target molecule-binding polypeptide comprises a light chain region sequence identical to SEQ ID NO: 36.
  • a target molecule-binding polypeptide can comprise a LCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%.
  • a LCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 15, and a LCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology to any one of SEQ ID NO: 17.
  • the LCDR1 sequence is identical to SEQ ID NO: 10.
  • the LCDR2 sequence is identical to SEQ ID NO: 15.
  • the LCDR3 sequence is identical to SEQ ID NO: 17.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 40.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence identical to SEQ ID NO: 40.
  • the target molecule-binding polypeptide comprises a light chain region sequence having 80%. 81%. 82%, 83%, 84%, 85%, 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology to SEQ ID NO: 39. In some cases, the target molecule-binding polypeptide comprises a light chain region sequence identical to SEQ ID NO: 39.
  • a target molecule-binding polypeptide can comprise a HCDR1 comprising a sequence having 80%, 81%, 82%, 83%. 84%, 85%. 86%. 87%. 88%. 89%. 90%.
  • HCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homolog ⁇ ' to SEQ ID NO: 6
  • a HCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NO: 7.
  • the HCDR1 sequence is identical to SEQ ID NO: 5.
  • the HCDR2 sequence is identical to SEQ ID NO: 6.
  • the HCDR3 sequence is identical to SEQ ID NO: 7.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 43.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence identical to SEQ ID NO: 43.
  • the target molecule-binding polypeptide comprises a heavy chain region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 41.
  • the target molecule-binding polypeptide comprises a heavy chain region sequence identical to SEQ ID NO: 41.
  • the target molecule-binding polypeptide comprises a heavy chain region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 45.
  • the target molecule-binding polypeptide comprises a heavy chain region sequence identical to SEQ ID NO: 45.
  • a target molecule-binding polypeptide can comprise a LCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%.
  • a LCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 13, and a LCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99%. or 100% homolog ⁇ ’ to any one of SEQ ID NO: 17.
  • the LCDR1 sequence is identical to SEQ ID NO: 11.
  • the LCDR2 sequence is identical to SEQ ID NO: 13.
  • the LCDR3 sequence is identical to SEQ ID NO: 17.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology to SEQ ID NO: 44.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence identical to SEQ ID NO: 44.
  • the target molecule-binding polypeptide comprises a light chain region sequence having 80%, 81%. 82%. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homology to SEQ ID NO: 42. In some cases, the target molecule-binding polypeptide comprises a light chain region sequence identical to SEQ ID NO: 42.
  • a target molecule-binding polypeptide can comprise a HCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 5, a HCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 6, and a HCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to
  • the HCDR1 sequence is identical to SEQ ID NO: 5.
  • the HCDR2 sequence is identical to SEQ ID NO: 6.
  • the HCDR3 sequence is identical to SEQ ID NO: 7.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 46.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence identical to SEQ ID NO: 46.
  • the target molecule-binding polypeptide comprises a heavy chain region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 32.
  • the target molecule-binding polypeptide comprises a heavy chain region sequence identical to SEQ ID NO: 32.
  • a target molecule-binding polypeptide can comprise a LCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
  • a LCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 16
  • a LCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NO: 20.
  • the LCDR1 sequence is identical to SEQ ID NO: 8.
  • the LCDR2 sequence is identical to SEQ ID NO: 16.
  • the LCDR3 sequence is identical to SEQ ID NO: 20.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homolog ⁇ ’ to SEQ ID NO: 47.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence identical to SEQ ID NO: 47.
  • a target molecule-binding polypeptide can comprise a HCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 5, a HCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 6, and a HCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to
  • the HCDR1 sequence is identical to SEQ ID NO: 5.
  • the HCDR2 sequence is identical to SEQ ID NO: 6.
  • the HCDR3 sequence is identical to SEQ ID NO: 7.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 48.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence identical to SEQ ID NO: 48.
  • the target molecule-binding polypeptide comprises a heavy chain region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 32.
  • the target molecule-binding polypeptide comprises a heavy chain variable region sequence identical to SEQ ID NO: 32.
  • a target molecule-binding polypeptide can comprise a LCDR1 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
  • a LCDR2 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to SEQ ID NO: 16
  • a LCDR3 comprising a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NO: 17.
  • the LCDR1 sequence is identical to SEQ ID NO: 12.
  • the LCDR2 sequence is identical to SEQ ID NO: 16.
  • the LCDR3 sequence is identical to SEQ ID NO: 17.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% homolog ⁇ ’ to SEQ ID NO: 49.
  • the target molecule-binding polypeptide comprises a light chain variable region sequence identical to SEQ ID NO: 49.
  • a target molecule-binding polypeptide disclosed herein binds to the target molecule with a KD value of 1.45 x 10' 8 M or less, e.g., as measured by surface plasmon resonance or other standard methods known to one skilled in the art.
  • the target molecule-binding polypeptide is a target molecule binding polypeptide or antigen-binding polypeptide described herein.
  • a target molecule-binding polypeptide disclosed herein has an EC50 value of 6.0 nM or less for binding to the target molecule, e.g., as measured by enzyme- linked immunosorbent assay (ELISA) or other standard methods known to one skilled in the art.
  • the target molecule-binding polypeptide is a target molecule binding polypeptide or antigen-binding polypeptide described herein.
  • a target molecule-binding polypeptide can be an antibody.
  • a target molecule-binding polypeptide can be an antibody fragment.
  • a target moleculebinding polypeptide can be a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a multi specific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a singledomain antibody, a diabody.
  • scFv single-chain Fvs
  • polypeptide described herein may be a multi-chain polypeptide.
  • the multi-chain polypeptide can be or can include an antibody, a Dual scFab, a F(ab’)2, a diabody, a crossMab, a DAF (two-in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in- holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a Fcab, a K/.-body.
  • an antibody a Dual scFab, a F(ab’)2, a diabody, a crossMab, a DAF (two-in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in- holes assembly, a charge pair, a Fab
  • an orthogonal Fab a DVD-IgG, a IgG(H)-scFv, a scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V.
  • a triple body a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-scFv, a F(ab’)2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a Diabody -Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a ImmTAC, an IgG-IgG conjugate, a Cov-X-Body, or a scFvl-PEG-scFv2.
  • the polypeptide may be a single-chain polypeptide.
  • the single-chain polypeptide can be or include a BiTe, a (scFv)2, a nanobody, a nanobody -HS A, a DART, a TandAb, a scDiabody, a scDiabody-CH3, scFv-CH-CL-scFv, a HSAbody, scDiabody-HS A, or a tandem-scFv.
  • the target molecule-binding polypeptide can be a humanized or human antibody. In some embodiments, the target molecule-binding polypeptide may not be a humanized or not human antibody.
  • the antibodies of use can be of various isotypes, preferably human IgGl, IgG2, IgG3 or IgG4, more preferably comprising human IgGl hinge and constant region sequences.
  • the antibodies or fragments thereof can be chimeric human-mouse, humanized (human framework and murine hypervariable (CDR) regions), or fully human, as well as variations thereof, such as half-IgG4 antibodies (referred to as “unibodies). More preferably, the antibodies or fragments thereof may be designed or selected to comprise human constant region sequences that belong to specific allotypes, which may result in reduced immunogenicity when administered to a human subject.
  • Preferred allotypes for administration include a non-Glml allotype (nGlml ), such as Gl m3, Glm3,l , Glm3,2 or Glm3,l,2. More preferably, the allotype is selected from the group consisting of the nGlml, Glm3, nGlml, 2 and Km3 allotypes.
  • nGlml non-Glml allotype
  • a target molecule-binding polypeptide can comprise a single-chain variable fragment (scFv), F(ab) fragment, a F(ab’)2 fragment, a monovalent IgG. a diabody, or a minibody.
  • a target molecule-binding polypeptide can be an scFv.
  • An scFv can comprise a linker.
  • an scFv can comprise a linker attached to a heavy chain variable region or portion thereof.
  • an scFv can comprise a linker attached to a light chain variable region or portion thereof.
  • an scFv can comprise a linker attached to a heavy chain variable region and a light chain variable region or portion thereof.
  • an scFv includes a linker at the C-terminus of a heavy chain variable region and the N-terminus of a light chain variable region.
  • an scFv includes a linker at the C-terminus of a light chain variable region and the N-terminus of a heavy chain variable region.
  • the linker may be a flexible linker, a rigid linker, or a cleavable linker.
  • the linker peptide may link a portion of a heavy chain variable domain to a portion of a light chain variable domain.
  • a heterodimeric Fc may comprise a first Fc polypeptide linked to the first antigen-binding polypeptide construct (e.g., P-selectin) with or without a first linker and a second Fc polypeptide linked to the second antigen-binding polypeptide construct (e.g., E-selectin) with or without a second linker.
  • the polypeptide linker can be about 2 amino acids to about 24 amino acids. In some embodiments, the polypeptide linker may about 2 amino acids to about 4 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 22 amino acids, about 2 amino acids to about 24 amino acids, about 4 amino acids to about 6 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 16 amino acids, about 4 amino acids to about 18 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 22 amino acids, about 2 amino acids to about 24 amino acids, about 4 amino acids to about 6 amino acids, about 4 amino acids
  • the polypeptide linker can be about 2 amino acids, about 4 amino acids, about 6 amino acids, about 8 amino acids, about 10 amino acids, about 12 amino acids, about 14 amino acids, about 16 amino acids, about 18 amino acids, about 20 amino acids, about 22 amino acids, or about 24 amino acids. In some embodiments, the polypeptide linker can be at least about 2 amino acids, about 4 amino acids, about 6 amino acids, about 8 amino acids, about 10 amino acids, about 12 amino acids, about 14 amino acids, about 16 amino acids, about 18 amino acids, about 20 amino acids, or about 22 amino acids.
  • the polypeptide linker may at most about 4 amino acids, about 6 amino acids, about 8 amino acids, about 10 amino acids, about 12 amino acids, about 14 amino acids, about 16 amino acids, about 18 amino acids, about 20 amino acids, about 22 amino acids, or about 24 amino acids.
  • the linker peptide may comprise (G 4 S) n [SEQ ID NO. 56], (SG 4 ) n [SEQ ID NO. 57].
  • a linker can be a (G 4 S)s linker.
