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WO2022161454A1 - 抗原结合蛋白及其用途 - Google Patents

抗原结合蛋白及其用途 Download PDF

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Publication number
WO2022161454A1
WO2022161454A1 PCT/CN2022/074550 CN2022074550W WO2022161454A1 WO 2022161454 A1 WO2022161454 A1 WO 2022161454A1 CN 2022074550 W CN2022074550 W CN 2022074550W WO 2022161454 A1 WO2022161454 A1 WO 2022161454A1
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WIPO (PCT)
Prior art keywords
seq
binding protein
amino acid
acid sequence
antigen binding
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PCT/CN2022/074550
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English (en)
French (fr)
Inventor
曹国庆
朱丽媛
施俊巍
Original Assignee
明慧医药（杭州）有限公司
明慧医药（上海）有限公司
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Application filed by 明慧医药（杭州）有限公司, 明慧医药（上海）有限公司 filed Critical 明慧医药（杭州）有限公司
Priority to US18/263,480 priority Critical patent/US20240309093A1/en
Priority to AU2022213745A priority patent/AU2022213745A1/en
Priority to JP2023546218A priority patent/JP2024504825A/ja
Priority to CA3206241A priority patent/CA3206241A1/en
Priority to CN202280011977.6A priority patent/CN116761889A/zh
Priority to EP22745326.3A priority patent/EP4286520A1/en
Publication of WO2022161454A1 publication Critical patent/WO2022161454A1/zh

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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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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

Definitions

  • the present application relates to the field of biomedicine, in particular to an antigen-binding protein and use thereof.
  • Human programmed cell death receptor-1 (PD-1) is one of the known major immune checkpoints, which is expressed on the surface of activated T lymphocytes, and it interacts with the ligand PD-L1 (programmed death receptor- The combination of ligand 1, programmed cell death-Ligand 1) and PD-L2 (programmed cell death-Ligand 2) can inhibit the activity of T lymphocytes and related in vivo cellular immune responses.
  • PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells. Numerous studies have shown that the interaction between PD-1 and PD-L1 is the main reason for PD-L1-positive tumor cells to evade immune surveillance. By blocking the negative regulation of PD-1/PD-L1 signaling pathway by tumor cells and activating the immune system, it can promote T cell-related tumor-specific cellular immune responses and treat tumors.
  • anti-PD-1 antibodies still have the defects of poor selectivity and low affinity. Therefore, it is necessary to develop novel anti-PD-1 antibodies with high affinity and specificity for PD-1.
  • the present application provides an isolated antigen-binding protein having one or more of the following properties: 1) in an ELISA assay, binds to PD-1 with an EC 50 of about 11 ⁇ g/mL or less; 2 ) inhibits human PD-L1 binding to human PD-1 with an EC50 of about 2 ⁇ g/mL or less in a competitive ELISA assay; and 3) is capable of binding primate (eg, human or monkey) PD-1 .
  • the isolated antigen binding protein binds the same or overlapping PD-1 epitope as the reference antibody.
  • the isolated antigen binding protein competes with the reference antibody for binding to PD-1.
  • the binding affinity of the isolated antigen binding protein to PD-1 is substantially the same as the binding affinity of the reference antibody to PD-1.
  • the reference antibody comprises a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:128.
  • the reference antibody comprises a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:127.
  • the reference antibody comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:126.
  • the reference antibody comprises a heavy chain variable region VH comprising the HCDR1, HCDR2 and HCDR3, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 128; the HCDR2 comprise the amino acid sequence shown in SEQ ID NO: 127; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 126.
  • the VH of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO:132.
  • the reference antibody comprises LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:131.
  • the reference antibody comprises LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:130.
  • the reference antibody comprises LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:129.
  • the reference antibody comprises a light chain variable region VL
  • the VL comprises the LCDR1, LCDR2 and LCDR3
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 131
  • the LCDR2 comprise the amino acid sequence shown in SEQ ID NO: 130
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 129.
  • the VL of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO:133.
  • the reference antibody comprises pembrolizumab or an antigen-binding fragment thereof.
  • the isolated antigen binding protein comprises HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:138.
  • the isolated antigen binding protein comprises HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:142.
  • the isolated antigen binding protein comprises HCDR3 comprising SEQ ID NOs: 3, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 and the amino acid sequence shown in any one of 67.
  • the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:137.
  • the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:141.
  • the isolated antigen binding protein comprises HCDR2 comprising SEQ ID NOs: 2, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and the amino acid sequence shown in any one of 54.
  • the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:136.
  • the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:140.
  • the isolated antigen binding protein comprises HCDR1 comprising SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and the amino acid sequence shown in any one of 41.
  • the isolated antigen binding protein comprises a heavy chain variable region VH comprising the HCDR1, HCDR2 and HCDR3, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 138; the The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 137; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 136.
  • the isolated antigen binding protein comprises a heavy chain variable region VH comprising the HCDR1, HCDR2 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 142; the The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 141; and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 140.
  • the VH of the isolated antigen binding protein comprises the HCDR1, HCDR2 and HCDR3 comprising SEQ ID NOs: 3, 55, 56, 57, 58, 59, 60, 61
  • the HCDR2 comprises SEQ ID NO: 2, 42, 43, 44, 45, 46, 47, 48, 49, 50
  • the HCDR1 comprises SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
  • the HCDR1, HCDR2 and HCDR3 of the isolated antigen binding protein comprise any one of the group of amino acid sequences selected from the group consisting of:
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 2
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 42
  • HCDR3 SEQ ID NO: 55;
  • HCDR1 SEQ ID NO: 4
  • HCDR2 SEQ ID NO: 43
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 5
  • HCDR2 SEQ ID NO: 44
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 6
  • HCDR2 SEQ ID NO: 45
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 7
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 8
  • HCDR2 SEQ ID NO: 47
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 10
  • HCDR2 SEQ ID NO: 45
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 11
  • HCDR2 SEQ ID NO: 44
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 12
  • HCDR2 SEQ ID NO: 43
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 13
  • HCDR2 SEQ ID NO: 48
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 14
  • HCDR2 SEQ ID NO: 45
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 15
  • HCDR2 SEQ ID NO: 47
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 16
  • HCDR2 SEQ ID NO: 43
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 17
  • HCDR2 SEQ ID NO: 49
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 19
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 20
  • HCDR2 SEQ ID NO: 50
  • HCDR3 SEQ ID NO: 56;
  • HCDR1 SEQ ID NO: 21, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 57;
  • HCDR1 SEQ ID NO: 22
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 51
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 58;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 24, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 59;
  • HCDR1 SEQ ID NO: 25
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 60;
  • HCDR1 SEQ ID NO: 26
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 61;
  • HCDR1 SEQ ID NO: 27
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 28
  • HCDR2 SEQ ID NO: 52
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 29, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 25
  • HCDR2 SEQ ID NO: 52
  • HCDR3 SEQ ID NO: 62;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 50
  • HCDR3 SEQ ID NO: 63;
  • HCDR1 SEQ ID NO: 30, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 31, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 32
  • HCDR2 SEQ ID NO: 53
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 33
  • HCDR2 SEQ ID NO: 52
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 34
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 64;
  • HCDR1 SEQ ID NO: 35
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 57;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 54
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 65;
  • HCDR1 SEQ ID NO:36
  • HCDR2 SEQ ID NO:46
  • HCDR3 SEQ ID NO:3;
  • HCDR1 SEQ ID NO: 37
  • HCDR2 SEQ ID NO: 52
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 59;
  • HCDR1 SEQ ID NO: 38
  • HCDR2 SEQ ID NO: 54
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 39
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 40
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 55;
  • HCDR1 SEQ ID NO: 41
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 3;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 46
  • HCDR3 SEQ ID NO: 66;
  • HCDR1 SEQ ID NO:9
  • HCDR2 SEQ ID NO:46
  • HCDR3 SEQ ID NO:67.
  • the isolated antigen binding protein comprises H-FR1
  • the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1
  • the H-FR1 comprises SEQ ID NO : the amino acid sequence shown at 68, 144-148, 230 and/or 235.
  • the isolated antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NOs: 69, 149- The amino acid sequence shown at 151 and/or 231.
  • the isolated antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NOs: 70, 152- The amino acid sequence shown at 161 and/or 232.
  • the isolated antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO : the amino acid sequence shown in 71, 162 and/or 233.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 comprises SEQ ID NOs: 68, 144-148, 230 and/or or the amino acid sequence shown in 235; the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 69, 149-151 and/or 231; the H-FR3 comprises SEQ ID NO: 70, 152-161 and/ or the amino acid sequence shown in 232; and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 71, 162 and/or 233.
  • the isolated antigen binding protein comprises a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO: 139 and/or 234.
  • the isolated antigen binding protein comprises a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO: 143 and/or 236.
  • the VH of the isolated antigen binding protein comprises the amino acid sequence set forth in any one of SEQ ID Nos: 72-120 and/or 163-227.
  • the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
  • the isolated antigen binding protein comprises a single domain antibody or antigen binding fragment thereof.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the antibodies include chimeric antibodies, humanized antibodies, and/or fully human antibodies.
  • the isolated antigen binding protein comprises the amino acid sequence set forth in any of SEQ ID Nos: 72-120 and/or 163-227.
  • the isolated antigen binding protein further comprises an immunoglobulin Fc region.
  • the VH of the antigen binding protein is directly or indirectly linked to the Fc region.
  • the C-terminus of the VH of the antigen binding protein is directly or indirectly linked to the N-terminus of the Fc region.
  • the VH of the antigen binding protein is fused in-frame to the Fc region.
  • the VH of the antigen binding protein is linked to the Fc region through a linker.
  • the linker comprises a peptide linker.
  • the linker comprises a flexible linker.
  • the isolated antigen binding protein comprises the VH, the linker and the Fc region of the antigen binding protein in order from N-terminus to C-terminus.
  • the Fc region comprises an IgGl-derived Fc or an IgG4-derived Fc.
  • the Fc region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 121-125.
  • the application provides one or more polypeptides comprising the isolated antigen binding protein.
  • the application provides one or more immunoconjugates comprising the isolated antigen binding protein or the polypeptide.
  • the application provides one or more isolated nucleic acid molecules encoding the isolated antigen binding protein, or the polypeptide.
  • the application provides one or more vectors comprising the isolated nucleic acid molecules.
  • the application provides one or more cells comprising and/or expressing the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule and /or said carrier.
  • the present application provides a method for preparing the isolated antigen-binding protein and/or the polypeptide, the method comprising expressing the isolated antigen-binding protein and/or the polypeptide The cells were cultured under the conditions.
  • the application provides one or more pharmaceutical compositions comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the The carrier, the cells, and optionally pharmaceutically acceptable adjuvants and/or excipients.
  • the application provides a method for detecting the presence and/or content of PD-1, comprising:
  • the isolated antigen binding protein or the polypeptide is administered.
  • the application provides one or more kits comprising the isolated antigen binding protein or the polypeptide.
  • the kit includes instructions for use describing methods for detecting the presence and/or content of PD-1.
  • the present application provides a use of the isolated antigen-binding protein or the polypeptide in the preparation of a kit for detecting the presence and/or content of PD-1.
  • the present application provides the use of one or more of the isolated antigen binding proteins and/or the polypeptides in the manufacture of a medicament for preventing and/or treating a disease or disorder.
  • the application provides one or more of the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell and/or said pharmaceutical composition for preventing, alleviating and/or treating diseases or conditions.
  • the disease or disorder comprises a tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a hematological tumor.
  • the tumor comprises a tumor associated with the expression of PD-L1.
  • the tumor is selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal cancer, and breast cancer.
  • the application provides a method of preventing and/or treating a disease or disorder, comprising administering to a subject in need thereof an effective amount of the isolated antigen-binding protein, the polypeptide, the The immunoconjugate, the isolated nucleic acid molecule, the carrier, and/or the cell.
  • the disease or disorder comprises a tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a hematological tumor.
  • the tumor comprises a tumor associated with the expression of PD-L1.
  • the tumor is selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal cancer, and breast cancer.
  • the present application provides a method for inhibiting the interaction of PD-1 and PD-L1, comprising administering to a subject in need thereof an effective amount of the isolated antigen-binding protein, the polypeptide, the The immunoconjugate, the isolated nucleic acid molecule, the carrier, and/or the cell.
  • the present application provides the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell and/or the The described pharmaceutical composition is used to inhibit the interaction between PD-1 and PD-L1.
  • the present application provides the use of the isolated antigen-binding protein and/or the polypeptide in the preparation of a medicament for inhibiting the interaction between PD-1 and PD-L1.
  • FIG 1 shows the SDS-PAGE protein expression results of the antigen-binding proteins described in this application.
  • Figure 2 shows the binding activity of the antigen-binding protein described in the present application to recombinant human PD-1, recombinant monkey PD-1 and recombinant mouse PD-1.
  • Figures 3A-3B show the kinetic detection results of the affinity between MHPD1-A Fc and Pembrolizumab described in the present application for antigenic proteins.
  • isolated generally refers to artificial means obtained from the natural state. If an "isolated" substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated of.
  • isolated does not exclude the admixture of artificial or synthetic materials, nor does it exclude the presence of other impurities that do not affect the activity of the material.
  • the term “antigen binding protein” generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen.