  • a (G 4 S)s linker can comprise an amino acid sequence of: GGGGSGGGGSGGGGS (SEQ ID NO: 21).
  • a linker can comprise a sequence having at least 80%, 85%, 90%, or 100% homology to SEQ ID NO: 21.
  • a target molecule-binding polypeptide further comprises an Fc fragment or an Fc region or domain of an antibody.
  • the Fc fragment or Fc region can be derived from IgGl, IgG2, IgG3, or IgG4.
  • the Fc region is a modified Fc fragment.
  • the Fc region or domain is mutated with a LALA mutation (L234A, L235A).
  • the Fc region is an Fc region variant containing one or more amino acid modifications.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) including an amino acid modification (e.g. a substitution) at one or more amino acid position.
  • the Fc region variant provides one or more characteristics and properties affecting in vivo serum half-life of the target molecule-binding polypeptide. Detailed descriptions of Fc region variants can be found in, for example, US Patent Nos. 7,317,091; 7,790,858; 8,969,526; and 11,059,892, which are incorporated by reference herein. Additional exemplar ⁇ ' Fc region modifications are described below.
  • the Fc region further comprises a triple M252Y/S254T/T256E mutation or YTE mutation. In other embodiments, the Fc region comprises a M428L/N434S or LS mutation.
  • a target molecule-binding polypeptide can bind to one or more amino acid residue of a target molecule. In some cases, the target molecule can have a sequence comprising one or more amino acid sequences set forth in SEQ ID NOs: 1-4 and 62-63.
  • the target molecule can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any one of SEQ ID NOs: 1-4 and 62-63.
  • the target molecule-binding polypeptide can bind to one or more amino acid residues set forth in one or more within any one of SEQ ID NOs: 1-4 and 62-63.
  • the target molecule-binding polypeptide can bind to one or more amino acid residues within a sequence having at least 80%.
  • the target molecule is a selectin. In some cases, the target molecule is a human selectin. In some cases, the target molecule is E-selectin or P-selectin. In some cases, the target molecule is P- selectin. In some cases, the target molecule is E-selectin. In some cases, the target moleculebinding protein binds to a first selectin but not a second selectin.
  • a target molecule-binding protein can bind to P-selectin but does not bind to E-selectin. In some cases, the target molecule-binding protein binds to both a first selectin and a second selectin. For instance, in some cases, a target molecule-binding protein described herein can bind both to P- selectin and to E-selectin. In some embodiments, the target molecule-binding protein binds to an antigen epitope of P-selectin. In some embodiments, the target molecule-binding protein binds to an antigen epitope of E-selectin.
  • the target molecule-binding protein binds separately to an antigen epitope of P-selectin and an epitope of E-selectin. In some embodiments, the target molecule-binding protein binds cell-surfaced expressed human P- selectin. In some embodiments, the target molecule-binding protein binds soluble human P- selectin. In some embodiments, the target molecule-binding protein binds cell-surfaced expressed human E-selectin. In some embodiments, the target molecule-binding protein binds soluble human E-selectin. In some embodiments, the target molecule-binding protein inhibits or reduces or blocks human P-selectin activity. In some embodiments, the target molecule-binding protein inhibits or reduces or blocks human E-selectin activity.
  • the target moleculebinding polypeptide such as an antibody in a host cell
  • post-translational modifications may occur. For example, this may include the cleavage of certain leader sequences, the addition of various sugar moieties in various glycosylation patterns, non-enzymatic glycation, deamidation, oxidation, disulfide bond scrambling and other cysteine variants such as free sulfhydryls, racemized disulfides, thioethers and trisulfide bonds, isomerization, C- terminal lysine clipping, and N-terminal glutamine cyclisation.
  • Glycation is a post-translational non-enzymatic chemical reaction between a reducing sugar, such as glucose, and a free amine group in the protein, and is typically observed at the epsilon amine of lysine side chains or at the N-Terminus of the protein. Glycation can occur during production and storage only in the presence of reducing sugars.
  • a reducing sugar such as glucose
  • Deamidation can occur during production and storage, is an enzymatic reaction primarily converting asparagine (N) to iso-aspartic acid (iso-aspartate) and aspartic acid (aspartate) (D) at approximately 3:1 ratio. This deamidation reaction is therefore related to isomerization of aspartate (D) to iso-aspartate.
  • the deamidation of asparagine and the isomerization of aspartate both involve the intermediate succinimide.
  • deamidation can occur with glutamine residues in a similar manner. Deamidation can occur in a CDR, in a Fab (non-CDR region), or in the Fc region.
  • Oxidation can occur during production and storage (for example in the presence of oxidizing conditions) and results in a covalent modification of a protein, induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation happens primarily with methionine residues, but may occur at try ptophan and free cysteine residues. Oxidation can occur in a CDR, in a Fab (non-CDR) region, or in the Fc region.
  • Disulfide bond scrambling can occur during production and basic storage conditions. Under certain circumstances, disulfide bonds can break or form incorrectly, resulting in unpaired cysteine residues (-SH). These free (unpaired) sulfhydryls (-SH) can promote shuffling.
  • thioether and racemization of a disulphide bond can occur under basic conditions, in production or storage, through a beta elimination of disulphide bridges back to cysteine residues via a dehydroalanine and persulfide intermediate. Subsequent crosslinking of dehydroalanine and cysteine results in the formation of a thioether bond or the free cysteine residues can reform a disulphide bond with a mixture of D- and /.-cysteine.
  • Trisulfides result from insertion of a sulfur atom into a disulphide bond (Cys-S-S- S-Cys ) and are formed due to the presence of hydrogen sulphide in production cell culture.
  • N-terminal glutamine (Q.) and glutamate (glutamic acid) (E) in the heavy chain and/or light chain is likely to form pyroglutamate (pGlu) via cyclization. Most pGlu formation happens in the production bioreactor, but it can be formed non-enzymatically, depending on pH and temperature of processing and storage conditions. Cyclization of N-terminal Q. or E is commonly observed in natural human antibodies.
  • C-terminal lysine clipping is an enzy matic reaction catalyzed by carboxypeptidases, and is commonly observed in recombinant and natural human antibodies. Variants of this process include removal of lysine from one or both heavy chains due to cellular enzymes from the recombinant host cell. Upon administration to the human subject/patient is likely to result in the removal of any remaining C- terminal lysines.
  • the target molecule-binding polypeptide may have a half-life (serum half-life) of at least 6 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 7 days, or at least 9 days in vivo in humans, or in a murine animal model.
  • a half-life serum half-life
  • Mutational changes to the Fc effector portion of the antibody can be used to change the affinity of the interaction between the FcRn and antibody to modulate antibody turnover.
  • the half-life of the antibody can be extended in vivo. This could be beneficial to patient populations as maximal dose amounts and maximal dosing frequencies could be achieved as a result of maintaining in vivo I C50 for longer periods of time.
  • the Fc effector function of the antibody may be removed, in its entirety or in part, since it may not be desirable to kill those cells expressing CD127. This removal may result in an increased safety profile.
  • a target molecule-binding polypeptide a constant region may have reduced ADCC and/or complement activation or effector functionality.
  • the constant domain may comprise a naturally disabled constant region of lgG2 or lgG4 isotype or a mutated IgGl constant domain.
  • the target molecule-binding polypeptide may be Fc disabled. Examples of suitable modifications are described in EP0307434.
  • One way to achieve Fc disablement comprises the substitutions of alanine residues at positions 235 and 237 (EU index numbering) of the heavy chain constant region, i.e., L235A and G237A (commonly referred to as "LAGA" mutations).
  • Another example comprises substitution with alanines at positions 234 and 235 (EU index numbering), i.e., L234A and L235A (commonly referred to as "LALA" mutations).
  • L234A and L235A commonly referred to as "LALA" mutations.
  • the Fc effector function of a target molecule-binding polypeptide disclosed herein has been disabled using the LAGA mutation.
  • the target molecule-binding polypeptide may be Fc enabled and not comprise the alanine substitutions at positions 235 and 237.
  • the Fc polypeptide or fragment thereof of a target molecule-binding polypeptide disclosed herein comprises a half-life-extending mutation comprising any one or more of M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I Q311I; D376V; T307A; E380A (EU numbering).
  • a half-life-extending mutation comprises M428L/N434S (also referred to as “MLNS” or “LS”).
  • a half-life-extending mutation comprises M252Y/S254T/T256E.
  • a half-life-extending mutation comprises T250Q/M428L. In some embodiments, a half-life-extending mutation comprises P257I/Q311I. In some embodiments, a half-life-extending mutation comprises P257I/N434H. In some embodiments, a half-life-extending mutation comprises D376V/N434H. In some embodiments, a half-life-extending mutation comprises T307A/E380A/N434A.
  • a target molecule-binding polypeptide disclosed herein, an antibody or antigen-binding fragment includes a Fc moiety that comprises the substitution mutation M428L/N434S.
  • a target molecule-binding polypeptide disclosed herein, an antibody or anti gen- binding fragment includes a Fc polypeptide or fragment thereof that comprises the substitution mutations G236A/A330L/I332E.
  • a target molecule-binding polypeptide disclosed herein, an antibody or antigen-binding fragment includes a (e.g., IgG) Fc moiety that comprises a G236A mutation, an A330L mutation, and a I332E mutation (GAALIE).
  • a target molecule-binding polypeptide disclosed herein, an antibody or antigen-binding fragment includes an Fc polypeptide or fragment thereof that comprises the substitution mutations: M428L/N434S and G236A/A330L/I332E, and optionally does not comprise S239D.
  • a target molecule-binding polypeptide disclosed herein, an antibody or antigen-binding fragment includes a Fc polypeptide or fragment thereof that comprises the substitution mutations: M428L/N434S and G236A/S239D/A330L/I332E.
  • a target moleculebinding polypeptide disclosed herein comprises the substitution mutations: LS (M428L/N434S), GAALIE (G236A/A330L/I332E), or GAALIE LS (G236A/A330L/I332E/M428L/N434S).
  • Additional alterations and mutations to decrease effector function include: (with reference to IgGl unless otherwise noted): a glycosylated N297A or N297Q orN297G; L235E; lgG4:F234A/L235A; and chimeric lgG2/lgG4.
  • lgG2 H268Q/V309L/A330S/P331S
  • lgG2 V234A/G237A/P238S/H268A/V309L/A330S/P331 S can reduce FcyR and Clq binding (Wang et al. 2018 and US 8,961,967).
  • L234F/L235E/P331S a chimeric antibody created using the CHI and hinge region from human lgG2 and the CH2 and CH3 regions from human lgG4; lgG2m4, based on the lgG2 isotype with four key amino acid residue changes derived from lgG4 (H268Q, V309L, A330S and P331S); lgG2o which contains V234A/G237A /P238S/H268A/V309L/A330S/P331 S substitutions to eliminate affinity for Fey receptors and Clq complement protein; lgG2m4 (H268Q/V309L/A330S/P331S, changes to lgG4); lgG4 (S228P/L234A/L235A); huIgGl L234A/L235A (AA); hulgG4
  • a target molecule-binding polypeptide disclosed herein may comprise one or more modifications selected from a mutated constant domain such that the antibody has enhanced effector functions/ ADCC and/or complement activation. Examples of suitable modifications are described in Shields et al. J. Biol. Chem (2001) 276:6591-6604, Lazar et al. PNAS (2006) 103:4005-4010 and US6737056, W02004063351 and W02004029207.
  • the target molecule-binding polypeptide may comprise a constant domain with an altered glycosylation profile such that the target molecule-binding polypeptide has enhanced effector functions/ ADCC and/or complement activation. Examples of suitable methodologies to produce a target molecule-binding polypeptide with an altered glycosylation profile are described in W02003/011878, W02006/014679 and EP1229125.
  • the binding affinity of the target molecule-binding polypeptide to a target may be about 2-fold to about 1000- fold (e.g., about 2-fold, 4-fold. 5-fold, 8-fold, 10-fold. 15-fold, 20-fold, 25-fold, 30-fold, 50-fold, 100-fold. 125-fold.
  • control target molecule-binding polypeptides comprising heavy chain regions according to SEQ ID NO: 54 and light chain regions according to SEQ ID NO: 55.
  • the binding affinity of the target molecule-binding polypeptide to a target antigen may be about 0. 1 fold to about 0.5 fold, about 0. 1 fold to about 1 fold, about 0. 1 fold to about 2 fold, about 0. 1 fold to about 3 fold, about 0.1 fold to about 4 fold, about 0. 1 fold to about 5 fold, about 0. 1 fold to about 10 fold, about 0. 1 fold to about 20 fold, about 0. 1 fold to about 30 fold, about 0.
  • 1 fold to about 40 fold about 0.1 fold to about 50 fold, about 0.5 fold to about 1 fold, about 0.5 fold to about 2 fold, about 0.5 fold to about 3 fold, about 0.5 fold to about 4 fold, about 0.5 fold to about 5 fold, about 0.5 fold to about 10 fold, about 0.5 fold to about 20 fold, about 0.5 fold to about 30 fold, about 0.5 fold to about 40 fold, about 0.5 fold to about 50 fold, about 1 fold to about 2 fold, about 1 fold to about 3 fold, about 1 fold to about 4 fold, about 1 fold to about 5 fold, about 1 fold to about 10 fold, about 1 fold to about 20 fold, about 1 fold to about 30 fold, about 1 fold to about 40 fold, about 1 fold to about 50 fold, about 2 fold to about 3 fold, about 2 fold to about 4 fold, about 2 fold to about 5 fold, about 2 fold to about 10 fold, about 2 fold to about 20 fold, about 2 fold to about 30 fold, about 2 fold to about 40 fold, about 2 fold to about 50 fold, about 2 fold to about 3 fold, about 2 fold to about
  • the binding affinity of the target molecule-binding polypeptide to a target antigen may be about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, about 20 fold, about 21 fold, about 22 fold, about 23 fold, about 24 fold, about 25 fold, about 26 fold, about 27 fold, about 28 fold, about 29 fold, about 30 fold, about 31 fold, about 32 fold, about 33 fold, about 34 fold, about 35 fold, about 36 fold, about 37 fold, about 38 fold, about 39 fold, about 40 fold, or about 50 fold greater than a binding affinity of the control target molecule-binding polypeptides comprising heavy chain regions according to SEQ ID NO: 54 and light chain regions according to SEQ
  • the target molecule-binding polypeptide described herein can cause a greater reduction in SCD WBC (sickle cell disease white blood cell) binding to P- selectin than inclacumab.
  • the target molecule-binding polypeptide described herein can reduce binding of SCD WBC to P-selectin by at least about 0.1 fold to about 0.5 fold, about 0.1 fold to about 1 fold, about 0. 1 fold to about 2 fold, about 0. 1 fold to about 3 fold, about 0. 1 fold to about 4 fold, about 0. 1 fold to about 5 fold, about 0. 1 fold to about 10 fold, about 0.1 fold to about 20 fold, about 0.1 fold to about 30 fold, about 0. 0.
  • 1 fold to about 40 fold about 0.1 fold to about 50 fold, about 0.5 fold to about 1 fold, about 0.5 fold to about 2 fold, about 0.5 fold to about 3 fold, about 0.5 fold to about 4 fold, about 0.5 fold to about 5 fold, about 0.5 fold to about 10 fold, about 0.5 fold to about 20 fold, about 0.5 fold to about 30 fold, about 0.5 fold to about 40 fold, about 0.5 fold to about 50 fold, about 1 fold to about 2 fold, about 1 fold to about 3 fold, about I fold to about 4 fold, about 1 fold to about 5 fold, about 1 fold to about 10 fold, about 1 fold to about 20 fold, about 1 fold to about 30 fold, about 1 fold to about 40 fold, about 1 fold to about 50 fold, about 2 fold to about 3 fold, about 2 fold to about 4 fold, about 2 fold to about 5 fold, about 2 fold to about 10 fold, about 2 fold to about 20 fold, about 2 fold to about 30 fold, about 2 fold to about 40 fold, about 2 fold to about 50 fold, about 3 fold to about 4 fold, about 2 fold to about
  • adhesion index was calculated as the ratio (%) of adherent cells per square millimeter for assays with 50 pg/ml of a target molecule-binding polypeptide described herein versus assays without the target molecule-binding polypeptide ('‘untreated”).
  • an adhesion index of the target molecule-binding polypeptide for a target cell may be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%.
  • the target molecule-binding polypeptide can have a KD for binding P-selectin of about 5 nM to about 10 nM, about 5 nM to about 20 nM, about 5 nM to about 30 nM, about 5 nM to about 40 nM, about 5 nM to about 50 nM, about 5 nM to about 60 nM, about 5 nM to about 70 nM, about 5 nM to about 80 nM, about 5 nM to about 90 nM, about 5 nM to about 100 nM, about 5 nM to about 150 nM, about 10 nM to about 20 nM, about 10 nM to about 30 nM, about 10 nM to about 40 nM, about 10 nM to about 50 nM, about 10 nM to about 60 nM, about 10 nM to about 70 nM, about 10 nM to about 80 nM, about 10 nM to about 100 nM, about
  • about 20 nM to about 90 nM about 20 nM to about 100 nM, about 20 nM to about 150 nM, about 30 nM to about 40 nM, about 30 nM to about 50 nM, about 30 nM to about 60 nM, about 30 nM to about 70 nM, about 30 nM to about 80 nM, about 30 nM to about 90 nM, about 30 nM to about 100 nM, about 30 nM to about 150 nM, about 40 nM to about 50 nM.
  • the target molecule binding peptide can have a KD for binding P- selectin of about 50 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM. about 900 nM, about 1,000 nM, or about 1,500 nM.
  • the target molecule-binding polypeptide can have a KD for binding E-selectin of about 5 nM to about 10 nM, about 5 nM to about 20 nM, about 5 nM to about 30 nM, about 5 nM to about 40 nM. about 5 nM to about 50 nM, about 5 nM to about 60 nM, about 5 nM to about 70 nM, about 5 nM to about 80 nM. about 5 nM to about 90 nM.
  • nM to about 150 nM about 20 nM to about 30 nM, about 20 nM to about 40 nM, about 20 nM to about 50 nM, about 20 nM to about 60 nM, about 20 nM to about 70 nM, about 20 nM to about 80 nM, about 20 nM to about 90 nM, about 20 nM to about 100 nM, about 20 nM to about 150 nM, about 30 nM to about 40 nM, about 30 nM to about 50 nM, about 30 nM to about 60 nM.
  • the target molecule binding peptide can have a KD for binding E- selectin of about 50 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM. about 900 nM, about 1,000 nM, or about 1,500 nM.
  • compositions comprising one or more of the target molecule-binding polypeptides disclosed herein along with a pharmaceutically acceptable carrier or excipient.
  • a composition can comprise a nucleic acid sequence encoding one or more of the target molecule-binding polypeptides disclosed herein.
  • a composition can comprise an expression vector comprising a nucleic acid sequence encoding one or more target molecule-binding polypeptides disclosed herein.
  • a composition can comprise a host cell comprising the nucleic acid or the expression vector encoding one or more of the target molecule-binding polypeptides disclosed herein.
  • compositions disclosed herein e.g.. a target molecule-binding polypeptide (for example, administered along with a pharmaceutically acceptable carrier or excipient) disclosed herein, a nucleic acid encoding a target molecule-binding polypeptide disclosed herein, an expression vector encoding a target molecule-binding polypeptide disclosed herein, or a host cell comprising a nucleic acid or expression vector disclosed herein
  • a pharmaceutical compositions disclosed herein e.g.. a target molecule-binding polypeptide (for example, administered along with a pharmaceutically acceptable carrier or excipient) disclosed herein, a nucleic acid encoding a target molecule-binding polypeptide disclosed herein, an expression vector encoding a target molecule-binding polypeptide disclosed herein, or a host cell comprising a nucleic acid or expression vector disclosed herein
  • therapy e.g.. of a subject in need thereof.
  • a target molecule-binding polypeptide disclosed herein can be used to treat a subject in need thereof.
  • a method can of treating a subject in need thereof can comprise administering to the subject any one of the target molecule-binding polypeptides disclosed herein, a pharmaceutical composition comprising a target molecule-binding polypeptide along with a pharmaceutically acceptable carrier or excipient, a nucleic acid sequence encoding a target molecule-binding polypeptide disclosed herein, an expression vector comprising the nucleic acid sequence encoding the target molecule-binding polypeptide, or a host cell comprising the nucleic acid.