  • the term “antigen binding protein” may include “antibody” or "antigen binding fragment”.
  • the antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and may comprise any molecule comprising an antigen-binding portion thereof.
  • antibody may include monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to camelid antibodies, fully human antibodies, chimeric antibodies, single chain antibodies (eg, scFv), and antibody fragments that bind to an antigen (eg, Fab, Fab' and (Fab)2 fragments).
  • antibody may also include all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein. In the present application, the "antibody” may include single domain antibodies.
  • single domain antibody generally refers to a class of antibodies that lack the antibody light chain and only have the variable region of the heavy chain.
  • hcAb heavy chain antibody
  • VHH variable domains of The hcAb
  • single-domain antibodies Compared with other antibodies, single-domain antibodies have the advantages of low immunogenicity, small molecules, and strong penetrating power, so they have broad application prospects in the fields of basic research, drug development, and disease treatment.
  • single domain antibodies can be from llamas.
  • Single domain antibodies may be composed of heavy chain variable regions (VH).
  • VH heavy chain variable region
  • the term "heavy chain variable region” generally refers to the amino-terminal domain of the heavy chain of an antigen-binding fragment. Heavy chain variable regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each heavy chain variable region can consist of three CDRs and four FR regions, which can be arranged from the amino terminus to the carboxy terminus in the following order: H-FR1, HCDR1, H-FR2, HCDR2, H-FR3, HCDR3 and H -FR4.
  • the heavy chain variable region contains a binding domain that interacts with an antigen (e.g., PD-1).
  • an antigen e.g., PD-1).
  • the exact boundaries of the CDRs have been defined differently from system to system.
  • the system described by Kabat Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) provides not only a method that can be applied to any variable region of an antigen binding protein.
  • the residue numbering system is defined, and precise residue boundaries are provided that define the CDRs.
  • These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that despite large diversity at the amino acid sequence level, certain subsections within the Kabat CDRs adopt nearly identical peptide backbone conformations. These subsections were designated L1 , L2 and L3 or H1, H2 and H3, where "L” and "H” refer to the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have overlapping boundaries with Kabat CDRs.
  • CDRs can be defined by the IMGT numbering system.
  • single domain antibody can be used interchangeably with “nanobody", "VHH".
  • amino acid range in the table refers to the amino acid numbering range from the N-terminus of the antigen-binding protein of the present application, for example, "26-33" refers to the amino acid sequence from the 26th to the 33rd position from the N-terminus.
  • the term "monoclonal antibody” generally refers to a population of substantially homologous antibodies, ie the individual antibodies comprising the population are identical except for possible naturally occurring mutations present in minor amounts.
  • Monoclonal antibodies are highly specific, directed against a single antigenic site.
  • the monoclonal antibodies can be prepared by hybridoma technology or produced in bacterial, eukaryotic, or plant cells by using recombinant DNA methods.
  • Monoclonal antibodies can also be obtained from phage antibody libraries, using techniques such as those described by Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991). conduct.
  • chimeric antibody generally refers to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class, while The remainder of the chain is then homologous to the corresponding sequence in another species.
  • variable regions of both light and heavy chains are derived from variable regions of antibodies from one animal species (eg, mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (eg, human) .
  • non-human B cells or hybridoma cells can be used to generate variable regions, and the constant regions combined therewith are derived from humans.
  • variable regions have the advantage of being easy to prepare and their specificity is not affected by the source of the constant regions with which they are combined.
  • the constant region of the chimeric antibody can be derived from humans, the possibility of eliciting an immune response when the chimeric antibody is injected is lower than that of using an antibody whose constant region is of non-human origin.
  • PD-1 generally refers to human programmed cell death receptor-1, a 288 amino acid type I membrane protein first described in 1992 (Ishida et al. ., EMBO J, 11 (1992), 3887-3895).
  • PD-1 is a member of the expanded CD28/CTLA-4 T cell regulator family and has two ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273).
  • the structure of the protein includes an extracellular IgV domain followed by a transmembrane region and an intracellular tail.
  • the intracellular tail contains two phosphorylation sites located in the immunoreceptor tyrosine-based inhibitory motif and the immunoreceptor tyrosine-based switch motif, suggesting that PD-1 negatively regulates TCR signaling. This is consistent with the binding of SHP-1 and SHP-2 phosphatases to the cytoplasmic tail of PD-1 upon ligand binding. PD-1 is upregulated after T cell receptor (TCR)-mediated activation and is observed on both activated and depleted T cells (Agata et al., Int. Immunology 8 (1996), 765-772) .
  • TCR T cell receptor
  • PD-1 protein Although PD-1 has a relatively broad expression pattern, its most important role is probably as a co-inhibitory receptor on T cells (Chinai et al., Trends in Pharmacological Sciences 36 (2015), 587-595). Thus, the focus is on blocking the interaction of PD-1 with its ligands to enhance T cell responses to treat tumors.
  • PD-1 protein "PD-1”, PD1”, PDCD1”, “hPD-1” and “hPD-I” are used interchangeably and include isoforms of human PD-1, species homologs, Functionally active fragments, and analogs that share at least one epitope with PD-1.
  • the "functionally active fragment” can include a fragment that retains the endogenous function of at least one naturally occurring protein (eg, binds to the antigen binding proteins described herein).
  • the "functionally active fragment” may include a domain that binds to the antigen binding protein of the present application.
  • An exemplary amino acid sequence of human PDl can be shown in UniProt Accession No. Q15116.
  • PD-L1 generally refers to programmed death receptor-ligand 1, which may also be referred to as B7 homologue 1, B7-H1, cluster of differentiation 274, (3) 274 or CD274, which downregulates T-cell activation and cytokine secretion upon binding to PD-1.
  • B7 homologue 1 B7-H1, cluster of differentiation 274, (3) 274 or CD274, which downregulates T-cell activation and cytokine secretion upon binding to PD-1.
  • PD-L1 includes any native PD-L1 from any vertebrate source, including mammals, such as primates (eg, humans and cynomolgus monkeys) and rodents (eg, mice and rats) ). The term encompasses "full length", unprocessed PD-L1 as well as any form of PD-L1 produced by cellular processing.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • PD-L1 includes complete PD-L1 and fragments thereof, as well as functional fragments, isoforms, species homologues, derivatives, and analogs of PD-L1, as well as having at least one epitope in common with PD-L1 analogs of .
  • the "functionally active fragments” can include fragments that retain the endogenous function of at least one naturally occurring protein (e.g., bind to the antigen binding proteins described herein).
  • the "functionally active fragment” may include a domain that binds to the antigen binding protein of the present application.
  • Exemplary human PD-L1 amino acid sequences can be found under NCBI Accession No. NP_001254653 or UniProt Accession No. Q9NZQ7.
  • the present application may also include functional variants, derivatives, analogs, homologues, and fragments thereof.
  • a variant of any given sequence refers to one in which a particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one Sequence of endogenous functions.
  • Variant sequences can be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide, so long as the The original functional activity is sufficient.
  • the term "derivative" generally refers to the polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and /or addition, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
  • analog generally refers to a polypeptide or polynucleotide and includes any mimetic of the polypeptide or polynucleotide, ie possessing at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics chemical compounds.
  • amino acid substitutions such as at least 1 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitutions, can be made, so long as the modified sequence remains substantially as desired activity or ability.
  • Amino acid substitutions can include the use of non-naturally occurring analogs.
  • homologue generally refers to an amino acid sequence or nucleotide sequence that has some homology to a naturally occurring sequence.
  • the term “homology” may be equivalent to sequence "identity”.
  • homologous sequences can include amino acid sequences that can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • a homologue will contain the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or it can be expressed in terms of sequence identity.
  • a reference to a sequence having a percent identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to that percent identity over the entire length of the referenced SEQ ID NO. the sequence of.
  • sequence alignments can be performed by various means known to those skilled in the art, eg, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment among the full-length sequences being compared.
  • proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphiphilic nature of the residues, so long as endogenous function is preserved.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids containing uncharged polar headgroups with similar hydrophilicity values include amino acids Paraparagine, Glutamine, Serine, Threonine and Tyrosine.
  • the term "tumor” generally refers to a neoplasm formed by the proliferation of local tissue cells under the action of various tumorigenic factors.
  • the tumor can include a solid tumor.
  • the tumor may comprise a hematological tumor.
  • the tumor can include a tumor associated with the expression of PD-L1.
  • the term "tumor associated with the expression of PD-L1" generally refers to tumors in which altered PD-L1 expression leads to disease progression or evasion of immune surveillance.
  • the "tumor associated with the expression of PD-L1" may be a tumor formed by up-regulation of PD-L1 expression, resulting in disease progression or evasion of immune surveillance.
  • the tumor associated with the protein expression of PD-L1 may be a PD-L1 positive tumor.
  • the protein expression of PD-L1 on the tumor cell surface or in the tumor microenvironment is approximately 1%, 5%, 10%, 15%, 20%, 25% higher than normal cells , 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.
  • solid tumor generally refers to a tangible mass that can be detected clinically (eg, X-ray, CT scan, ultrasound, or palpation).
  • the solid tumor can be selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal cancer, and breast cancer.
  • the term "blood tumor” generally refers to tumors that cannot be seen or palpable on X-ray, CT scan, B-ultrasound, and palpation.
  • the blood tumor can include leukemia.
  • the hematological tumor may comprise a lymphoma.
  • the blood tumor can include multiple myeloma.
  • the term "immunoconjugate” generally refers to the conjugation of such other agents (eg, chemotherapeutic agents, radioactive elements, cytostatic and cytotoxic agents) to the isolated antigen-binding protein (eg, A conjugate formed by covalently linking a linker molecule) that can deliver the other agent to the target cell through the specific binding of the isolated antigen-binding protein to the antigen on the target cell (eg, tumor cells).
  • the immunoconjugate is then internalized and finally enters the interior of the target cell (eg, into vesicles such as lysosomes), at which point the linker molecule in the immunoconjugate can be cleaved, releasing the other
  • the agent thus exerts its cytotoxic effect.
  • the antigen may also be secreted by the target cell and located in the space outside the target cell.
  • the term "subject” generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, isolated from their natural environment or artificially synthesized, or analogs thereof.
  • the term "vector” generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • the vector can transfer the inserted nucleic acid molecule into and/or between cells.
  • the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell.
  • the vector can produce the desired expression product by culturing suitable cells containing the vector.
  • the vector may comprise a lentiviral vector.
  • the term "cell” generally refers to a plasmid or vector that can or has contained a nucleic acid molecule described herein, or an individual cell capable of expressing a polypeptide described herein or an antigen binding protein described herein , cell lines or cell cultures.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the polypeptides or antigen-binding proteins described herein.
  • the cells can be obtained by transfecting cells in vitro using the vectors described herein.
  • the cells may be prokaryotic cells (eg E.
  • the cells can be immune cells.
  • the immune cells can be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and /or peripheral blood mononuclear cells.
  • treating generally refers to: (i) preventing the occurrence of a disease, disorder or condition in a patient who may be susceptible to a disease, disorder and/or condition but has not been diagnosed with the disease; (ii) inhibiting the disease , disease or condition, i.e. arresting its development; and (iii) alleviating the disease, disorder or condition, i.e. causing the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition subsided.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • proteins are used interchangeably and generally refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (eg, binding to labeling components).
  • amino acid includes natural and/or non-natural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.
  • polynucleotide used interchangeably and generally refer to nucleosides of any length Polymeric forms of acids, such as deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined by ligation analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence of isolated DNA, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • Transfer RNA Transfer RNA
  • ribosomal RNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro-RNA
  • ribozyme ribozyme
  • cDNA recombinant polynucleotide
  • branched polynucleotide plasmid
  • vector any sequence
  • a polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation to labeled components.
  • K D (likewise, “K D " or “KD”) generally refers to "affinity constant” or "equilibrium dissociation constant”, and refers to in titration measurements at equilibrium, or by The value obtained by dividing the dissociation rate constant (k d ) by the association rate constant ( ka ). Association rate constants ( ka ), dissociation rate constants (k d ), and equilibrium dissociation constants (K D ) are used to represent the effect of binding proteins (eg, isolated antigen-binding proteins described herein) on antigens (eg, PD-1 ). protein) binding affinity. Methods for determining association and dissociation rate constants are well known in the art.
  • the K D value can be determined by Octet (Sartorius Corporation), or can be determined using other experimental approaches and instruments such as BIAcore (Biomolecular Interaction Analysis System, GE Healthcare Life Science).
  • the K D value can also be measured using KinExA (Kinetic Exclusion Assay) from Sapidyne Instruments (Boise, Idaho), or other surface plasmon resonance (SPR) instruments .