  • the administering can be intravenously, subcutaneously, or intramuscularly.
  • a target molecule-binding polypeptide having a high binding affinity e.g., low KD
  • a target molecule-binding polypeptide such as one comprising one or more sequences of SEQ ID NOs: 5-49
  • the administering increases blood oxygen saturation or decreases frequency of chest pain, severity of chest pain, frequency of limb pain, severity of limb pain, risk of stroke, and/or risk of heart attack in a subject (e.g., a subject having sickle cell disease, “SCD’ ).
  • a subject has a blood hemoglobin level of from about 6 g/dL to about 11 g/dL or 5 g/dL to about 10 g/dL. In some cases, a subject has a blood hemoglobin level of from about 6 g/dL to about 11 g/dL or higher. Tn some cases, a subject has a blood hemoglobin level of from about 6 g/dL to about 11 g/dL or lower. In some cases, the subject has sickle cell anemia, lupus, or primary myelofibrosis. In some cases, the subject has sickle cell disease.
  • the subject has sickle cell disease and a genotype selected from the group consisting of a hemoglobin SS (Hb SS) genotype, a hemoglobin SC (Hb SC) genotype, a hemoglobin SB + thalassemia (Hb Sbeta + - thalassemia) genotype, a hemoglobin SB 0 thalassemia (Hb Sbeta 0 -thalassemia) genotype, a hemoglobin SD (Hb SD) genoty pe, a hemoglobin SE (Hb SE) genotype, and a hemoglobin SO (Hb SO) genotype.
  • HBB hemoglobin beta gene
  • a target molecule-binding polypeptide described herein is administered to a subject with SCD to reduce the frequency of vaso-occlusive crises.
  • a target molecule-binding polypeptide, a pharmaceutical composition, a nucleic acid sequence, the expression vector, or a host cell disclosed herein can be used in the manufacture of a medicament for a blood disease, an autoimmune condition, a cardiovascular condition, or a cancer.
  • kits comprising one or more target molecule-binding polypeptides, pharmaceutical compositions, nucleic acid sequences, expression vectors, or host cells disclosed herein along with instructions for use.
  • Also provided herein are methods of producing an polypeptide that include: (a) culturing a cell (e.g., any of the cells described herein) including any of the nucleic acids encoding any of the polypeptides described herein, or any of the expression vectors described herein that include nucleic acid encoding any of the polypeptides described herein, in a culture medium under conditions sufficient to allow for the production of the polypeptide; and (b) harvesting the polypeptide from the host cell or the culture medium.
  • a cell e.g., any of the cells described herein
  • a cell including any of the nucleic acids encoding any of the polypeptides described herein, or any of the expression vectors described herein that include nucleic acid encoding any of the polypeptides described herein
  • the method further includes isolating the polypeptide (e.g., through performance of one or more column chromatography steps, ultrafiltration/diafiltration, and/or viral inactivation). In some embodiments of any of the methods described herein, the method further includes formulating the isolated polypeptide into a composition (e.g., a pharmaceutical composition).
  • a composition e.g., a pharmaceutical composition.
  • Non-limiting examples of a mammalian cell include: a human cell, a rodent cell (e.g., a rat cell or a mouse cell), a rabbit cell, a dog cell, a cat cell, a porcine cell, or a nonhuman primate cell.
  • a host cell can be a CHO cell or a HEK cell.
  • Cells can be maintained in vitro under conditions that favor cell proliferation, cell growth, and/or cell differentiation.
  • cells can be cultured by contacting a cell (e.g., any of the cells described herein) with a cell culture medium that includes supplemental growth factors to support cell viability and cell growth.
  • nucleic acids e.g., any of the exemplary nucleic acids described herein
  • expression vectors e.g., any of the exemplary 7 expression vectors described herein (e.g., an AAV vector)
  • cells e.g., mammalian cells
  • Non-limiting examples of methods that can be used to introduce a nucleic acid (e.g., DNA, RNA, ssRNA, siRNA, microRNA, or mRNA) and/or an expression vector (e.g., any of the exemplary expression vectors described herein (e.g., an AAV vector) include: electroporation, lipofection, transfection, microinjection, calcium phosphate transfection, dendrimer-based transfection, anionic polymer transfection, cationic polymer transfection, transfection using highly branched organic compounds, cell-squeezing, sonoporation, optical transfection, magnetofection, particlebased transfection (e.g., nanoparticle transfection), transfection using liposomes (e.g., cationic liposomes), and viral transduction (e.g., lentiviral transduction, adenoviral transduction).
  • a nucleic acid e.g., DNA, RNA, ssRNA, siRNA, microRNA, or m
  • Some methods described herein further include isolating or purifying the polypeptide from cell culture medium or from a cell (e.g.. a mammalian cell) using techniques well-known in the art (e.g., ion exchange chromatography (anionic or cation), metal-affinity chromatography, ligand-affinity 7 chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and precipitation (e.g., ammonium sulfate precipitation, polyethylene glycol precipitation).
  • ion exchange chromatography anionic or cation
  • metal-affinity chromatography e.g., metal-affinity chromatography
  • ligand-affinity 7 chromatography ligand-affinity 7 chromatography
  • size exclusion chromatography e.g., size exclusion chromatography
  • hydrophobic interaction chromatography e.g., ammonium sulfate precipitation, polyethylene glycol precipitation
  • compositions for use in a therapy includes an antibody monotherapy, antibody-drug conjugate (ADC) therapy, T cell-engaging immunotherapy, or CAR-T therapy.
  • the pharmaceutical compositions may further comprise at least one additional therapeutic agent.
  • compositions comprising a target molecule-binding polypeptide described herein can be provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus. 20th ed. (2003); Ansel et al..
  • Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • compositions comprising a target molecule-binding polypeptide described herein may be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • an aqueous or nonaqueous solvent such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol
  • solubilizers isotonic agents
  • suspending agents emulsifying agents
  • stabilizers and preservatives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • compositions may be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • the compositions may also be formulated, in various embodiments, into sustained release microcapsules, such as with biodegradable or non-biodegradable polymers.
  • a non-limiting exemplary biodegradable formulation includes poly lactic acid-glycolic acid polymer.
  • a nonlimiting exemplary non-biodegradable formulation includes a poly glycerin fatty acid ester.
  • composition described herein may be manufactured as a medicament for treatment of cancer.
  • compositions that include any of the polypeptides, vectors, or nucleic acids described herein.
  • Any of the pharmaceutical compositions can include any of the polypeptides, vectors, or nucleic acids described herein and one or more (e.g., 1, 2. 3, 4, or 5) pharmaceutically or physiologically acceptable carriers, diluents, or excipients.
  • any of the pharmaceutical compositions described herein can include one or more buffers (e.g., a neutral-buffered saline, a phosphate-buffered saline (PBS)), one or more carbohydrates (e.g., glucose, mannose, sucrose, dextran, or mannitol), one or more proteins, polypeptides, or amino acids (e.g., glycine), one or more antioxidants, one or more chelating agents (e.g.. glutathione or EDTA). one or more preservatives, and/or a pharmaceutically acceptable carrier (e.g., PBS, saline, or bacteriostatic water).
  • buffers e.g., a neutral-buffered saline, a phosphate-buffered saline (PBS)
  • carbohydrates e.g., glucose, mannose, sucrose, dextran, or mannitol
  • proteins e.g., glycine
  • antioxidants
  • any of the pharmaceutical compositions described herein can further include one or more (e.g., 1, 2. 3, 4, or 5) agents that promote the entry of any of the vectors or nucleic acids descnbed herein into a cell (e.g., a mammalian cell) (e.g., a liposome or cationic lipid).
  • a cell e.g., a mammalian cell
  • agents that promote the entry of any of the vectors or nucleic acids descnbed herein into a cell e.g., a mammalian cell
  • a liposome or cationic lipid e.g., 1, 2. 3, 4, or 5
  • any of the vectors or nucleic acids described herein can be formulated using natural and/or synthetic polymers.
  • Non-limiting examples of polymers that can be included in any of the pharmaceutical compositions described herein can include, but are not limited to: poloxamer, chitosan, dendrimers and poly(lactic-co-gly colic acid) (PLGA) polymers.
  • PLGA poly(lactic-co-gly colic acid)
  • the pharmaceutical compositions provided herein can be, e.g., formulated to be compatible with their intended route of administration. In some embodiments, the compositions are formulated for subcutaneous, intramuscular, or intravenous administration.
  • compositions include a therapeutically effective amount of any of the polypeptides, vectors, or nucleic acids described herein.
  • Single or multiple administrations of any of the pharmaceutical compositions described herein can be given (e.g., administered) to a subject depending on, for example, the frequency and the dosage required and tolerated by the subject.
  • a dosage of the pharmaceutical composition including any of the polypeptides described herein, any of the vectors described herein, or any of the nucleic acids described herein should provide a sufficient quantity to effectively ameliorate or treat symptoms, conditions or diseases.
  • kits-of-parts comprising a pharmaceutical composition together with instructions for use is further provided.
  • the kit-of-parts may comprise reagents in predetermined amounts with instructions for use.
  • kits comprising a target moleculebinding polypeptide disclosed herein.
  • a kit can be a diagnostic kit.
  • a kit comprises a target molecule-binding polypeptide disclosed herein and instructions for use.
  • a kit comprises means for measuring a target molecule-binding polypeptide level in a sample and instructions for use.
  • a kit may provide a unit or device for obtaining a sample from a subject (e.g., a device with a needle coupled to an aspirator).
  • a kit may include a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a kit component described herein.
  • Containers of a kit may be airtight, waterproof (e.g., impermeable to changes in moistures or evaporation), and/or lighttight.
  • a kit may include a device suitable for administration of the components, e.g., a syringe, inhalant, pipette, forceps, measured spoon, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such deliver ⁇ ' device.
  • the device may be a medical implant device, e.g., packaged for surgical insertion.
  • a kit disclosed herein may comprise one or more reagents or instruments which enable the method to be carried out.
  • reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions), a support comprising wells on which quantitative reactions can be done.
  • a kit may be a specific kit for a specific tissue sample.
  • a kit disclosed herein may comprise a control.