  • the K D value can be determined by BIAcore.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides an isolated antigen-binding protein that can have an EC 50 of about 11 ⁇ g/mL or less in an ELSA assay (eg, the EC 50 is not greater than about 11 ⁇ g/mL, not greater than about 10 ⁇ g/mL, not more than about 9.5 ⁇ g/mL, not more than about 9.0 ⁇ g/mL, not more than about 8.5 ⁇ g/mL, not more than about 8.0 ⁇ g/mL, not more than about 7.5 ⁇ g/mL, Not higher than about 7.0 ⁇ g/mL, not higher than 6.5 ⁇ g/mL, not higher than about 6.0 ⁇ g/mL, not higher than 5.5 ⁇ g/mL, not higher than about 5.0 ⁇ g/mL, not higher than about 4.5 ⁇ g/mL mL, not higher than about 4.0 ⁇ g/mL, not higher than about 3.5 ⁇ g/mL, not higher than about 3.0 ⁇ g/mL
  • the isolated antigen-binding proteins described herein can exhibit an EC50 of about 2 ⁇ g/mL or less (eg, the EC50 is not higher than about 1.9 ⁇ g/mL, not higher than about 1.8 ⁇ g/mL) mL, not higher than about 1.7 ⁇ g/mL, not higher than about 1.5 ⁇ g/mL, not higher than about 1.3 ⁇ g/mL, not higher than about 1.1 ⁇ g/mL, not higher than about 1.0 ⁇ g/mL, not higher than About 0.9 ⁇ g/mL, not more than 0.8 ⁇ g/mL, not more than about 0.7 ⁇ g/mL, not more than 0.6 ⁇ g/mL, not more than about 0.5 ⁇ g/mL, not more than about 0.4 ⁇ g/mL, not more than greater than about 0.3 ⁇ g/mL, not greater than about 0.2 ⁇ g/mL, or not greater than about 0.1 ⁇ g
  • the isolated antigen binding protein may bind the same or overlapping PD-1 epitope as the reference antibody.
  • the isolated antigen binding protein can compete with the reference antibody for binding to PD-1.
  • the binding affinity of the isolated antigen-binding protein to PD-1 may be substantially the same as the binding affinity of the reference antibody to PD-1.
  • binding affinity can be determined by an ELISA assay.
  • the binding affinity can be characterized by EC50 .
  • the EC50 of the isolated antigen-binding protein for PD-1 is approximately equal to or lower than the EC50 of the reference antibody for PD-1.
  • the reference antibody may comprise a heavy chain variable region VH which may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.
  • the exact boundaries of the HCDRs may be defined differently according to different systems.
  • the HCDRs described herein can be defined by the Kabat system (see the system described by Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))), which defines The method not only provides an unambiguous residue numbering system applicable to any variable region of an antigen binding protein, but also provides precise residue boundaries that define the CDRs. These CDRs may be referred to as Kabat CDRs.
  • the HCDRs described in this application can also be defined by the Chothia system (see studies by Chothia and colleagues (Chothia and Lesk, J. Mol.
  • HCDRs can be determined according to the IMGT definition rules.
  • the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 138.
  • the sequence of the HCDR3 of the reference antibody can be determined according to the IMGT definition rules.
  • AADX 4 DX 6 X 7 GFDY (SEQ ID NO: 138), wherein X 4 can be I or L, X 6 can be H, R or V, and X 7 can be A, D, G, H, L, N , R, S, T or V.
  • the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 142.
  • the sequence of the HCDR3 of the reference antibody can be determined according to the IMGT definition rules.
  • AADX 4 DX 6 X 7 GFDY (SEQ ID NO: 142), wherein X 4 can be I or L, X 6 can be H or R, and X 7 can be A, H or S.
  • the HCDR3 of the reference antibody may comprise any one of SEQ ID NOs: 3, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 and 67. the amino acid sequence described.
  • the sequence of the HCDR3 of the reference antibody can be determined according to the IMGT definition rules.
  • the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 137.
  • the sequence of the HCDR2 of the reference antibody can be determined according to the IMGT definition rules.
  • X 1 GWX 4 X 5 X 6 TX 8 (SEQ ID NO: 137), wherein X 1 can be I or L, X 4 can be A, N, R or S, and X 5 can be A, F, W or Y, X6 can be A , G or S, X8 can be E, Q, S or T.
  • the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 141.
  • the sequence of the HCDR2 of the reference antibody can be determined according to the IMGT definition rules.
  • IGWX 4 X 5 X 6 TX 8 (SEQ ID NO: 141), wherein X 4 can be A or N, X 5 can be F or Y, X 6 can be A or S, X 8 can be E, Q or T.
  • the HCDR2 of the reference antibody may comprise any one of SEQ ID NOs: 2, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and 54. the amino acid sequence described.
  • the sequence of the HCDR2 of the reference antibody can be determined according to the IMGT definition rules.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:136.
  • the sequence of HCDR1 of the reference antibody can be determined according to the IMGT definition rules.
  • X 1 X 2 X 3 X 4 X 5 IYA (SEQ ID NO: 136), wherein X 1 can be A, G, H, K, S or V, and X 2 can be A, G, H, L, N , P, R, S, T or V, X 3 can be D, E, F, G, I, K, Q, R, S or V, X 4 can be D, G, H, K, L, P , Q, R, S, T, V or Y, X 5 can be A, F, N, Q, R, S or T.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 140.
  • the sequence of HCDR1 of the reference antibody can be determined according to the IMGT definition rules.
  • X 1 X 2 X 3 X 4 X 5 IYA (SEQ ID NO: 140), wherein X 1 can be G, H or K, X 2 can be A, G, L, N, S or V, X 3 can be is D, R or V, X4 can be G, H, P, Q, R or T, X5 can be R, S or T.
  • the HCDR1 of the reference antibody may comprise SEQ ID NO: 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, Any of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41 the amino acid sequence described.
  • the sequence of HCDR1 of the reference antibody can be determined according to the IMGT definition rules.
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 138; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 137; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO: 136 may be included.
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 142; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 141; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO: 140 may be included.
  • HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 1; HCDR2 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 2; and HCDR3 of the reference antibody The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody may comprise the single domain antibody MHPD1-A or an antibody having the same HCDR1-3.
  • HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 1; HCDR2 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 42; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:55 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-1 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:4; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:43; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-2 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:5; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:44; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-3 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:6; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:45; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-4 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:7; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-5 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:8; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:47; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-6 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:9; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-7 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 10; HCDR2 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 45; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-8 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 11; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 44; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-9 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 12; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 43; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-10 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 13; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 48; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-11 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 14; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 45; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-12 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 15; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 47; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-13 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 16; HCDR2 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 43; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-14 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 17; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 49; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-15 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 18; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-16 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 19; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-17 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 20; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 50; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:56 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-18 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 21; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:57 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-19 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 22; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-20 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:9; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:51; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-21 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:9; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:58 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-22 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 23; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-23 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 24; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:59 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-24 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:25; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:60 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-25 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 26; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:61 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-26 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 27; HCDR2 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-27 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 28; HCDR2 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 52; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-28 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 29; HCDR2 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-29 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 25; HCDR2 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 52; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:62 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-30 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:9; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:50; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:63 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-31 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:30; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-32 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:31; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-33 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:32; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:53; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-35 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:33; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:52; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-36 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:34; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:64 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-37 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:35; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:57 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-38 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:9; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:54; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-39 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:9; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:65 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-40 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:36; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-41 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:37; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:52; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-42 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:9; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:59 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-43 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:38; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:54; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-44 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:39; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-45 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:40; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:55 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-46 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:41; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:3 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-47 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:9; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:66 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-48 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:9; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO:46; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO:67 may be included.
  • the reference antibody can include the single domain antibody MHPD1-A-49 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the VH of the reference antibody may comprise the framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68, 144-148, 230 and/or 235.
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69, 149-151 and/or 231.
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70, 152-161 and/or 232.
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71, 162 and/or 233.
  • the H-FR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 70; and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 71.
  • the reference antibody may comprise the single domain antibody MHPD1-A or an antibody having the same H-FR1-4.
  • the reference antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:139.
  • X 27 can be A, G, H, L, N, P, R, S, T or V
  • X 28 can be D, E, F, G, I, K, Q, R, S or V
  • X 29 can be D, G, H, K, L, P, Q, R, S, T, V or Y
  • X 30 can be A, F, N, Q, R, S or T
  • X 51 can Is I or L
  • X 54 can be A, N, R or S
  • X 55 can be A, F, W or Y
  • X 56 can be A, G or S
  • X 58 can be E, Q, S or T
  • the reference antibody may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:143.
  • the heavy chain variable region of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NOs: 72-120 and/or 163-227.
  • the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 128.
  • the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:127.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:126.
  • HCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 126; HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 127; and HCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO: 128 may be included.
  • the reference antibody may comprise pembrolizumab or an antibody having the same HCDR1-3 therewith.
  • the reference antibody may comprise a light chain variable region VL, which may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 131.
  • the LCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 130.
  • the LCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 129.
  • LCDR1 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 129; LCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO: 130; and LCDR3 of the reference antibody
  • the amino acid sequence shown in SEQ ID NO: 131 may be included.
  • the reference antibody may comprise pembrolizumab or an antibody having the same LCDR1-3 therewith.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 126; the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 127; the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 127; comprise the amino acid sequence shown in SEQ ID NO: 128; the LCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 129; the LCDR2 of the reference antibody may comprise the amino acid shown in SEQ ID NO: 130 Sequence; LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 131.
  • the reference antibody can include pembrolizumab or an antigen binding protein having the same HCDR1-3 and LCDR1-3.
  • the VH of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:132.
  • the VL of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:133.
  • the reference antibody may comprise an antigen binding protein having the same VH and VL as or with pembrolizumab.
  • the reference antibody may comprise a heavy chain and a light chain
  • the heavy chain of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO: 134.
  • the light chain of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:135.
  • the reference antibody may comprise an antigen binding protein having the same heavy and light chains as or with pembrolizumab.
  • the antigen binding protein may comprise a heavy chain variable region VH, and the VH may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.
  • the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 138.
  • sequence of the HCDR3 of the antigen binding protein can be determined according to the IMGT definition rules.
  • AADX 4 DX 6 X 7 GFDY (SEQ ID NO: 138), wherein X 4 can be I or L, X 6 can be H, R or V, and X 7 can be A, D, G, H, L, N , R, S, T or V.
  • the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 142.
  • sequence of the HCDR3 of the antigen binding protein can be determined according to the IMGT definition rules.
  • AADX4DX6X7GFDY (SEQ ID NO: 142 ) , wherein X4 can be I or L, X6 can be H or R, and X7 can be A , H or S.
  • the HCDR3 of the antigen binding protein may comprise any one of SEQ ID NOs: 3, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 and 67. the amino acid sequence described.
  • the sequence of the HCDR3 of the antigen binding protein can be determined according to the IMGT definition rules.
  • the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 137.
  • sequence of the HCDR2 of the antigen binding protein can be determined according to the IMGT definition rules.
  • X 1 GWX 4 X 5 X 6 TX 8 (SEQ ID NO: 137), wherein X 1 can be I or L, X 4 can be A, N, R or S, and X 5 can be A, F, W or Y, X6 can be A , G or S, X8 can be E, Q, S or T.
  • the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 141.
  • sequence of the HCDR2 of the antigen binding protein can be determined according to the IMGT definition rules.
  • IGWX 4 X 5 X 6 TX 8 (SEQ ID NO: 141), wherein X 4 can be A or N, X 5 can be F or Y, X 6 can be A or S, X 8 can be E, Q or T.
  • the HCDR2 of the antigen binding protein may comprise any one of SEQ ID NOs: 2, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and 54. the amino acid sequence described.
  • the sequence of the HCDR2 of the antigen binding protein can be determined according to the IMGT definition rules.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 136.
  • sequence of HCDR1 of the antigen binding protein can be determined according to the IMGT definition rules.
  • X 1 X 2 X 3 X 4 X 5 IYA (SEQ ID NO: 136), wherein X 1 can be A, G, H, K, S or V, and X 2 can be A, G, H, L, N , P, R, S, T or V, X 3 can be D, E, F, G, I, K, Q, R, S or V, X 4 can be D, G, H, K, L, P , Q, R, S, T, V or Y, X 5 can be A, F, N, Q, R, S or T.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 140.
  • the sequence of HCDR1 of the antigen binding protein can be determined according to the IMGT definition rules.
  • X 1 X 2 X 3 X 4 X 5 IYA (SEQ ID NO: 140), wherein X 1 can be G, H or K, X 2 can be A, G, L, N, S or V, X 3 can be is D, R or V, X4 can be G, H, P, Q, R or T, X5 can be R, S or T.
  • the HCDR1 of the antigen binding protein may comprise SEQ ID NO: 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, Any of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41 the amino acid sequence described.
  • the sequence of HCDR1 of the antigen binding protein can be determined according to the IMGT definition rules.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 138; HCDR2 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 137; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO: 136 may be included.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 142; HCDR2 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 141; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO: 140 may be included.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 2; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A or an antibody having the same HCDR1-3.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 42; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:55 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-1 or an antibody having the same HCDR1-3.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:4; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:43; and the HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-2 or an antibody having the same HCDR1-3.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 5; HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 44; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-3 or an antibody having the same HCDR1-3.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 6; HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 45; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-4 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 7; HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-5 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 8; HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 47; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-6 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 9; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-7 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 10; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 45; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-8 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 11; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 44; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-9 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 12; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 43; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-10 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 13; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 48; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-11 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 14; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 45; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-12 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 15; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 47; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-13 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 16; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 43; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-14 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 17; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 49; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-15 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 18; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-16 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 19; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-17 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 20; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 50; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:56 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-18 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 21; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:57 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-19 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 22; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-20 or an antibody having the same HCDR1-3 therewith.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 9; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 51; and the HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-21 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 9; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:58 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-22 or an antibody having the same HCDR1-3 therewith.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 23; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and the HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-23 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 24; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:59 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-24 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 25; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:60 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-25 or an antibody having the same HCDR1-3 therewith.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 26; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and the HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:61 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-26 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 27; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-27 or an antibody having the same HCDR1-3 therewith.
  • the HCDR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 28; the HCDR2 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 52; and the HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-28 or an antibody having the same HCDR1-3 therewith.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 29; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and the HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-29 or an antibody having the same HCDR1-3 therewith.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 25; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 52; and the HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:62 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-30 or an antibody having the same HCDR1-3 therewith.
  • the HCDR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 9; the HCDR2 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 50; and the HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:63 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-31 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 30; HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-32 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 31; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-33 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 32; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 53; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-35 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 33; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 52; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-36 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 34; HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:64 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-37 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 35; HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:57 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-38 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 9; HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 54; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-39 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 9; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:65 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-40 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 36; HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-41 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 37; HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO: 52; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-42 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 9; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:59 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-43 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 38; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 54; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-44 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 39; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-45 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 40; HCDR2 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 46; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:55 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-46 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 41; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:3 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-47 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 9; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein
  • the amino acid sequence shown in SEQ ID NO:66 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-48 or an antibody having the same HCDR1-3 therewith.
  • HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 9; HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 46; and HCDR3 of the antigen binding protein The amino acid sequence shown in SEQ ID NO:67 may be included.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A-49 or an antibody having the same HCDR1-3 therewith.
  • the VH of the antigen binding protein may comprise the framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 230 and/or 235.
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:231.
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:232.
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:233.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68, 144-148, and/or 230.
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69, and/or 149-151.
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70, and/or 152-161.
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71, and/or 162.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 70; and the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 71.
  • the antigen binding protein may comprise the single domain antibody MHPD1-A or an antibody having the same H-FR1-4 therewith.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:234.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO: 236.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:139.
  • X 27 can be A, G, H, L, N, P, R, S, T or V
  • X 28 can be D, E, F, G, I, K, Q, R, S or V
  • X 29 can be D, G, H, K, L, P, Q, R, S, T, V or Y
  • X 30 can be A, F, N, Q, R, S or T
  • X 51 can Is I or L
  • X 54 can be A, N, R or S
  • X 55 can be A, F, W or Y
  • X 56 can be A, G or S
  • X 58 can be E, Q, S or T
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise the amino acid sequence shown in SEQ ID NO:143.
  • the heavy chain variable region may comprise the amino acid sequence shown in any one of SEQ ID Nos: 72-120 and/or 163-227.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A or have the same HCDR1-3 and H-FR1-4 antigen-binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:72.
  • the antigen binding protein can include the single domain antibody MHPD1-A or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:1; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:42; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:55;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-1 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:73.
  • the antigen binding protein can include the single domain antibody MHPD1-A-1 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-1.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:4; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:43; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-2 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:74.
  • the antigen binding protein can include the single domain antibody MHPD1-A-2 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-2.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:5; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:44; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-3 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:75.
  • the antigen binding protein can include the single domain antibody MHPD1-A-3 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-3.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:45; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-4 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:76.
  • the antigen binding protein can include the single domain antibody MHPD1-A-4 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-4.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:45; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 147; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-4 H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO:182.
  • the antigen binding protein can include the single domain antibody MHPD1-A-4H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-4H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:7; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-5 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:77.
  • the antigen binding protein can include the single domain antibody MHPD1-A-5 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:8; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:47; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-6 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:78.
  • the antigen binding protein can include the single domain antibody MHPD1-A-6 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-6.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-7 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:79.
  • the antigen binding protein can include the single domain antibody MHPD1-A-7 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-7.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 148; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-7 H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:185.
  • the antigen binding protein can include the single domain antibody MHPD1-A-7H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-7H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-8 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:80.
  • the antigen binding protein can include the single domain antibody MHPD1-A-8 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-8.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 147; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-8 H or have the same HCDR1-3 and H- Antigen binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO:186.
  • the antigen binding protein can include the single domain antibody MHPD1-A-8H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) that has the same VH as the MHPD1-A-8H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 11; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 44; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-9 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:81.
  • the antigen binding protein can include the single domain antibody MHPD1-A-9 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-9.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 12; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 43; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-10 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:82.
  • the antigen binding protein can include the single domain antibody MHPD1-A-10 or an antigen binding protein (e.g., a single domain antibody) that has the same HCDR3 (e.g., the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-10.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 48; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-11 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:83.
  • the antigen binding protein can include the single domain antibody MHPD1-A-11 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-11.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 14; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 45; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-12 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:84.
  • the antigen binding protein can include the single domain antibody MHPD1-A-12 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-12.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO: 15; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO: 47; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-13 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:85.
  • the antigen binding protein can include the single domain antibody MHPD1-A-13 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-13.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO: 15; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO: 47; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 147; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-13H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:191.
  • the antigen binding protein can include the single domain antibody MHPD1-A-13H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-13H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 16; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 43; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-14 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:86.
  • the antigen binding protein can include the single domain antibody MHPD1-A-14 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-14.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 17; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 49; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-15 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:87.
  • the antigen binding protein can include the single domain antibody MHPD1-A-15 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-15.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 17; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 49; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 148; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-15H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:193.
  • the antigen binding protein can include the single domain antibody MHPD1-A-15H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-15H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-16 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:88.
  • the antigen binding protein can include the single domain antibody MHPD1-A-16 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-16.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-17 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:89.
  • the antigen binding protein can include the single domain antibody MHPD1-A-17 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-17.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:20; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:50; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:56;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-18 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:90.
  • the antigen binding protein can include the single domain antibody MHPD1-A-18 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-18.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 21; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 57;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-19 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:91.
  • the antigen binding protein can include the single domain antibody MHPD1-A-19 or an antigen binding protein (e.g., a single domain antibody) that has the same HCDR3 (e.g., the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-19.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:22; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-20 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:92.
  • the antigen binding protein can include the single domain antibody MHPD1-A-20 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-20.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:51; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-21 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:93.
  • the antigen binding protein can include the single domain antibody MHPD1-A-21 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-21.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:58;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-22 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:94.
  • the antigen binding protein can include the single domain antibody MHPD1-A-22 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-22.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 23; the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 46; the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-23 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:95.
  • the antigen binding protein can include the single domain antibody MHPD1-A-23 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-23.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:24; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:59;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-24 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:96.
  • the antigen binding protein can include the single domain antibody MHPD1-A-24 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-24.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 24; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 59;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 148; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-24H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:202.
  • the antigen binding protein can include the single domain antibody MHPD1-A-24H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-24H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:25; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:60;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-25 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:97.
  • the antigen binding protein can include the single domain antibody MHPD1-A-25 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-25.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:25; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:60;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 148; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-25H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:203.
  • the antigen binding protein can include the single domain antibody MHPD1-A-25H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (e.g., a single domain antibody) having the same VH as the MHPD1-A-25H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:26; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:61;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-26 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:98.
  • the antigen binding protein can include the single domain antibody MHPD1-A-26 or an antigen binding protein (eg, a single domain antibody) having the same HCDR3 therewith (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-26.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:27; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-27 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:99.
  • the antigen binding protein can include the single domain antibody MHPD1-A-27 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-27.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:28; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:52; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-28 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO:100.
  • the antigen binding protein can include the single domain antibody MHPD1-A-28 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-28.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:29; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-29 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:101.
  • the antigen binding protein can include the single domain antibody MHPD1-A-29 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-29.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:25; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:52; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:62;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-30 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:102.
  • the antigen binding protein can include the single domain antibody MHPD1-A-30 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-30.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:25; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:52; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:62;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 148; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-30 H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:208.
  • the antigen binding protein can include the single domain antibody MHPD1-A-30H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-30H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:50; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:63;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of ; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-31 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:103.
  • the antigen binding protein can include the single domain antibody MHPD1-A-31 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-31.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:30; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-32 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:104.
  • the antigen binding protein can include the single domain antibody MHPD1-A-32 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-32.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:30; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 148; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen-binding protein may comprise the single domain antibody MHPD1-A-32H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:210.
  • the antigen binding protein can include the single domain antibody MHPD1-A-32H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-32H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:31; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-33 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:105.
  • the antigen binding protein can include the single domain antibody MHPD1-A-33 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-33.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:32; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:53; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-35 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:106.
  • the antigen binding protein can include the single domain antibody MHPD1-A-35 or an antigen binding protein (e.g., a single domain antibody) that has the same HCDR3 (e.g., the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-35.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:33; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:52; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-36 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:107.
  • the antigen binding protein can include the single domain antibody MHPD1-A-36 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-36.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:34; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:64;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-37 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:108.
  • the antigen binding protein can include the single domain antibody MHPD1-A-37 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-37.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:35; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:57;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-38 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:109.
  • the antigen binding protein can include the single domain antibody MHPD1-A-38 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-38.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:9; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:54; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-39 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 110.
  • the antigen binding protein can include the single domain antibody MHPD1-A-39 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-39.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:9; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:54; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 148; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-39H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:217.
  • the antigen binding protein can include the single domain antibody MHPD1-A-39H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-39H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:65;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-40 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 111.
  • the antigen binding protein can include the single domain antibody MHPD1-A-40 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-40.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:36; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-41 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 112.
  • the antigen binding protein can include the single domain antibody MHPD1-A-41 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-41.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:52; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-42 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 113.
  • the antigen binding protein can include the single domain antibody MHPD1-A-42 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein may include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-42.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:37; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:52; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 148; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-42H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:223.
  • the antigen binding protein can include the single domain antibody MHPD1-A-42H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-42H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:59;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-43 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 114.
  • the antigen binding protein can include the single domain antibody MHPD1-A-43 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-43.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO: 38; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO: 54; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-44 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 115.
  • the antigen binding protein can include the single domain antibody MHPD1-A-44 or an antigen binding protein (e.g., a single domain antibody) that has the same HCDR3 (e.g., the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-44.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO: 38; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO: 54; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 148; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-44H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO:222.
  • the antigen binding protein can include the single domain antibody MHPD1-A-44H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-44H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:39; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-45 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 116.
  • the antigen binding protein can include the single domain antibody MHPD1-A-45 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-45.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:39; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 148; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 150; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 161
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 162; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-45H or have the same HCDR1-3 and H- Antigen-binding proteins of FR1-4.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:223.
  • the antigen binding protein can include the single domain antibody MHPD1-A-45H or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, has the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-45H.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:40; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:55;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-46 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 117.
  • the antigen binding protein can include the single domain antibody MHPD1-A-46 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-46.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 41; the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 46; the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 3;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-47 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 118.
  • the antigen binding protein can include the single domain antibody MHPD1-A-47 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-47.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:9; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:46; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:66;
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-48 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 119.
  • the antigen binding protein can include the single domain antibody MHPD1-A-48 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-48.
  • the antigen binding protein may comprise a heavy chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9; the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:46; the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:67;
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68;
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69;
  • the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 70
  • the amino acid sequence of the H-FR4; the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 71; for example, the antigen binding protein may comprise the single domain antibody MHPD1-A-49 or have the same HCDR1-3 and H-FR1 -4 antigen binding protein.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:120.
  • the antigen binding protein can include the single domain antibody MHPD1-A-49 or an antigen binding protein (eg, a single domain antibody) that has the same HCDR3 (eg, the same HCDR1-3).
  • the antigen binding protein can include an antigen binding protein (eg, a single domain antibody) having the same VH as the MHPD1-A-49.
  • the isolated antigen binding protein further comprises an immunoglobulin Fc region.
  • the present application provides a polypeptide comprising the antigen binding protein of the present application and the Fc region of an immunoglobulin.
  • the VH of the antigen binding protein is directly or indirectly linked to the Fc region.
  • the C-terminus of the VH of the antigen binding protein is directly or indirectly linked to the N-terminus of the Fc region.
  • the VH of the antigen binding protein is fused in frame with the Fc region.
  • the VH of the antigen binding protein is linked to the Fc region through a linker.
  • the linker includes a peptide linker.
  • the linker includes a flexible linker.
  • the isolated antigen-binding protein comprises the VH, the linker and the Fc region of the antigen-binding protein in order from the N-terminus to the C-terminus.
  • the Fc region includes IgG1-derived Fc or IgG4-derived Fc.
  • the Fc region comprises the amino acid sequence set forth in any one of SEQ ID NOs: 121-125.
  • the application provides one or more polypeptides that may comprise the isolated antigen binding proteins of the application.
  • the polypeptide may comprise a fusion protein.
  • the application provides one or more immunoconjugates that can comprise the isolated antigen binding proteins of the application.
  • the immunoconjugate may further comprise a pharmaceutically acceptable therapeutic agent.
  • the present application also provides isolated one or more nucleic acid molecules that encode the isolated antigen-binding proteins described herein.
  • each of the one or more nucleic acid molecules may encode the entire antigen binding protein, or may encode a portion thereof (eg, HCDR1-3, one of the heavy chain variable regions, or variety).
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis.
  • the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA techniques.
  • nucleic acids encoding the isolated antigen-binding proteins can be prepared by various methods known in the art, including but not limited to, using reverse transcription PCR and PCR to obtain the isolated antigen-binding proteins described herein protein nucleic acid molecules.