  • the kit may comprise any of the compositions (e.g., pharmaceutical compositions) described herein which include any of the nucleic acids, any of the polypeptides, or any of the vectors described herein.
  • a kit can include a solid composition (e.g., a lyophilized composition including any of the vectors, polypeptides, or nucleic acids described herein) and a liquid for solubilizing the lyophilized composition.
  • the kit includes a vial including any of the pharmaceutical compositions described herein (e.g., formulated as an aqueous pharmaceutical composition).
  • instructions for use may be provided in a kit. These instructions may be presented in the kit in a variety' of forms, such as printed information on a suitable medium or substate (e.g., a piece or pieces of paper on which the information is printed), in the packaging of the kit, in a package insert, etc.
  • instructions for use can be provided on a computer readable medium (e.g., jump/thumb drive, CD, etc.), or which the information has been recorded, or at a website address which may be used via the internet to access the information at a website.
  • Another aspect of the disclosure provides a pre-filled syringe or autoinjector device, comprising a target molecule-binding polypeptide or a composition described herein.
  • a composition stored in a container, pre-fdled syringe, injector or autoinjector device contains a target molecule-binding polypeptide disclosed herein.
  • a support can be a solid support.
  • a support may take a variety of configurations ranging from simple to complex, depending on the intended use of the support.
  • a support can have an overall slide or plate configuration, such as a rectangular or disc configuration.
  • a standard microplate configuration can be used.
  • the surface may be smooth or substantially planar, or have irregularities, such as depressions or elevations.
  • a support may have a rectangular cross-sectional shape, having a length of from about 10-200 mm, 40-150 mm, or 75- 125 mm; a width of from about 10-200 mm, 20-120 mm, or 25-80 mm, and a thickness of from about 0.01-5.0 mm, 0.1-2 mm, or 0.2 to 1 mm.
  • a support can be organic or inorganic; may be metal (e.g., copper or silver) or non- metal; may be a polymer or nonpolymer; may be conducting. semiconducting or nonconducting (insulating); may be reflecting or nonreflecting; may be porous or nonporous; etc.
  • a support as described herein can be formed of any suitable material, including metals, metal oxides, semiconductors, polymers (particularly organic polymers in any suitable form including woven, nonwoven, molded, extruded, cast, etc.), silicon, silicon oxide, and composites thereof.
  • a support can be an array.
  • a support comprises an array.
  • An array can comprise an ordered spatial arrangement of two or more discrete regions.
  • An array can comprise the target molecule-binding polypeptide described herein located at known or unknown discrete regions. Row and column arrangements of arrays can be selected due to the relative simplicity in making such arrangements. The spatial arrangement can, however, be essentially any form selected by the user, and optionally, in a pattern. Areas of an array may be any convenient shape, including circular, ellipsoid, oval, annular, or some other analogously- curved shape, where the shape may, in certain embodiments, be a result of the particular method employed to produce the array.
  • a support can be planar. In some instances, a support can be spherical. In some instances, a support can be a bead. In some instances, a support can be magnetic. In some embodiments, a magnetic support can comprise magnetite, maghemitite, FePt, SrFe. iron, cobalt, nickel, chromium dioxide, ferrites, or mixtures thereof. In some embodiments, a support can be nonmagnetic. In some embodiments, the nonmagnetic support can comprise a polymer, metal, glass, alloy, mineral, or mixture thereof. In some instances, a nonmagnetic material can be a coating around a magnetic support.
  • a magnetic material may be distributed in the continuous phase of a magnetic material.
  • the support comprises magnetic and nonmagnetic materials.
  • a support can comprise a combination of a magnetic material and a nonmagnetic material.
  • the target molecule-binding polypeptide disclosed herein is directly or indirectly associated with a support disclosed herein.
  • a computer system is programmed or otherwise configured to interface with an apparatus that is configured to detect P-selectin, E- selectin, or both and/or binding of an antibody or antigen-binding fragment thereof disclosed herein to moiety.
  • the computer system can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device.
  • the electronic device can be a mobile electronic device.
  • a computer system includes a central processing unit (CPU, also "processor” and “computer processor” herein, which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • CPU central processing unit
  • processor also "processor” and “computer processor” herein, which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • a computer system also includes memory or memory location (e.g., random-access memory. read-only memory, flash memory'), electronic storage unit (e.g., hard disk), communication interface (e.g., network adapter) for communicating with one or more other systems, and peripheral devices, such as cache, other memory, data storage and/or electronic display adapters.
  • the memory, storage unit, interface and peripheral devices are in communication with the CPU through a communication bus, such as a motherboard.
  • the storage unit can be a data storage unit (or data repository) for storing data.
  • the computer system is operatively coupled to a computer network (“network") with the aid of the communication interface.
  • the network can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
  • the netw ork is a telecommunication and/or data network.
  • the netw ork can include one or more computer servers, which can enable distributed computing, such as cloud computing.
  • the network in some cases with the aid of the computer system can implement a peer-to-peer network, which may enable devices coupled to the computer system to behave as a client or a server.
  • the CPU executes a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory’.
  • the instructions can be directed to the CPU, which can subsequently program or otherwise configure the CPU to implement methods of the present disclosure. Examples of operations performed by the CPU can include fetch, decode, execute, and writeback.
  • the CPU can be part of a circuit, such as an integrated circuit. One or more other components of the system can be included in the circuit.
  • the circuit is an application specific integrated circuit (ASIC).
  • the storage unit can store files, such as drivers, libraries, and saved programs.
  • the storage unit can store user data, e.g., user preferences and user programs.
  • the computer system in some cases can include one or more additional data storage units that are external to the computer system, such as located on a remote server that is in communication with the computer system through an intranet or the Internet.
  • the computer system communicates with one or more remote computer systems through the network.
  • the computer system can communicate with a remote computer system of a user.
  • remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's telephones, Smart phones, or personal digital assistants.
  • the user can access the computer system via the network.
  • Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system, such as, for example, on the memory or electronic storage unit.
  • the machine executable or machine-readable code can be provided in the form of software.
  • the code can be executed by the processor.
  • the code can be retrieved from the storage unit and stored on the memory for ready access by the processor.
  • the electronic storage unit can be precluded, and machine-executable instructions are stored on memory’.
  • the code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code or can be compiled during runtime.
  • the code can be supplied in a programming language that can be selected to enable the code to execute in a precompiled or as-compiled fashion.
  • aspects of the systems and methods provided herein can be embodied in programming.
  • Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a ty pe of machine readable medium.
  • Machine- executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory. random-access memory. flash memory ) or a hard disk.
  • Storage ty pe media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • the phy sical elements that carry such waves such as wired or wireless links, optical links, or the like, also may be considered as media bearing the software.
  • terms such as computer or machine "readable medium” refer to any medium that participates in providing instructions to a processor for execution.
  • a machine readable medium such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium.
  • Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings.
  • Volatile storage media include dynamic memory', such as main memory of such a computer platform.
  • Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system.
  • Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications.
  • RF radio frequency
  • IR infrared
  • Common forms of computer- readable media therefore include for example: hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other phy sical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory’ chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data.
  • a computer system disclosed herein can include or be in communication with an electronic display that comprises a user interface (UI) for providing, for example, one or more results (immediate results or archived results from a previous experiment), one or more user inputs, reference values from a library or database, or a combination thereof.
  • UI user interface
  • Uls include, without limitation, a graphical user interface (GUI) and web-based user interface.
  • Antibody affinity was measured with standard biolayer interferometry (BLI) approaches using the Octet® Platform (Sartorius). Briefly, antibodies were first immobilized on anti-hlgG Fc capture (AHC) sensors. After, these antibody -loaded tips were dipped into serial dilutions of soluble recombinant antigen solutions (human, rat, or cynomolgus P-selectin or E- selectin) followed by transfer to antigen-free buffer wells to observe the association and dissociation of the antigen to the antibody.
  • Raw data were processed with standard 1 : 1 kinetic models in the Octet® software, according to manufacturer’s recommendations.
  • FIGs. 1-6 and Table 4 show antibody affinity of PDL huEP5C7 antibodies to Human P-Selectin-His, Rhesus P-Selectin-His, Rat P-Selectin-His, Human E-Selectin-His, Cyno E-Selectin-His, and Rat P-Selectin-His.
  • FIGs. 22-27 and Table 5 show antibody affinity of Ab_0296, Ab_00349, Ab_0350, Ab_0352, Ab_0354, or Ab_0357 antibodies to Human P-Selectin. As shown in FIGs. 22-27 and Table 5. Ab 0352, Ab 0354. and Ab 0357 antibodies exhibit comparable binding affinity for Human P-selectin, compared to PDL huEP5C7.
  • FIGs. 28-33 and Table 6 show antibody affinity of Ab_0296, Ab_00349, Ab_0350, Ab_0352, Ab_0354, or Ab_0357 antibodies to Human E-Selectin. As shown in FIG. 28-33 and Table 6. Ab_0296, Ab_00349, Ab_0350. Ab_0352, Ab_0354, and Ab_0357 antibodies exhibit improved binding affinity (e.g., four-fold greater) for Human E-selectin, compared to PDL huEP5C7.
  • Example 2 Determination of antibody aggregation after thermal stress
  • Antibody solubility was studied after incubation under thermal stress conditions. Briefly, antibodies were buffer-exchanged into citrate buffer (25 mM sodium citrate-citric acid, 6% (w/v) sucrose, pH 6.0) and diluted to 1 mg/ml in citrate buffer. Antibody preparations w ere then incubated at 37°C and samples were taken at 0 weeks, 1 week, 2 weeks, and 4 weeks.