  • the application provides one or more vectors comprising one or more nucleic acid molecules described herein.
  • One or more of the nucleic acid molecules may be included in each vector.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary depending on species or cell type function, but typically comprises 5' untranslated and 5' and 3' untranslated sequences involved in transcription and translation initiation, respectively, such as the TATA box, plus Cap sequences, CAAT sequences, etc.
  • a 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid.
  • the expression control sequences may also include enhancer sequences or upstream activator sequences.
  • suitable promoters may include, for example, the promoters for SP6, T3 and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (such as CMV), wherein the promoter's A portion may be fused to a portion of the gene promoter for other cellular proteins (eg, human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not contain additional introns.
  • One or more nucleic acid molecules described herein can be operably linked to the expression control element.
  • the vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering.
  • the vector can be an expression vector.
  • the vector can be a viral vector.
  • the viral vector can be administered directly to the patient (in vivo) or can be administered in an indirect form, eg, by treating cells with the virus in vitro, and then administering the treated cells to the patient (ex vivo).
  • Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers. Lentiviral vectors may comprise long terminal repeats 5'LTR and truncated 3'LTRs, RREs, rev response elements (cPPT), central termination sequences (CTS) and/or post-translational regulatory elements (WPRE). The vectors described herein can be introduced into cells.
  • the application provides a cell.
  • the cells may comprise the isolated antigen binding proteins described herein, the polypeptides, the immunoconjugates, one or more nucleic acid molecules and/or one or more vectors described herein .
  • each or each cell may contain one or one nucleic acid molecule or vector described herein.
  • each or each cell can comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into such host cells, such as prokaryotic cells (eg, bacterial cells), CHO cells, NS/0 cells, HEK293T cells, 293F cells, or HEK293A cells, or other eukaryotic cells, Such as cells from plants, fungi or yeast cells, etc.
  • the vectors described herein can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the cells can include yeast cells.
  • the cells can include E. coli cells.
  • the cells can include mammalian cells.
  • the cells can include immune cells.
  • the cells may include immune cells.
  • the cells can include immune cells.
  • the cells can include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells cell.
  • NK natural killer
  • the application provides a pharmaceutical composition.
  • the pharmaceutical composition may comprise the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and/or the isolated antigen-binding protein described herein. or pharmaceutically acceptable adjuvants and/or excipients.
  • the pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and /or nonionic surfactants. Unless incompatible with the cells described herein, any conventional medium or agent is contemplated for use in the pharmaceutical compositions of the present application.
  • the pharmaceutically acceptable excipients may include additives other than the main drug in the pharmaceutical preparation, and may also be referred to as excipients.
  • the excipients may include binders, fillers, disintegrants, lubricants in the tablet.
  • the excipients may include wine, vinegar, medicinal juice, etc. in Chinese medicine pills.
  • the excipient may comprise a base part of a semisolid formulation ointment, cream.
  • the excipients may include preservatives, antioxidants, flavors, fragrances, solubilizers, emulsifiers, solubilizers, osmo-regulators, colorants in liquid formulations.
  • the present application provides a method for detecting the presence and/or content of PD-1, which may comprise administering the isolated antigen binding protein or the polypeptide.
  • the methods may include in vitro methods, ex vivo methods, methods not for diagnostic or therapeutic purposes.
  • the method may include a method for detecting the presence and/or amount of PD-1 for non-diagnostic purposes, which may include the steps of:
  • the present application provides a kit, which can comprise the isolated antigen binding protein or the polypeptide.
  • the kit may further comprise instructions for use describing the method for detecting the presence and/or content of PD-1.
  • the methods may include in vitro methods, ex vivo methods, methods not for diagnostic or therapeutic purposes.
  • the present application provides the use of the isolated antigen-binding protein or the polypeptide in the preparation of a kit, which can be used for a method for detecting the presence and/or content of PD-1.
  • the methods may include in vitro methods, ex vivo methods, methods not for diagnostic or therapeutic purposes.
  • the present application provides the use of the isolated antigen-binding protein and/or the polypeptide in preparing a medicament, which can be used for preventing and/or treating a disease or disorder.
  • the present application provides the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell and/or or said pharmaceutical composition for preventing, alleviating and/or treating diseases or conditions.
  • the disease or disorder can include a tumor.
  • the tumor can include a solid tumor.
  • the tumor may comprise a hematological tumor.
  • the tumor can include a tumor associated with the expression of PD-L1.
  • the heavy chain can include tumors associated with upregulated expression of PD-L1.
  • the tumor can be selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal cancer, and breast cancer.
  • the present application provides a method of preventing and/or treating a disease or disorder, which may comprise administering to a subject in need thereof an effective amount of the isolated antigen-binding protein, the polypeptide, the The immunoconjugate, the isolated nucleic acid molecule, the carrier, and/or the cell.
  • the disease or disorder can include a tumor.
  • the tumor can include a solid tumor.
  • the tumor may comprise a hematological tumor.
  • the tumor can include a tumor associated with the expression of PD-L1.
  • the heavy chain can include tumors associated with upregulated expression of PD-L1.
  • the tumor can be selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal cancer, and breast cancer.
  • the present application provides a method for inhibiting the interaction of PD-1 and PD-L1, comprising administering to a subject in need thereof an effective amount of the isolated antigen-binding protein, the polypeptide, the The immunoconjugate, the isolated nucleic acid molecule, the carrier, and/or the cell.
  • the method can be an ex vivo or in vitro method.
  • the method may comprise contacting the biological sample with the antigen binding protein and/or PD-L1 described herein under conditions that allow the antigen binding protein and/or PD-L1 to bind PD-1, It is detected whether a complex is formed between the antigen binding protein and PD-1, and whether a complex is formed between PD-1 and PD-L1.
  • the present application provides the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell and/or the The described pharmaceutical composition is used to inhibit the interaction between PD-1 and PD-L1.
  • the present application provides the use of the isolated antigen-binding protein and/or the polypeptide in the preparation of a medicament for inhibiting the interaction between PD-1 and PD-L1.
  • a healthy female adult alpaca (Alpaca) was immunized with recombinant human PD-1 protein.
  • 1 mg of recombinant human PD-1 protein was emulsified with equal volume of Freund's complete adjuvant and then injected subcutaneously at multiple points;
  • 0.5 mg of recombinant human PD-1 protein was emulsified with equal volume of incomplete Freund's adjuvant and then emulsified at multiple points.
  • a total of 3 booster immunizations were performed.
  • lymphocyte separation medium Solarbio, Cat. P8900
  • total RNA was extracted by TRIzol TM Reagent (Thermo Fisher Scientific, Cat. 15596018).
  • PrimeScript TM II 1st Strand cDNA Synthesis Kit (TAKARA, Code No. 6210B) was used to obtain cDNA by reverse transcription, and the VHH gene was amplified by nested PCR.
  • VHH gene fragment was recovered with a gel purification kit (TAKARA, Code No. 9761) and digested with restriction endonuclease sfil (TAKARA, Code No. 1244A), and then cloned into the phagemid vector pComb 3XSS, The constructed cloned product was transformed into E.coli TG1 electrotransformed competent cells to obtain a PD-1 single domain antibody library.
  • TAKARA gel purification kit
  • TAKARA restriction endonuclease sfil
  • the library capacity was determined by plate gradient dilution method to be 3.2 ⁇ 10 8 , and the results of colony PCR showed that the clone rate of the library was 98%.
  • Take an appropriate amount of the library bacterial solution (the number of bacterial cells is about 3.2 ⁇ 10 9 ) and inoculate it in 100 mL LB medium (containing ampicillin) to cultivate to mid-logarithmic phase. After adding an appropriate amount of helper phage M13KO7 to infect for 60 minutes, the medium is replaced.
  • Solid-phase affinity screening was used to screen PD-1 nanobodies, and three rounds of screening were carried out:
  • Binding and washing In the first round of screening, after removing the blocking solution, add 100 ⁇ L of the nanobody phage display library constructed in Example 2 to the microplate, incubate at 37°C for 1 hour and then remove the unbound library. Microplates were washed with PBST and PBS (5 times each) to remove unbound or less binding phage; the second round of PBST and PBS was washed 10 times each, and the third round of PBST and PBS was washed 20 times each.
  • Positive control wells enriched library for each round
  • negative control helper phage M13KO7
  • blank wells PBS
  • the clones corresponding to the positive wells were protected, plasmid extracted, sequenced, and the DNA sequences were translated into amino acid sequences and compared.
  • the clones with the same HCDR1, HCDR2, and HCDR3 sequences were regarded as the same antibody strain, and the clones with different HCDR sequences were regarded as different antibody strains.
  • the positive clones screened in Example 3 were transformed into Top10F', and IPTG induced VHH expression.
  • the cell pellet was collected by centrifugation, and the supernatant was collected after ultrasonication.
  • Metal Affinity Resins were purified and buffer exchanged to phosphate buffer.
  • the purified VHH was quantified by measuring the OD280 value and stored at 4°C for later use.
  • the SDS PAGE of the VHH that has high affinity for the antigen (human recombinant PD-1) and can compete with human recombinant PD-L1 for binding to human recombinant PD-1 is shown in Figure 1, and the obtained VHH is named MHPD1-A .
  • Recombinant human PD-1 antigen (2 ⁇ g/mL) was added to a 96-well plate and coated overnight at 4°C. Blocked with 1% casein.
  • MHPD1-A samples were serially diluted 2-fold with 0.1% BSA/PBS, starting at 20 ⁇ g/mL.
  • 4 ⁇ g/mL recombinant human PD-L1-Fc and different concentrations of VHH were added to the antigen (recombinant human PD-1 protein)-coated 96-well plate, and incubated at room temperature for 1 hour. Plates were washed with PBST between different incubations.
  • the pTT5 recombinant vector containing the nucleic acid sequence encoding MHPD1-A and human Fc segment (the C-terminus of VHH is directly connected to the N-terminus of the human Fc hinge region) was synthesized, and the liposome-DNA complex was prepared by the following steps: 15 ⁇ g of plasmid DNA was 1 was diluted to a total volume of 0.5 mL; 30 ⁇ L of 293fectin TM reagent was I was diluted to a total volume of 0.5 mL; mixed gently and incubated for 5 minutes at room temperature.
  • Example 7 Species cross-validation of fusion protein with human, monkey and mouse PD-1
  • 96-well Greiner plates were coated with 2 ⁇ g/mL recombinant human PD-1-his protein, recombinant monkey PD-1-his protein and recombinant mouse PD-1-his protein at 4°C overnight. After blocking with 1% casein the next day, 5 ⁇ g/mL and 1.67 ⁇ g/mL Nanobody-Fc fusion protein samples were added to the antigen-coated plates, respectively, and incubated for 2 hours. Plates were then washed with PBST and anti-human IgG-HRP (1:5000 dilution in 1% casein) was added for 1 hour for binding detection. As shown in Figure 2, MHPD1-A Fc can bind to recombinant human PD-1-his and recombinant monkey PD-1-his, but has no binding activity to recombinant mouse PD-1-his.
  • Example 8 Affinity detection of fusion protein to recombinant human PD-1
  • CM5 chip About 250 RU of recombinant human PD-1-his protein was coupled to a CM5 chip at 25°C using an amine coupling kit (GE Healthcare, Cat No. BR100050) following standard amine coupling procedures. 2-fold serial dilutions of MHPD1-A Fc samples (detecting 5 concentrations) were sequentially injected at a flow rate of 30 ⁇ L/min at 25°C, the binding time was 120s, and the dissociation time was 600s. use The analysis software (BIAevaluation) used the 1:1 Langmuir binding model to fit the curve to determine the kinetic constants such as the binding rate constant, the dissociation rate constant and the binding dissociation constant.
  • Recombinant human PD-L1 (C-Fc, 0.2 ⁇ g/well) was added to a 96-well plate and coated overnight at 4°C. Blocked with 1% casein.
  • MHPD1-A Fc was serially diluted 2-fold with 0.1% BSA/PBS, starting at 10 ⁇ g/mL.
  • 0.5 ⁇ g/mL recombinant human PD-1 (C-Fc&C-Avi&C-6 ⁇ His) and different concentrations of MHPD1-A Fc were added to the antigen-coated 96-well plate, and incubated at room temperature for 1 hour. Plates were washed with PBST between different incubations.
  • Horseradish peroxidase-labeled streptavidin (1:10000) was used for detection, and Graphpad Prism software was used to fit the curve and calculate the EC 50 value. The results are shown in Table 3.
  • the results show that the tested MHPD1-A Fc can competitively inhibit the binding of recombinant human PD-L1 to recombinant human PD-1, and the activity is comparable to or better than the positive control Nivolumab.
  • HEK293 cells overexpressing human PD-1 were cultured in DMEM medium containing 10% FBS in a CO 2 incubator (37°C, 5% CO 2 ). Cells were harvested and washed once with assay buffer. For cell counting and viability detection, add 3 ⁇ 10 5 live cells to each test tube, and add 100 ⁇ L of MHPD1-A Fc at different concentrations (3000ng/mL, 1000ng/mL, 300ng/mL, 100ng/mL, 30ng/mL) , 10 ng/mL, 3 ng/mL, 1 ng/mL and 0.3 ng/mL), incubate at 4°C for 1 hour.
  • Example 11 Fusion protein competes with human PD-L1 for binding to HEK293 cells overexpressing human PD-1
  • HEK293 cells overexpressing human PD-1 were cultured with DMEM medium containing 10% FBS and 1 mg/mL G418 in a CO 2 incubator (37° C., 5% CO 2 ). Cells were harvested and washed once with assay buffer. Cell counts and viability assays were performed, and 3 x 105 viable cells (cell viability > 95%) were added to each tube.
  • MHPD1-A Fc could competitively inhibit the binding of recombinant human PD-L1 to HEK293 cells overexpressing hPD-1, and the competitive binding activity of MHPD1-A Fc was slightly better than that of the positive control, Nivolumab.
  • Table 5 shows the EC50 values of MHPD1-A Fc competing with human PD-L1 for binding to HEK293/hPD1.
  • the fusion protein competes with human PD-L1 for binding to HEK293 cells overexpressing human PD-1
  • VHH variant sequences were designed by mutating some amino acid sites of HCDR1-3 based on structural prediction and experience.
  • complete gene synthesis (His tag) was carried out, the pcDNA3.4 plasmid was digested with NotI/XbaI (NEB, Cat. R0189L, R0145L), and recombined with the VHH variant gene sequence.
  • the plasmids were transformed into TOP10 competent cells and coated with bacterial liquid, and cultured at 37°C for 16-20h.
  • HEK293 cells were transiently transfected with lipofection reagent, placed in a cell incubator at 37°C with 5% CO2, and incubated on an orbital shaker rotating at 125 rpm.
  • the supernatant was collected by centrifugation, purified by Ni affinity chromatography ((Ni Smart Beads 6FF, GE#17531802), and the buffer was replaced by phosphate buffer by dialysis to obtain a series of VHH variants, named respectively MHPD1-A-4, MHPD1-A-7, MHPD1-A-8, MHPD1-A-13, MHPD1-A-15, MHPD1-A-24, MHPD1-A-25, MHPD1-A-30, MHPD1 -A-32, MHPD1-A-39, MHPD1-A-42, MHPD1-A-44 and MHPD1-A-45.
  • Recombinant human PD-1 protein (2 ⁇ g/mL) was added to a 96-well plate and coated overnight at 4°C. Blocked with 1% casein. VHH variants were serially diluted 2-fold in 0.1% BSA/PBS, starting at 20 ⁇ g/mL. 4 ⁇ g/mL recombinant human PD-L1-Fc and VHH variants at different concentrations were added to the antigen (recombinant human PD-1 protein)-coated 96-well plate, and incubated at room temperature for 1 hour. Plates were washed with PBST between different incubations.
  • a horseradish peroxidase-conjugated anti-human IgG secondary antibody (1:5000, containing 1% casein) was used for detection. After analysis, the results showed that the VHH variant can effectively inhibit the binding of recombinant human PD-L1 to recombinant human PD-1.
  • VHH variant with higher affinity synthesize the full-length sequence with its gene sequence and Fc sequence, carry out NotI/XbaI digestion (NEB, Cat.R0189L, R0145L) to the pcDNA3.4 plasmid, and recombine with the above-mentioned full-length sequence
  • NEB NotI/XbaI digestion
  • the recombinant plasmids were transformed into TOP10 competent cells and coated with bacterial liquid, and cultured at 37°C for 16-20h. Single colonies in the plate were selected for colony PCR and electrophoresis to identify positive clones, and positive clones were selected for sequencing and the clones with correct sequencing were expanded and cultured to extract plasmids.
  • HEK293 cells were transiently transfected with lipofection reagent, placed in a cell incubator at 37°C with 5% CO2, and incubated on an orbital shaker rotating at 125 rpm. On the 5th day, the supernatant was collected by centrifugation, purified by a Protein A affinity chromatography column, and the buffer was replaced with phosphate buffer by dialysis to obtain a fusion protein containing the VHH variant.
  • Example 16 Competitive binding ELISA experiments of fusion proteins comprising VHH variants and recombinant human PD-L1 to antigenic proteins
  • Recombinant human PD-L1 (C-Fc, 0.2 ⁇ g/well) was added to a 96-well plate and coated overnight at 4°C. Blocked with 1% casein.
  • the VHH variant fusion proteins were serially diluted 2-fold with 0.1% BSA/PBS, with an initial concentration of 10 ⁇ g/mL.
  • 0.5 ⁇ g/mL recombinant human PD-1 (C-Fc&C-Avi&C-6 ⁇ His) and different concentrations of VHH variant fusion proteins were added to the antigen-coated 96-well plate, and incubated at room temperature for 1 hour. Plates were washed with PBST between different incubations.
  • VHH variant fusion protein can effectively inhibit the binding of recombinant human PD-L1 to recombinant human PD-1.
  • the referenced human sequence Germeline was identified, and the humanized sequence was designed.
  • the degree of humanization changed from 86.44% to 90.68%. etc., it was constructed into a Nanobody-Fc fusion protein, which was transiently expressed in ExpiCHO-s, and the second round of humanization was performed according to the degree of humanization and affinity.
  • the degree of humanization was greater than 90%, and it was constructed into a Nanobody-Fc fusion protein, which was transiently expressed in ExpiCHO-s to verify its binding activity.
  • Nanobody-Fc fusion proteins with different degrees of humanization to antigen protein (human PD-1) were verified by ELISA.
  • 2 ⁇ g/mL recombinant human PD-1-his antigen protein was added, 30 ⁇ L/well, and coated overnight at 4°C. Wash the plate 3 times with PBST. 5% PBS-Milk was added and incubated at room temperature for 2 h. Wash the plate 3 times with PBST. All Nanobody-Fc fusion proteins were diluted 3-fold with the initial concentration of 10 ⁇ g/mL; 30 ⁇ L/well was added to each well, and incubated at room temperature for 1 h.
  • Example 18 Affinity maturation of humanized Nanobodies and detection of the binding activity of Nanobody-Fc fusion protein to antigen (first round)
  • the VHH of the parental antibody MHPD1-A-H19 was cloned into the phage display vector of Party B; a series of primers were designed to perform single-point or continuous three-point mutation of its HCDR to construct an affinity mature phage display library.
  • immune tube screening i.e. solid phase screening
  • use the antigen protein hPD-1-His to coat the immune tube
  • affinity maturation screening candidate antibodies with higher affinity for hPD-1-His antigen were screened at the ELISA level, and 14 candidate molecules were selected for the construction of Nanobody-Fc fusion protein according to the affinity and sequence at the VHH level.
  • Nanobody-Fc fusion protein The binding activity of the affinity-matured Nanobody-Fc fusion protein to the antigen protein (hPD-1-his) was verified by ELISA: 2 ⁇ g/mL recombinant human PD-1-his was added to the 96-well microtiter plate. Antigen protein, 30 ⁇ L/well, coated overnight at 4°C. Wash the plate 3 times with PBST. 5% PBS-Milk, incubated at room temperature for 2h. Wash the plate 3 times with PBST. All Nanobody-Fc fusion proteins were diluted 3-fold with the initial concentration of 10 ⁇ g/mL; 30 ⁇ L/well was added to each well, and incubated at room temperature for 1 h. Wash the plate 3 times with PBST.
  • Recombinant human PD-L1-mFc (8 ⁇ g/mL) was added to a 96-well plate and coated overnight at 4°C. Wash the plate 3 times with PBST. 5% PBS-Milk at room temperature for 1 hour. All Nanobody-Fc fusion proteins were diluted 3-fold with the initial concentration of 10 ⁇ g/mL; 30 ⁇ L/well was added to each well, and incubated at room temperature for 1 hour. Add 0.5 ⁇ g/mL recombinant human PD-1-Fc-Biotin to the antigen-coated 96-well plate, 30 ⁇ L/well, and incubate at room temperature for 1 hour. Plates were washed with PBST between different incubations.
  • Antibody code IC50 (nM) Antibody code IC50 (nM) MHPD1-A-H19 Fc 1.310 MHPD1-A-8 H Fc 0.323 MHPD1-A-1 H Fc 0.572 MHPD1-A-9 H Fc 0.256 MHPD1-A-2 H Fc 0.419 MHPD1-A-10 H Fc 0.287 MHPD1-A-3 H Fc 0.275 MHPD1-A-11 H Fc 0.365 MHPD1-A-4 H Fc 0.281 MHPD1-A-12 H Fc 0.293 MHPD1-A-5 H Fc 0.306 MHPD1-A-13 H Fc 0.395 MHPD1-A-6 H Fc 0.407 MHPD1-A-14 H Fc 0.852 MHPD1-A-7 H Fc 0.316 Pembrolizumab 0.410 Nivolumab 0.402
  • Hep3B-OS8-PDL1 cells were resuspended in PRMI1640 medium supplemented with 10% FBS, inoculated in a 96-well cell culture plate, and placed in a cell culture incubator for overnight culture; the next day, the culture in the cell culture plate was removed Add 50uL pre-prepared medium containing different concentrations of Nanobody-Fc fusion protein and 50uL Jurkat-NFAT-PD1 cell suspension to each well, and incubate in a carbon dioxide incubator (37°C, 5% CO 2 ) for 6 hours .
  • a carbon dioxide incubator 37°C, 5% CO 2
  • Example 21 Affinity maturation of nanobodies and detection of nanobody-Fc fusion protein binding to human PD-1 and competition binding activity with human PD-L1 to human PD-1 (second round)
  • the VHH of the parent MHPD1-A-7 H was cloned into the phage display vector of Party B; a series of primers were designed to perform single-point or continuous three-point mutation of its HCDR to construct an affinity mature phage display library.
  • immune tube screening i.e. solid phase screening
  • use the antigen protein hPD-1-His to coat the immune tube
  • affinity maturation screening candidate antibodies with higher affinity for hPD-1-His antigen were screened at the ELISA level, and 29 candidate molecules were selected for nanobody-Fc fusion protein construction according to the affinity and sequence at the VHH level.
  • Nanobody-Fc fusion protein The binding activity of the affinity-matured Nanobody-Fc fusion protein to the antigen protein (hPD-1-his) was verified by ELISA: 2 ⁇ g/mL recombinant human PD-1-his was added to the 96-well microtiter plate. Antigen protein, 30 ⁇ L/well, coated overnight at 4°C. Wash the plate 3 times with PBST. 5% PBS-Milk, incubated at room temperature for 2h. Wash the plate 3 times with PBST. All Nanobody-Fc fusion proteins were diluted 3-fold with the initial concentration of 10 ⁇ g/mL; 30 ⁇ L/well was added to each well, and incubated at room temperature for 1 h. Wash the plate 3 times with PBST.
  • Some fusion proteins with higher affinity to human PD-1 were selected to detect the competitive binding activity of recombinant PD-L1 to human PD-1.
  • Recombinant human PD-L1-mFc (8 ⁇ g/mL) was added to a 96-well plate and coated overnight at 4°C. Wash the plate 3 times with PBST. 5% PBS-Milk at room temperature for 1 hour. All Nanobody-Fc fusion proteins were diluted 3-fold with the initial concentration of 10 ⁇ g/mL; 30 ⁇ L/well was added to each well, and incubated at room temperature for 1 hour.