  • citrate buffer 25 mM sodium citrate-citric acid, 6% (w/v) sucrose, pH 6.0
  • antibody preparations were incubated at 50°C and samples were taken at 0 days. 1 day, 2 days, 4 days, and 7 days. Samples were analyzed on either an Agilent AdvanceBio SEC 300 (4.6x300 mm, 2.7 pm LC Column, Potassium Phosphate, pH 6.9 mobile phase, isocratic flow gradient at 0.35 mL/min) or an Agilent AdvanceBio SEC 200 with the same operating conditions. Mass percent of the injected sample contained in each observed peak was calculated and summarized using AdvanceBio instrument software according to manufacturer recommendations. The resulting time-dependent change in high molecular weight species/aggregates (HMWS), monomeric antibody species, and low molecular weight species/truncated products (LMWS). Similar studies were performed at 37 C for up to 4 weeks with antibody preparations diluted to 20 mg/ml
  • FIGs. 7 and 10 show time-dependent aggregation of PDL huEP5C7 antibodies after incubation under thermal stress conditions. No time-dependent changes to binding of PDL huEP5C7 antibodies to Human P-Selectin were observed following thermal stress conditions (FIGs. 9 and 12). However, binding of PDL huEP5C7 antibodies to Human E-Selectin decreased following thermal stress conditions (FIGs. 8 and 11)
  • FIGs. 34-35, 40-41, and 46-47 show time-dependent aggregation of Ab_0296, Ab_0352, and Ab_0357 antibodies after incubation under thermal stress conditions. Timedependent changes to binding of Ab_0296, Ab_0352, and Ab_0357 antibodies to Human P- Selectin or Human E-Selectin were observed following thermal stress conditions (FIGs. 36-37, 42-43, and 48-49).
  • FIG. 52 shows off-target binding of Ab_0296, Ab_0352, Ab_0357, and PDL huEP5C7 after incubation under thermal stress conditions.
  • Example 3 Determination of antibody aggregation after forced isomerization/acidic stress [00328] To evaluate aggregation after forced isomerization, antibody preparations were buffer exchanged into 100 mM sodium citrate, pH 3.5 and incubated at 25 °C for 2, 4, or 24 hours. Samples w ere then neutralized with addition of a predetermined volume of IM Tris and analyzed for aggregation by size exclusion chromatography as described above.
  • Example 4 Determination of antibody aggregation after forced deamidation/basic stress [00331] To evaluate aggregation after forced deamidation, antibody preparations were buffer exchanged into 1% (w/v) ammonium bicarbonate, pH 9.0 and incubated at 37°C for 2, 4, 24. 48. or 96 hours. Samples were then neutralized by buffer exchange into water and analyzed for aggregation by size exclusion chromatography as described above.
  • Example 5 Antibody inhibition of endothelial cell-RBC aggregates under venous flow conditions
  • SCD sickle cell disease
  • adhesion of blood cells to inflamed endothelium is driven by interactions between various blood cell and endothelial surface proteins, including P and E-selectin and the associated selectin ligands.
  • whole blood was collected from patients with SCD undergoing exchange transfusion protocols under IRB- approved protocols.
  • RBCs were isolated from citrate-anticoagulated blood, and these cells were incubated with PBS or 40 pM heme for 15 minutes or 4 hours to simulate acute or chronic hemolysis, commonly found in patients with sickle cell disease.
  • EP5C7 achieves statistically significant reduction in adhesion events compared to Crizalizumab and isotype control under both acute and chronic inflammatory' (FIGs. 19-21).
  • Example 6 Antibody inhibition of platelet-leukocyte aggregates under static conditions
  • Platelet-leukocy te aggregates are driven in part by P-selectin interactions on activated platelets with PSGL1 on leukocytes.
  • citrate-anticoagulated whole blood from healthy donors was mixed with various concentrations of antibody.
  • 25 pM TRAP-6 (Fisher Scientific, 34-975) was added at room temperature. Samples are promptly processed for FACS to detect platelet leukocyte adhesion events.
  • Crizanlizumab and Ab_0357 achieved similar reduction of P-selectin-dependent platelet-leukocyte aggregates across several unique blood donors (FIGs. 71-76).
  • Example 7 Antibody inhibition of platelet-leukocyte aggregates under static conditions
  • OASis public repository 7 was assessed to describe % relativeness, where 100% is perfectly human and 37-40% is typical to germline encoded antibody.
  • Detailed descriptions of OASis and the BioPhi platform are found in Prihoda et al., Mabs, 2022 Jan-Dec; 14(1): 020203. doi: 10.1080/19420862.2021.2020203, which is hereby incorporated by reference in its entirety.
  • an OASis identity score was assessed by converting the identity' score to the 0-100% range based on 544 therapeutic mAbs from IMGT mAb DB.
  • the 0% OASis percentile score corresponded to the least human and the 100% OASis percentile score corresponded to the most human antibody in the clinical or preclinical stage (including terminated mAbs).
  • the 0-100% percentile range provided a more intuitive scale that is robust to changes in the prevalence threshold.
  • the median OASis percentile was 5-7% for murine mAbs, 37-40% for humanized mAbs and 80-81% for human mAbs.
  • BioPhi humanness is reported using OASis, as well as using traditional methods based on positional residue frequency and germline sequence identity (Table 7).
  • Example 8 Antibody inhibition of red blood cell adhesion to endothelial cells under physiological flow conditions
  • Microchannels functionalized with endothelial cells mimic the intravascular environment and thereby allow the study of various pathological conditions in vitro.
  • a new microfluidic approach was introduced to the fabrication of microfluidic channels seeded with human endothelial cells utilizing a lamination-based technique and laser micro-machined components (endothelium-on-a-chip).
  • the endothelium-on-a-chip microfluidic platform allowed for assessment of HbSS red blood cell (RBC) adhesion to human endothelial cells that were stimulated with pathophysiologic levels of heme in a clinically diverse adult sickle cell disease (SCD) population.
  • RBC HbSS red blood cell
  • FIG. 77 depicts baseline RBC adhesion in non-SCD subjects on heme-activated HUVECs under physiologic flow conditions.
  • HUVECs were pretreated for 4 hours with 40 pM heme and basal culture media, after which RBC samples supplemented +/- 100 pg/ml of the test antibodies injected through microfluidic chambers pre-adhered with a HUVEC cell monolayer.
  • Adherent blood cells were counted per high power microscopic field and compared versus isolype control-treated samples.
  • test antibodies were crizanlizumab. PF-07209326, and BHB0007.
  • Treatment with lOOpg/ml of BHB0070 showed a statistically significant decrease in RBC adhesion to endothelium under physiological flow conditions in both chronic long term and acute short term heme activation conditions (FIGS. 78 and 79).
  • Embodiment 1 A target molecule-binding polypeptide, comprising: (a) a first light chain complementarity determining region (LCDR1) comprising a sequence having a sequence identical to the amino acid sequence set out in any one of SEQ ID NOs: 8-12; (b) a second light chain complementarity determining region (LCDR2) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 13-16; and (c) a third light chain complementarity determining region (LCDR3) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 17- 20.
  • LCDR1 first light chain complementarity determining region
  • LCDR2 second light chain complementarity determining region
  • LCDR3 a third light chain complementarity determining region
  • Embodiment 2 A target molecule-binding polypeptide, comprising: (a) a first light chain complementarity determining region (LCDR1) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 8-12; (b) a second light chain complementarity determining region (LCDR2) comprising a sequence having a sequence identical to the amino acid sequence set out in any one of SEQ ID NOs: 13-16; and (c) a third light chain complementarity determining region (LCDR3) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 17-20.
  • LCDR1 first light chain complementarity determining region
  • LCDR2 second light chain complementarity determining region
  • LCDR3 a third light chain complementarity determining region
  • Embodiment 3 A target molecule-binding polypeptide, comprising: (a) a first light chain complementarity determining region (LCDR1) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 8-12; (b) a second light chain complementarity determining region (LCDR2) comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in any one of SEQ ID NOs: 13- 1 ; and (c) a third light chain complementarity determining region (LCDR3) comprising a sequence having a sequence identical to the amino acid sequence set out in any one of SEQ ID NOs: 17-20.
  • LCDR1 first light chain complementarity determining region
  • LCDR2 second light chain complementarity determining region
  • LCDR3 a third light chain complementarity determining region
  • Embodiment 4 The target molecule-binding polypeptide of any one of embodiments 1-3, wherein the LCDR1 comprises a sequence identical to SEQ ID NO: 8.
  • Embodiment 5 The target molecule-binding polypeptide of any one of embodiments 1-4, wherein the LCDR2 comprises a sequence identical to SEQ ID NO: 13.
  • Embodiment 6 The target molecule-binding polypeptide of any one of embodiments 1-5, wherein the LCDR3 comprises a sequence identical to SEQ ID NO: 18.
  • Embodiment 7. The target molecule-binding polypeptide of any one of embodiments 1 -6, wherein the target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 25.
  • Embodiment 8. The target molecule-binding polypeptide of any one of embodiments 1-7, wherein the target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 25.
  • Embodiment 9 The target molecule-binding polypeptide of any one of embodiments 1-8, wherein the target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 23.
  • Embodiment 10 The target molecule-binding polypeptide of any one of embodiments 1-9, wherein the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 23.
  • Embodiment 1 1 The target molecule-binding polypeptide of any one of embodiments 1-3, wherein the LCDR1 comprises a sequence identical to SEQ ID NO: 9.
  • Embodiment 12 The target molecule-binding polypeptide of any one of embodiments 1-3 or 11, wherein the LCDR2 comprises a sequence identical to SEQ ID NO: 13.
  • Embodiment 13 The target molecule-binding polypeptide of any one of embodiments 1-3, 11 or
  • LCDR3 comprises a sequence identical to SEQ ID NO: 17.
  • Embodiment 14 The target molecule-binding polypeptide of any one of embodiments 1-3 or 11-
  • target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 31.
  • Embodiment 15 The target molecule-binding polypeptide of any one of embodiments 1-3 or 11-
  • target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 31.
  • Embodiment 16 The target molecule-binding polypeptide of any one of embodiments 1-3 or 11-
  • target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 29.
  • Embodiment 17 The target molecule-binding polypeptide of any one of embodiments 1-3 or 11-16, wherein the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 29.
  • Embodiment 18 The target molecule-binding polypeptide of any one of embodiments 1-4, wherein the LCDR2 comprises a sequence identical to SEQ ID NO: 13.
  • Embodiment 19 The target molecule-binding polypeptide of any one of embodiments 1-4 or 18, wherein the LCDR3 comprises a sequence identical to SEQ ID NO: 19.
  • Embodiment 20 The target molecule-binding polypeptide of any one of embodiments 1-4 or 18-
  • target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 35.
  • Embodiment 21 The target molecule-binding polypeptide of any one of embodiments 1-4 or 18-
  • target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 35.
  • Embodiment 22 The target molecule-binding polypeptide of any one of embodiments 1-4 or 18-
  • target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 33.
  • Embodiment 23 The target molecule-binding polypeptide of any one of embodiments 1-4 or 18-22, wherein the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 33.
  • Embodiment 24 The target molecule-binding polypeptide of any one of embodiments 1-4, wherein the LCDR2 comprises a sequence identical to SEQ ID NO: 14.
  • Embodiment 25 The target molecule-binding polypeptide of any one of embodiments 1-4 or 24, wherein the LCDR3 comprises a sequence identical to SEQ ID NO: 17.
  • Embodiment 26 The target molecule-binding polypeptide of any one of embodiments 1-4 or 24-
  • target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 37.
  • Embodiment 27 The target molecule-binding polypeptide of any one of embodiments 1-4 or 24-
  • target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 37.
  • Embodiment 28 The target molecule-binding polypeptide of any one of embodiments 1-4 or 24-
  • the target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 36.
  • Embodiment 29. The target molecule-binding polypeptide of any one of embodiments 1 -4 or 24-28, wherein the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 36.
  • Embodiment 30 The target molecule-binding polypeptide of any one of embodiments 1-3, wherein the LCDR1 comprises a sequence identical to SEQ ID NO: 10.
  • Embodiment 31 The target molecule-binding polypeptide of any one of embodiments 1-3 or 30, wherein the LCDR2 comprises a sequence identical to SEQ ID NO: 15.
  • Embodiment 32 The target molecule-binding polypeptide of any one of embodiments 1-3 or 30-
  • LCDR3 comprises a sequence identical to SEQ ID NO: 17.
  • Embodiment 33 The target molecule-binding polypeptide of any one of embodiments 1-3 or 30-
  • target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 40.
  • Embodiment 34 The target molecule-binding polypeptide of any one of embodiments 1-3 or 30-
  • target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 40.
  • Embodiment 35 The target molecule-binding polypeptide of any one of embodiments 1-3 or 30-
  • target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 39.
  • Embodiment 36 The target molecule-binding polypeptide of any one of embodiments 1-3 or 30-35, wherein the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 39.
  • Embodiment 37 The target molecule-binding polypeptide of any one of embodiments 1-3, wherein the LCDR1 comprises a sequence identical to SEQ ID NO: 11.
  • Embodiment 38 The target molecule-binding polypeptide of any one of embodiments 1-3 or 37, wherein the LCDR2 comprises a sequence identical to SEQ ID NO: 13.
  • Embodiment 39 The target molecule-binding polypeptide of any one of embodiments 1-3 or 37-
  • LCDR3 comprises a sequence identical to SEQ ID NO: 17.
  • Embodiment 40 The target molecule-binding polypeptide of any one of embodiments 1-3 or 37-
  • the target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 44.
  • Embodiment 41 The target molecule-binding polypeptide of any one of embodiments 1-3 or 37-
  • target molecule-binding polypeptide comprises a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 44.
  • Embodiment 42 The target molecule-binding polypeptide of any one of embodiments 1-3 or 37-
  • target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 42.
  • Embodiment 43 The target molecule-binding polypeptide of any one of embodiments 1-3 or 37-42, wherein the target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 42.
  • Embodiment 44 The target molecule-binding polypeptide of any one of embodiments 1-4, wherein the LCDR2 comprises a sequence identical to SEQ ID NO: 16.
  • Embodiment 45 The target molecule-binding polypeptide of any one of embodiments 1-4 or 44, wherein the LCDR3 comprises a sequence identical to SEQ ID NO: 20.
  • Embodiment 46 The target molecule-binding polypeptide of any one of embodiments 1-4 or 44-
  • target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 47.
  • Embodiment 47 The target molecule-binding polypeptide of any one of embodiments 1-4 or 44-
  • target molecule-binding polypeptide comprises a light chain variable domain having at least 90% sequence identity the amino acid sequence set out in SEQ ID NO: 47.
  • Embodiment 48 The target molecule-binding polypeptide of any one of embodiments 1-4 or 44-
  • target molecule-binding polypeptide comprises a light chain variable region comprising a sequence having at least 95% sequence identity to the amino acid sequence set out in SEQ ID NO: 47.
  • Embodiment 49 The target molecule-binding polypeptide of any one of embodiments 1-4 or 44-
  • target molecule-binding polypeptide comprises a light chain variable region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 47.
  • Embodiment 50 The target molecule-binding polypeptide of any one of embodiments 1-3, wherein the LCDR1 comprises a sequence identical to SEQ ID NO: 12.
  • Embodiment 51 The target molecule-binding polypeptide of any one of embodiments 1-3 or 50, wherein the LCDR2 comprises a sequence identical to SEQ ID NO: 16.
  • Embodiment 52 The target molecule-binding polypeptide of any one of embodiments 1-3 or 50- 51, wherein the LCDR3 comprises a sequence identical to SEQ ID NO: 17.
  • Embodiment 53 The target molecule-binding polypeptide of any one of embodiments 1-3 or SO-
  • target molecule-binding polypeptide comprises a light chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 49.
  • Embodiment 54 The target molecule-binding polypeptide of any one of embodiments 1-3 or SO-
  • target molecule-binding polypeptide comprises a light chain variable domain having at least 90% sequence identity to the amino acid sequence set out in SEQ ID NO: 49.
  • Embodiment 55 The target molecule-binding polypeptide of any one of embodiments 1-3 or SO-
  • target molecule-binding polypeptide comprises a light chain region comprising a sequence having at least 95% sequence identity to the amino acid sequence set out in SEQ ID NO: 49.
  • Embodiment 56 The target molecule-binding polypeptide of any one of embodiments 1-3 or SO-
  • target molecule-binding polypeptide comprises a light chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 49.
  • Embodiment 57 The target molecule-binding polypeptide of any one of embodiments 1-56, wherein the target molecule-binding polypeptide comprises: (a) a first heavy chain complementarity determining region (HCDR1) comprising a sequence having at least 80% homology relative to SEQ ID NO: 5; (b) a second heavy chain complementarity determining region (HCDR2) comprising a sequence having at least 80% homology relative to SEQ ID NO: 6; (c) a third heavy chain complementarity determining region (HCDR3) comprising a sequence having at least 80% homology relative to SEQ ID NO: 7; or (d) a combination thereof.
  • HCDR1 first heavy chain complementarity determining region
  • HCDR2 second heavy chain complementarity determining region
  • HCDR3 a third heavy chain complementarity determining region
  • Embodiment 58 The target molecule-binding polypeptide of any one of embodiments 1-57, wherein the target molecule-binding polypeptide comprises: (a) a HCDRl comprising a sequence identical to the amino acid sequence set out in SEQ ID NO: 5; (b) a HCDR2 comprising a sequence identical to the amino acid sequence set out in SEQ ID NO: 6; (c) a HCDR3 comprising a sequence identical to the amino acid sequence set out in SEQ ID NO: 7; or (d) a combination thereof.
  • Embodiment 59 The target molecule-binding polypeptide of any one of embodiments 1-58, wherein the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 24.
  • Embodiment 60 The target molecule-binding polypeptide of any one of embodiments 1-59. wherein the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 24.
  • Embodiment 62 The target molecule-binding polypeptide of any one of embodiments 1-61, wherein the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 22.
  • Embodiment 63 The target molecule-binding polypeptide of any one of embodiments 1-58. wherein the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 30.
  • Embodiment 64 The target molecule-binding polypeptide of any one of embodiments 1-58 or 63, wherein the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 30.
  • Embodiment 65 The target molecule-binding polypeptide of any one of embodiments 1-58 or 63-64, wherein the target molecule-binding polypeptide comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 28.
  • Embodiment 68 The target molecule-binding polypeptide of any one of embodiments 1-58 or 67, wherein the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 34.
  • Embodiment 69 The target molecule-binding polypeptide of any one of embodiments 1-58 or 67-68, wherein the target molecule-binding polypeptide comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 32.
  • Embodiment 70 The target molecule-binding polypeptide of any one of embodiments 1-58 or 67-69, wherein the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 32.
  • Embodiment 71 The target molecule-binding polypeptide of any one of embodiments 1-58 or 67-68, wherein the target molecule-binding polypeptide comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 38.
  • Embodiment 72 The target molecule-binding polypeptide of any one of embodiments 1-58 or 67-68 or 71, wherein the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 38
  • Embodiment 73 The target molecule-binding polypeptide of any one of embodiments 1-58. wherein the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 43.
  • Embodiment 74 The target molecule-binding polypeptide of any one of embodiments 1-58 or 73, wherein the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 43.
  • Embodiment 75 The target molecule-binding polypeptide of any one of embodiments 1-58 or 73-74, wherein the target molecule-binding polypeptide comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 41.
  • Embodiment 76 The target molecule-binding polypeptide of any one of embodiments 1-58 or 73-75, wherein the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 41.
  • Embodiment 77 The target molecule-binding polypeptide of any one of embodiments 1-58. wherein the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 46.
  • Embodiment 78 The target molecule-binding polypeptide of any one of embodiments 1-58 or 77, wherein the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 46.
  • Embodiment 79 The target molecule-binding polypeptide of any one of embodiments 1-58 or 77-78, wherein the target molecule-binding polypeptide comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 32.
  • Embodiment 80 The target molecule-binding polypeptide of any one of embodiments 1-58 or 77-79, wherein the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 32.
  • Embodiment 81 The target molecule-binding polypeptide of any one of embodiments 1-58, wherein the target molecule-binding polypeptide comprises a heavy chain variable domain having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 48.
  • Embodiment 82 The target molecule-binding polypeptide of any one of embodiments 1-58 or 81, wherein the target molecule-binding polypeptide comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 48.
  • Embodiment 83 The target molecule-binding polypeptide of any one of embodiments 1-58 or 81-82, wherein the target molecule-binding polypeptide comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 32.
  • Embodiment 84 The target molecule-binding polypeptide of any one of embodiments 1-58 or 81-83, wherein the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 32.
  • Embodiment 85 The target molecule-binding polypeptide of any one of embodiments 1-60, wherein the target molecule-binding polypeptide comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 26.
  • Embodiment 86 The target molecule-binding polypeptide of any one of embodiments 1-60 or 85, wherein the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 26.