  • Antibody code IC50 (nM) Antibody code IC50 (nM) MHPD1-A-7 H Fc 0.778 MHPD1-A-32 H Fc 1.718 MHPD1-A-15H Fc 0.517 MHPD1-A-37 H Fc 1.544 MHPD1-A-20 H Fc 1.211 MHPD1-A-39 H Fc 0.850 MHPD1-A-21 H Fc 1.609 MHPD1-A-45H Fc 0.944 MHPD1-A-23 H Fc 1.202 MHPD1-A-49 H Fc 1.262 MHPD1-A-30 H Fc 1.078 Pembrolizumab 1.248

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Abstract

一种抗原结合蛋白及其用途，其可与帕博利珠单抗竞争结合相同或重叠的PD-1表位。所述抗原结合蛋白能够以约2μg/mL或更低的EC 50有效抑制人PD-L1与人PD-1的结合。

Description

抗原结合蛋白及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种抗原结合蛋白及其用途。
背景技术
人程序性细胞死亡受体-1(PD-1)是已知的主要免疫检查点之一，其表达在已经激活的T淋巴细胞表面，它与配体PD-L1(程序性死亡受体-配体1，programmed cell death-Ligand 1)和PD-L2(程序性死亡受体-配体2，programmed cell death-Ligand 2)结合可以抑制T淋巴细胞的活性及相关的体内细胞免疫反应。PD-L1广泛表达于B、T淋巴细胞及外周细胞如微血管上皮细胞，肺、肝、心等组织细胞中。大量研究表明，PD-1和PD-L1的相互作用是导致PD-L1表达阳性肿瘤细胞规避免疫监视的主要原因。通过阻断肿瘤细胞对PD-1/PD-L1信号通路的负调控，激活免疫系统，能够促进T细胞相关的肿瘤特异性细胞免疫反应，治疗肿瘤。
目前，抗PD-1抗体还存在选择性不强、亲和力较低的缺陷。因此，有必要开发对PD-1亲和力高、特异性强的新型抗PD-1抗体。
发明内容
本申请提供了一种分离的抗原结合蛋白，其具有下述性质中的一种或多种：1)在ELISA检测中，以约11μg/mL或更低的EC 50与PD-1结合；2)在竞争ELISA检测中，以约2μg/mL或更低的EC 50抑制人PD-L1与人PD-1结合；以及3)能够结合灵长类动物(例如，人或猴)的PD-1。
在某些实施方式中，所述分离的抗原结合蛋白与参比抗体结合相同或重叠的PD-1表位。
在某些实施方式中，所述分离的抗原结合蛋白与所述参比抗体竞争结合PD-1。
在某些实施方式中，所述分离的抗原结合蛋白对PD-1的结合亲和力与所述参比抗体对PD-1的结合亲和力基本相同。
在某些实施方式中，所述参比抗体包含HCDR3，所述HCDR3包含SEQ ID NO:128所示的氨基酸序列。
在某些实施方式中，所述参比抗体包含HCDR2，所述HCDR2包含SEQ ID NO:127所示的氨基酸序列。
在某些实施方式中，所述参比抗体包含HCDR1，所述HCDR1包含SEQ ID NO:126所 示的氨基酸序列。
在某些实施方式中，所述参比抗体包含重链可变区VH，所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:128所示的氨基酸序列；所述HCDR2包含SEQ ID NO:127所示的氨基酸序列；且所述HCDR1包含SEQ ID NO:126所示的氨基酸序列。
在某些实施方式中，所述参比抗体的VH包含SEQ ID NO:132所示的氨基酸序列。
在某些实施方式中，所述参比抗体包含LCDR3，所述LCDR3包含SEQ ID NO:131所示的氨基酸序列。
在某些实施方式中，所述参比抗体包含LCDR2，所述LCDR2包含SEQ ID NO:130所示的氨基酸序列。
在某些实施方式中，所述参比抗体包含LCDR1，所述LCDR1包含SEQ ID NO:129所示的氨基酸序列。
在某些实施方式中，所述参比抗体包含轻链可变区VL，所述VL包含所述LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:131所示的氨基酸序列；所述LCDR2包含SEQ ID NO:130所示的氨基酸序列；且所述LCDR1包含SEQ ID NO:129所示的氨基酸序列。
在某些实施方式中，所述参比抗体的VL包含SEQ ID NO:133所示的氨基酸序列。
在某些实施方式中，所述参比抗体包括帕博利珠单抗或其抗原结合片段。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR3，所述HCDR3包含SEQ ID NO：138所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR3，所述HCDR3包含SEQ ID NO：142所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR3，所述HCDR3包含SEQ ID NO：3、55、56、57、58、59、60、61、62、63、64、65、66和67中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO：137所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO：141所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO：2、42、43、44、45、46、47、48、49、50、51、52、53和54中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，所述HCDR1包含SEQ ID NO： 136所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，所述HCDR1包含SEQ ID NO：140所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，所述HCDR1包含SEQ ID NO：1、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40和41中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO：138所示的氨基酸序列；所述HCDR2包含SEQ ID NO：137所示的氨基酸序列；且所述HCDR1包含SEQ ID NO：136所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO：142所示的氨基酸序列；所述HCDR2包含SEQ ID NO：141所示的氨基酸序列；且所述HCDR1包含SEQ ID NO：140所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO：3、55、56、57、58、59、60、61、62、63、64、65、66和67中任一项所示的氨基酸序列；所述HCDR2包含SEQ ID NO：2、42、43、44、45、46、47、48、49、50、51、52、53和54中任一项所示的氨基酸序列；且所述HCDR1包含SEQ ID NO：1、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40和41中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的HCDR1、HCDR2和HCDR3包含选自下述的任意一组氨基酸序列：
1)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:3；
2)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:42，和HCDR3：SEQ ID NO:55；
3)HCDR1：SEQ ID NO:4，HCDR2：SEQ ID NO:43，和HCDR3：SEQ ID NO:3；
4)HCDR1：SEQ ID NO:5，HCDR2：SEQ ID NO:44，和HCDR3：SEQ ID NO:3；
5)HCDR1：SEQ ID NO:6，HCDR2：SEQ ID NO:45，和HCDR3：SEQ ID NO:3；
6)HCDR1：SEQ ID NO:7，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
7)HCDR1：SEQ ID NO:8，HCDR2：SEQ ID NO:47，和HCDR3：SEQ ID NO:3；
8)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
9)HCDR1：SEQ ID NO:10，HCDR2：SEQ ID NO:45，和HCDR3：SEQ ID NO:3；
10)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:44，和HCDR3：SEQ ID NO:3；
11)HCDR1：SEQ ID NO:12，HCDR2：SEQ ID NO:43，和HCDR3：SEQ ID NO:3；
12)HCDR1：SEQ ID NO:13，HCDR2：SEQ ID NO:48，和HCDR3：SEQ ID NO:3；
13)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:45，和HCDR3：SEQ ID NO:3；
14)HCDR1：SEQ ID NO:15，HCDR2：SEQ ID NO:47，和HCDR3：SEQ ID NO:3；
15)HCDR1：SEQ ID NO:16，HCDR2：SEQ ID NO:43，和HCDR3：SEQ ID NO:3；
16)HCDR1：SEQ ID NO:17，HCDR2：SEQ ID NO:49，和HCDR3：SEQ ID NO:3；
17)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
18)HCDR1：SEQ ID NO:19，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
19)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:56；
20)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:57；
21)HCDR1：SEQ ID NO:22，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
22)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:51，和HCDR3：SEQ ID NO:3；
23)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:58；
24)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
25)HCDR1：SEQ ID NO:24，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:59；
26)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:60；
27)HCDR1：SEQ ID NO:26，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:61；
28)HCDR1：SEQ ID NO:27，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
29)HCDR1：SEQ ID NO:28，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:3；
30)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
31)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:62；
32)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:63；
33)HCDR1：SEQ ID NO:30，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
34)HCDR1：SEQ ID NO:31，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
35)HCDR1：SEQ ID NO:32，HCDR2：SEQ ID NO:53，和HCDR3：SEQ ID NO:3；
36)HCDR1：SEQ ID NO:33，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:3；
37)HCDR1：SEQ ID NO:34，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:64；
38)HCDR1：SEQ ID NO:35，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:57；
39)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:54，和HCDR3：SEQ ID NO:3；
40)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:65；
41)HCDR1：SEQ ID NO:36，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
42)HCDR1：SEQ ID NO:37，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:3；
43)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:59；
44)HCDR1：SEQ ID NO:38，HCDR2：SEQ ID NO:54，和HCDR3：SEQ ID NO:3；
45)HCDR1：SEQ ID NO:39，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
46)HCDR1：SEQ ID NO:40，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:55；
47)HCDR1：SEQ ID NO:41，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
48)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:66；
49)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:67。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO：68、144-148、230和/或235所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO：69、149-151和/或231所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO：70、152-161和/或232所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO：71、162和/或233所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO：68、144-148、230和/或235所示的氨基酸序列；所述H-FR2包含SEQ ID NO：69、149-151和/或231所示的氨基酸序列；所述H-FR3包含SEQ ID NO：70、152-161和/或232所示的氨基酸序列；以及所述H-FR4包含SEQ ID NO：71、162和/或233所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，所述VH包含SEQ ID NO：139和/或234所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，所述VH包含SEQ ID NO：143和/或236所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白的VH包含SEQ ID NO：72-120和/或163-227中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述分离的抗原结合蛋白包括单域抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和/或dAb。
在某些实施方式中，所述抗体包括嵌合抗体、人源化抗体和/或全人源抗体。
在某些实施方式中，所述分离的抗原结合蛋白包含SEQ ID NO：72-120和/或163-227中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白还包含免疫球蛋白Fc区。
在某些实施方式中，所述抗原结合蛋白的所述VH与所述Fc区直接或间接地连接。
在某些实施方式中，所述抗原结合蛋白的所述VH的C端与所述Fc区的N端直接或间接地连接。
在某些实施方式中，所述抗原结合蛋白的所述VH与所述Fc区在框内融合。
在某些实施方式中，所述抗原结合蛋白的所述VH通过连接子与所述Fc区连接。
在某些实施方式中，所述连接子包括肽连接子。
在某些实施方式中，所述连接子包括柔性连接子。
在某些实施方式中，所述分离的抗原结合蛋白从N端到C端依次包含所述抗原结合蛋白的所述VH，所述连接子和所述Fc区。
在某些实施方式中，所述Fc区包括源自IgG1的Fc或源自IgG4的Fc。
在某些实施方式中，所述Fc区包含SEQ ID NO：121-125中任一项所示的氨基酸序列。
另一方面，本申请提供了一种或多种多肽，其包含所述分离的抗原结合蛋白。
另一方面，本申请提供了一种或多种免疫缀合物，其包含所述分离的抗原结合蛋白或所述的多肽。
另一方面，本申请提供了一种或多种分离的核酸分子，其编码所述分离的抗原结合蛋白，或者所述的多肽。
另一方面，本申请提供了一种或多种载体，其包含所述分离的核酸分子。