  • Embodiment 87 The target molecule-binding polypeptide of any one of embodiments 1-58 or 71-72, wherein the target molecule-binding polypeptide comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 38.
  • Embodiment 88 The target molecule-binding polypeptide of any one of embodiments 1-58. 71- 72 or 87, wherein the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 38.
  • Embodiment 89 The target molecule-binding polypeptide of any one of embodiments 1-58 or 73-74, wherein the target molecule-binding polypeptide comprises a heavy chain region comprising a sequence having at least 80% sequence identity to the amino acid sequence set out in SEQ ID NO: 45.
  • Embodiment 90 The target molecule-binding polypeptide of any one of embodiments 1-58, 73- 74 or 89, wherein the target molecule-binding polypeptide comprises a heavy chain region having a sequence identical to the amino acid sequence set out in SEQ ID NO: 45.
  • Embodiment 91 The target molecule-binding polypeptide of any one of embodiments 1-58. wherein the target molecule-binding comprises a half-life extension mutation.
  • Embodiment 92 The target molecule-binding polypeptide of any one of embodiments 1-91, wherein the target molecule-binding polypeptide comprises a scaffold selected from IgGl or IgG4.
  • Embodiment 93 The target molecule-binding polypeptide of any one of embodiments 1-92. wherein the target molecule-binding polypeptide is an antibody fragment and the antibody fragment comprises a single-chain variable fragment (scFv), F(ab) fragment, a F(ab’)2 fragment, a monovalent IgG, a diabody, or a minibody.
  • scFv single-chain variable fragment
  • F(ab) fragment F(ab’)2 fragment
  • monovalent IgG a diabody, or a minibody.
  • Embodiment 94 The target molecule-binding polypeptide of embodiment 93. wherein the target molecule-binding polypeptide comprises an scFv.
  • Embodiment 95 The target molecule-binding polypeptide of any one of embodiments 1-94, wherein the target molecule-binding polypeptide is a monoclonal antibody.
  • Embodiment 96 The target molecule-binding polypeptide of any one of embodiments 1-94. wherein the target molecule-binding polypeptide is human, humanized, or chimeric.
  • Embodiment 97 The target molecule-binding polypeptide of any one of embodiments 1-96, wherein the target molecule-binding polypeptide binds to one or more amino acid residue within the amino acid sequence set forth in SEQ ID NO: 1.
  • Embodiment 98 The target molecule-binding polypeptide of any one of embodiments 1-96. wherein the target molecule-binding polypeptide binds to one or more amino acid residue within the amino acid sequence set forth in SEQ ID NO: 2.
  • Embodiment 99 The target molecule-binding polypeptide of any one of embodiments 1-96. wherein the target molecule-binding polypeptide binds to one or more amino acid residue within the amino acid sequence set forth in SEQ ID NO: 3.
  • Embodiment 100 The target molecule-binding polypeptide of any one of embodiments 1-96, wherein the target molecule-binding polypeptide binds to one or more amino acid residue within the amino acid sequence set forth in SEQ ID NO: 4.
  • Embodiment 101 The target molecule-binding polypeptide of any one of embodiments 1-100, wherein the target molecule is a selectin molecule.
  • Embodiment 102 The target molecule-binding polypeptide of embodiment 101 , wherein the selectin molecule is P-selectin, E-selectin, or both.
  • Embodiment 103 A pharmaceutical composition comprising the target molecule-binding polypeptide of any one of embodiments 1-102 and a pharmaceutically acceptable carrier or excipient.
  • Embodiment 104 A nucleic acid sequence encoding the target molecule-binding polypeptide of any one of embodiments 1-102.
  • Embodiment 105 An expression vector comprising a nucleic acid sequence encoding target molecule-binding polypeptide of any one of embodiments 1-102.
  • Embodiment 106 A host cell comprising the nucleic acid of embodiment 104 or the expression vector of embodiment 105.
  • Embodiment 107 The target molecule-binding polypeptide according to any one of embodiments 1-102. the pharmaceutical composition of embodiment 103, the nucleic acid sequence of embodiment 104, the expression vector of embodiment 105, or the host cell of embodiment 106 for use in therapy.
  • Embodiment 108 A method of treating a subject in need thereof comprising, administering to the subject the target molecule-binding polypeptide according to any one of embodiments 1-102, the pharmaceutical composition of embodiment 103, the nucleic acid sequence of embodiment 104, the expression vector of embodiment 105, or the host cell of embodiment 106.
  • Embodiment 109 The method of embodiment 108, wherein the administering is subcutaneous or intramuscular.
  • Embodiment 110 The method of embodiment 108 or 109, wherein administering increases blood oxygen saturation or decreases frequency of chest pain, severity of chest pain, frequency of limb pain, severity of limb pain, a risk of stroke, or a risk of heart attack, in the subject.
  • Embodiment 111 The method of any one of embodiments 108-110, wherein the subj ect has a blood hemoglobin level of from about 6 g/dL to about 11 g/dL.
  • Embodiment 112. The method of any one of embodiments 108-111, wherein the subject has sickle cell anemia, lupus, or primary myelofibrosis.
  • Embodiment 113 The method of any one of embodiments 108-112, wherein the subject has a mutation in the hemoglobin beta gene (HBB).
  • HBB hemoglobin beta gene
  • Embodiment 114 Use of the target molecule-binding polypeptide according to any one of embodiments 1-102, the pharmaceutical composition of embodiment 103, the nucleic acid sequence of embodiment 104, the expression vector of embodiment 105, or the host cell of embodiment 106, in the manufacture of a medicament for a blood disease, an autoimmune condition, a cardiovascular condition or a cancer.
  • Embodiment 115 The use of embodiment 114, wherein the cardiovascular condition is sickle cell disease, systemic lupus erythematosus, or primary’ myelofibrosis.
  • Embodiment 116 A kit comprising the target molecule-binding polypeptide according to any one of embodiments 1-102, the pharmaceutical composition of embodiment 103, the nucleic acid sequence of embodiment 104, the expression vector of embodiment 105, or the host cell of embodiment 106 and instructions for use.
  • Embodiment 117 A target molecule-binding polypeptide, wherein the target molecule-binding polypeptide has increased humanness compared to humanness of a control antibody, as defined by Observed Antibody Space identity’ search (OASis) percentile score, optionally wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 and/or a light chain according to SEQ ID NO: 55.
  • OASis Observed Antibody Space identity’ search
  • Embodiment 1 18. The target molecule-binding polypeptide of embodiment 1 17, wherein the target molecule-binding polypeptide has humanness of more than 40%, 50%, 60%, 70%, 80%, or 90% as defined by OASis percentile score.
  • OASis Detailed descriptions of OASis and the BioPhi platform are found in Prihoda et al., Mabs, 2022 Jan-Dec; 14(1): 020203. doi:
  • a target molecule-binding polypeptide wherein the target molecule-binding polypeptide has increased stability compared to stability' of a control antibody, as measured bymelting temperature (Tm), optionally wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 and/or a light chain according to SEQ ID NO: 55.
  • Embodiment 120 The target molecule-binding polypeptide of embodiment 119, wherein the target molecule-binding polypeptide has a Tm of more than 65 °C, 70 °C, 75 °C or 80 °C.
  • Embodiment 121 A target molecule-binding polypeptide, wherein the target molecule-binding polypeptide has increased stability under an acidic condition compared to stability under acidic condition of a control antibody, as measured by aggregation temperature (T agg ), optionally wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 and/or a light chain according to SEQ ID NO: 55.
  • Embodiment 122 The target molecule-binding polypeptide of embodiment 121. wherein the target molecule-binding polypeptide has a T agg of more than 60°C or 65 °C.
  • Embodiment 123 The target molecule-binding polypeptide of embodiment 121 or 122, wherein the acidic condition comprises a pH of less than 7.0, a pH of about 2.0 to about 6.0, a pH of about 2.0 to about 5.0, a pH of about 2.0 to about 4.0 or a pH of about 3.0.
  • Embodiment 124 A target molecule-binding polypeptide, wherein the target molecule-binding polypeptide has an increased binding affinity to human E-selectin compared to a binding affinity of a control antibody, optionally wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 and/or a light chain according to SEQ ID NO: 55.
  • Embodiment 125 The target molecule-binding polypeptide of embodiment 124. wherein the binding affinity of the target molecule-binding polypeptide to human E-selectin is at least 2-fold, 3-fold, or 4-fold greater than the binding affinity of a control antibody, optionally wherein the control antibody comprises a heavy chain according to SEQ ID NO: 54 and/or a light chain according to SEQ ID NO: 55.
  • Embodiment 126 The target molecule-binding polypeptide of any one of embodiments 124-125, wherein the target molecule-binding polypeptide has a KD for binding to human E-selectin of less than 4.0 x 10-8 M, less than 3.0 x 10-8 M, or less than 2.0 x 10-8 M.
  • Embodiment 127 The target molecule-binding polypeptide of any one of embodiments 117-125, wherein the control antibody comprises a heavy chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 54 and a light chain variable domain having a sequence identical to the amino acid sequence set out in SEQ ID NO: 55.
  • Embodiment 128 The target molecule-binding polypeptide of any one of embodiments 117-127, wherein the target molecule-binding polypeptide is the target molecule-binding polypeptide of any one of embodiments 1-102.

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Abstract

La présente divulgation concerne des polypeptides et des variants capables de se lier à des molécules de sélectine telles que la sélectine P, la sélectine E, ou les deux.
PCT/US2024/038872 2023-07-21 2024-07-19 Compositions de liaison à la sélectine p et e et leurs procédés d'utilisation Pending WO2025024323A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995034324A1 (fr) * 1994-06-14 1995-12-21 Protein Design Labs, Inc. Anticorps monoclonaux croises se liant specifiquement a la selectine e et a la selectine p
WO2008076487A2 (fr) * 2006-12-20 2008-06-26 Verenium Corporation Anticorps: procédé de fabrication et d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995034324A1 (fr) * 1994-06-14 1995-12-21 Protein Design Labs, Inc. Anticorps monoclonaux croises se liant specifiquement a la selectine e et a la selectine p
WO2008076487A2 (fr) * 2006-12-20 2008-06-26 Verenium Corporation Anticorps: procédé de fabrication et d'utilisation

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