另一方面，本申请提供了一种或多种细胞，其包含和/或表达所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述的分离的核酸分子和/或所述的载体。
另一方面，本申请提供了一种制备所述分离的抗原结合蛋白和/或所述的多肽的方法，所述方法包括在使得所述的分离的抗原结合蛋白和/或所述的多肽表达的条件下，培养所述的细胞。
另一方面，本申请提供了一种或多种药物组合物，其包含所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述分离的核酸分子，所述的载体，所述的细胞，以及任选地药学上可接受的佐剂和/或赋形剂。
另一方面，本申请提供了一种用于检测PD-1的存在和/或含量的方法，其包括：
施用所述分离的抗原结合蛋白或所述的多肽。
另一方面，本申请提供了一种或多种试剂盒，其包含所述的分离的抗原结合蛋白或所述的多肽。
在某些实施方式中，所述的试剂盒包含使用说明，所述使用说明记载用于检测PD-1的存在和/或含量的方法。
另一方面，本申请提供了一种所述分离的抗原结合蛋白或所述的多肽在制备试剂盒中的用途，所述试剂盒用于检测PD-1的存在和/或含量的方法。
另一方面，本申请提供了一种或多种所述分离的抗原结合蛋白和/或所述多肽在制备药物中的用途，所述药物用于预防和/或治疗疾病或病症。
另一方面，本申请提供了一种或多种所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述分离的核酸分子，所述的载体，所述的细胞和/或所述的药物组合物，其用于预防、缓解和/或治疗疾病或病症。
在某些实施方式中，所述疾病或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括血液瘤。
在某些实施方式中，所述肿瘤包括与PD-L1的表达相关的肿瘤。
在某些实施方式中，所述肿瘤选自下组：黑色素瘤，肺癌，头颈鳞状细胞癌，淋巴瘤，肝细胞癌，肾细胞癌，尿路上皮癌，结直肠癌和乳腺癌。
另一方面，本申请提供了一种预防和/或治疗疾病或病症的方法，其包括向有需要的受试者施用有效量的所述的分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述的分离 的核酸分子，所述的载体，和/或所述的细胞。
在某些实施方式中，所述疾病或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括血液瘤。
在某些实施方式中，所述肿瘤包括与PD-L1的表达相关的肿瘤。
在某些实施方式中，所述肿瘤选自下组：黑色素瘤，肺癌，头颈鳞状细胞癌，淋巴瘤，肝细胞癌，肾细胞癌，尿路上皮癌，结直肠癌和乳腺癌。
另一方面，本申请提供了一种抑制PD-1与PD-L1相互作用的方法，其包括向有需要的受试者施用有效量的所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述的分离的核酸分子，所述的载体，和/或所述的细胞。
另一方面，本申请提供了所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述的分离的核酸分子，所述的载体，所述的细胞和/或所述的药物组合物，其用于抑制PD-1与PD-L1的相互作用。
另一方面，本申请提供了所述分离的抗原结合蛋白和/或所述的多肽在制备药物中的用途，所述药物用于抑制PD-1与PD-L1的相互作用。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是本申请所述抗原结合蛋白的SDS-PAGE蛋白表达结果。
图2显示的是本申请所述抗原结合蛋白与重组人PD-1、重组猴PD-1及重组小鼠PD-1的结合活性。
图3A-3B显示的是本申请所述MHPD1-A Fc与帕博利珠单抗(Pembrolizumab)对抗原蛋白的亲和动力学检测结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
在本申请中，术语“抗原结合蛋白”通常是指一种能够特异性识别和/或中和特定抗原的多肽分子。在本申请中，术语“抗原结合蛋白”可包括“抗体”或“抗原结合片段”。例如，所述抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链组成的免疫球蛋白，并且可包括任何包含其抗原结合部分的分子。术语“抗体”可包括单克隆抗体、抗体片段或抗体衍生物，包括但不限于驼源抗体、全人源抗体、嵌合抗体、单链抗体(例如，scFv)，以及与抗原结合的抗体片段(例如，Fab、Fab’和(Fab)2片段)。术语“抗体”还可包括抗体的所有重组体形式，例如在原核细胞中表达的抗体、未糖基化的抗体以及本文所述的任何与抗原结合的抗体片段及其衍生物。在本申请中，所述“抗体”可以包括单域抗体。
在本申请中，术语“单域抗体”通常是指缺失抗体轻链而只有重链可变区的一类抗体。研究发现，双峰驼、单峰驼、羊驼和美洲驼中存在一种仅由重链组成、但具有完整功能的重链抗体(heavy chain antibody，hcAb)，其可变区(variable domains of the hcAb，简称为VHH)分子量仅为常规抗体的1/10，是目前可以得到的具有完整抗体功能的最小分子片段，称为单域抗体(single domain antibody,sdAb)。单域抗体与其它抗体相比，具有免疫原性低、分子小、穿透力强等优点，因此其在基础研究、药物开发、疾病治疗等领域有广阔的应用前景。例如，单域抗体可以来自羊驼。单域抗体可由重链可变区(VH)构成。术语“重链可变区”通常是指抗原结合片段的重链的氨基末端结构域。重链可变区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在成为框架区(FR)的更保守的区域中。每个重链可变区可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：H-FR1、HCDR1、H-FR2、HCDR2、H-FR3、HCDR3和H-FR4。重链可变区含有与抗原(例如，PD-1)相互作用的结 合结构域。所述CDRs的确切边界已根据不同系统不同地限定。由Kabat(Kabat等人，Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987)和(1991))描述的系统，不仅提供了可应用于抗原结合蛋白的任何可变区的明确残基编号系统，还提供了限定CDRs的精确残基边界。这些CDRs可以被称为Kabat CDRs。Chothia和同事(Chothia和Lesk，J.Mol.Biol.196：901-917(1987)以及Chothia等人，Nature 342：877-883(1989))发现尽管在氨基酸序列水平上具有大的多样性，但是Kabat CDRs内的某些亚部分采取几乎相同的肽主链构象。这些亚部分命名为L1、L2和L3或H1、H2和H3，其中“L”和“H”分别指轻链和重链区域。这些区域可以被称为Chothia CDRs，所述Chothia CDRs具有与Kabat CDRs重叠的边界。与Kabat CDRs重叠的限定CDRs的其他边界已由Padlan(FASEB J.9：133-139(1995))和MacCallum(J Mol Biol 262(5)：732-45(1996))描述。另外，其他的CDR边界定义可能不严格地遵循上述系统之一，但仍将与Kabat CDRs重叠，尽管按照特定残基或残基组或甚至整个CDRs并不显著影响抗原结合的预测或实验发现，它们可以缩短或加长。在本申请中，CDRs可用IMGT编号系统定义。在本申请中，术语“单域抗体”可与“纳米抗体”、“VHH”互换使用。虽然本申请中请求保护的范围是基于IMGT编号系统定义所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本申请的保护范围中。示例性的定义规则如下表所示。
  IMGT AbM Kabat Chothia
HCDR1 26-33 26-35 31-35 26-32
HCDR2 51-58 50-59 50-66 52-57
HCDR3 97-107 99-107 99-107 99-107
其中，表中氨基酸范围指从本申请的抗原结合蛋白的N端开始的氨基酸编号范围，例如“26-33”指N端起第26位到第33位的氨基酸序列。
在本申请中，术语“单克隆抗体”通常是指一群基本同源的抗体，即包含该群的各个抗体除了可能的以微量存在的天然发生的突变之外是相同的。单克隆抗体是高度特异性的，直接针对单个抗原性位点。例如，所述单克隆抗体可以通过杂交瘤技术制备或者通过使用重组DNA方法在细菌、真核动物或植物细胞中产生。单克隆抗体也可以得自噬菌体抗体文库，使用例如Clackson etal.,Nature,352:624-628(1991)和Marks et al.,Mol.Biol.，222:581-597(1991)所述的技术进行。
在本申请中，术语“嵌合抗体”通常是指这样的抗体，其中每个重链或轻链氨基酸序列的一部分与来自特定物种的抗体中相应氨基酸序列同源，或者属于特定的类别，而该链的其余 区段则与另一物种中的相应序列同源。例如，轻链和重链的可变区均来自一个动物物种(如小鼠、大鼠等)的抗体的可变区，而恒定部分则与来自另一物种(如人)的抗体序列同源。例如，为获得嵌合抗体，可利用非人源的B细胞或杂交瘤细胞产生可变区，而与其组合的恒定区则来自人。所述可变区具有易于制备的优点，并且其特异性不受与其组合的恒定区的来源的影响。同时，由于嵌合抗体的恒定区可来源于人类，因此嵌合在注射时抗体引发免疫应答的可能性会低于使用恒定区为非人来源的抗体。
在本申请中，术语“PD-1”或“PD1”通常是指人程序性细胞死亡受体-1，是一种288个氨基酸的I型膜蛋白，首次在1992年有描述(Ishida et al.,EMBO J,11(1992)，3887-3895)。PD-1是扩大的CD28/CTLA-4 T细胞调节物家族的一个成员且具有两种配体，PD-L1(B7-H1，CD274)和PD-L2(B7-DC，CD273)。该蛋白质的结构包括一个胞外IgV域，接着是一个跨膜区和一个胞内尾。胞内尾含有位于免疫受体基于酪氨酸的抑制性基序和免疫受体基于酪氨酸的转换基序中的两个磷酸化位点，提示PD-1阴性负调节TCR信号。这与配体结合后SHP-1和SHP-2磷酸酶对PD-1的胞质尾的结合一致。PD-1在T细胞受体(TCR)介导的活化后上调且在活化的和耗尽的T细胞二者上观察到(Agata et al.,Int.Immunology 8(1996),765-772)。虽然PD-1具有相对广泛的表达样式，但是它最重要的作用很可能是作为T细胞上的共抑制性受体(Chinai et al.,Trends in Pharmacological Sciences 36(2015),587-595)。因而聚焦于阻断PD-1与它的配体的相互作用以增强T细胞应答，从而治疗肿瘤。“PD-1蛋白”，“PD-1”，PD1”，PDCD1”，“hPD-1”和“hPD-I”可以互换使用，而且包括人PD-1的同等型，物种同源物，功能活性片段，和与PD-1具有至少一个共同表位的类似物。例如，所述“功能活性片段”可以包括保留至少一种天然存在的蛋白质的内源功能(例如，与本申请所述的抗原结合蛋白结合)的片段。例如，所述“功能活性片段”可以包括与本申请的抗原结合蛋白结合的结构域。示例性的人PD1的氨基酸序列可以在UniProt登录号Q15116中显示。
在本申请中，术语“PD-L1”或“PDL1”通常是指程序性死亡受体-配体1，也可称为B7同源物1、B7-H1、分化簇274、(3)274或CD274，其与PD-1结合后下调T细胞活化和细胞因子分泌。“PD-L1”包括任何脊椎动物来源的任何天然PD-L1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人和食蟹猴)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-L1以及由细胞加工所产生的任何形式的PD-L1。PD-L1可作为跨膜蛋白或作为可溶性蛋白存在。“PD-L1”包括完整的PD-L1及其片段，还包括PD-L1的功能性片段、同等型、物种同源物、衍生物、类似物，以及具有至少一个与PD-L1共同表位的类似物。例如，所述“功能活性片段”可以包括保留至少一种天然存在的蛋白质的内源功能(例如，与本 申请所述的抗原结合蛋白结合)的片段。例如，所述“功能活性片段”可以包括与本申请的抗原结合蛋白结合的结构域。示例性的人PD-L1氨基酸序列可在NCBI登录号NP_001254653或UniProt登录号Q9NZQ7下找到。
除了本文提到的特定蛋白质和核苷酸之外，本申请还可包括其功能性变体、衍生物、类似物、同源物及其片段。
术语“功能性变体”指与天然存在序列具有基本上同一的氨基酸序列或由基本上同一的核苷酸序列编码并能够具有天然存在序列的一种或多种活性的多肽。在本申请的上下文中，任何给定序列的变体是指其中残基的特定序列(无论是氨基酸或核苷酸残基)已经经过修饰而使得所述多肽或多核苷酸基本上保留至少一种内源功能的序列。可以通过天然存在的蛋白质和/或多核苷酸中存在的至少一个氨基酸残基和/或核苷酸残基的添加、缺失、取代、修饰、替换和/或变异来获得变体序列，只要保持原来的功能活性即可。
在本申请中，术语“衍生物”通常是指本申请的多肽或多核苷酸而言包括自/对序列的一个(或多个)氨基酸残基的任何取代、变异、修饰、替换、缺失和/或添加，只要所得的多肽或多核苷酸基本上保留其至少一种内源功能。
在本申请中，术语“类似物”通常对多肽或多核苷酸而言，包括多肽或多核苷酸的任何模拟物，即拥有该模拟物模拟的多肽或多核苷酸的至少一种内源功能的化学化合物。
通常，可以进行氨基酸取代，例如至少1个(例如，1、2、3、4、5、6、7、8、9、10或20个以上)氨基酸取代，只要经修饰的序列基本上保持需要的活性或能力。氨基酸取代可包括使用非天然存在的类似物。
在本申请中，术语“同源物”通常是指与天然存在序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。
用于本申请的蛋白质或多肽也可以具有氨基酸残基的缺失、插入或取代，所述氨基酸残基产生沉默的变化并导致功能上等同的蛋白质。可以根据残基的极性、电荷、溶解性、疏水性、亲水性和/或两性性质的相似性进行有意的氨基酸取代，只要保留内源性功能即可。例如，带负电荷的氨基酸包括天冬氨酸和谷氨酸；带正电荷的氨基酸包括赖氨酸和精氨酸；并且含具有相似亲水性值的不带电极性头基的氨基酸包括天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸和酪氨酸。
在本申请中，术语“肿瘤”通常是指在各种致瘤因子作用下，局部组织细胞增生所形成的赘生物。例如，所述肿瘤可包括实体瘤。例如，所述肿瘤可包括血液瘤。例如，所述肿瘤可包括与PD-L1的表达相关的肿瘤。术语“与PD-L1的表达相关的肿瘤”通常是指PD-L1表达改变导致疾病进展或逃避免疫监视而形成的肿瘤。例如，所述“与PD-L1的表达相关的肿瘤”可以是PD-L1表达量上调导致疾病进展或逃避免疫监视而形成的肿瘤。所述与PD-L1的蛋白表达相关的肿瘤可以是PD-L1阳性的肿瘤。在PD-L1阳性的肿瘤中，与正常细胞相比，肿瘤细胞表面或肿瘤微环境中的PD-L1的蛋白表达量高约1％，5％，10％，15％，20％，25％，30％，35％，40％，50％，60％，70％，80％或更高。
在本申请中，术语“实体瘤”通常是指可通过临床检测(例如，X线射片，CT扫描，B超或触诊)到的有形肿块。例如，所述实体瘤可选自下组：黑色素瘤，肺癌，头颈鳞状细胞癌，肝细胞癌，肾细胞癌，尿路上皮癌，结直肠癌和乳腺癌。
在本申请中，术语“血液瘤”通常是指X线射片、CT扫描、B超以及触诊无法看到或扪及到的肿瘤。例如，所述血液瘤可以包括白血病。例如，所述血液瘤可以包括淋巴瘤。例如，所述血液瘤可包括多发性骨髓瘤。
在本申请中，术语“免疫缀合物”通常是指所述其他试剂(例如，化疗剂、放射性元素、细胞生长抑制剂和细胞毒性剂)与所述分离的抗原结合蛋白缀合(例如，通过连接分子共价相连)而形成的缀合物，该缀合物可以通过所述分离的抗原结合蛋白与靶细胞上的抗原的特异性结合，将所述其他试剂递送至靶细胞(例如，肿瘤细胞)。然后所述免疫缀合物经所述内化，最终进入靶细胞内部(例如，进入溶酶体等泡囊体)，此时所述免疫缀合物中的连接分子可以裂解，释放所述其他试剂从而发挥其细胞毒性效应。此外，所述抗原也可以由所述靶细胞分泌，并位于所述靶细胞外的间隙。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的 分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指一种核酸分子，该种核酸分子能够转运与其连接的另一核酸。所述载体可将插入的核酸分子转移到细胞中和/或细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体可以是当引入合适的细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的细胞，所述载体可以产生期望的表达产物。在本申请中，所述载体可包含慢病毒载体。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的多肽或本申请所述的抗原结合蛋白的个体细胞，细胞系或细胞培养物。所述细胞可以包括单个细胞的子代。由于天然的，意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的多肽或抗原结合蛋白即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在一些实施方式中，所述细胞可以是免疫细胞。例如，所述免疫细胞可以选自下组：T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
在本申请中，术语“治疗”通常是指：(i)预防可能易患疾病、病症和/或病状、但尚未诊断出患病的患者出现该疾病、病症或病状；(ii)抑制该疾病、病症或病状，亦即遏制其发展；以及(iii)缓解该疾病、病症或病状，亦即使得该疾病、病症和/或病状和/或与该疾病、病症和/或病状相关联的症状消退。
在本申请中，术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换地使用，通常是指具有任何长度的氨基酸的聚合物。该聚合物可以是直链或支链的，它可以包含修饰的氨基酸，并且可以被非氨基酸中断。这些术语还涵盖已经被修饰的氨基酸聚合物。这些修饰可以包含：二硫键形成、糖基化、脂化(lipidation)、乙酰化、磷酸化、或任何其他操纵(如与标记组分结合)。术语“氨基酸”包括天然的和/或非天然的或者合成的氨基酸，包括甘氨酸以及D和L旋光异构体、以及氨基酸类似物和肽模拟物。
在本申请中，术语“多核苷酸”、“核苷酸”、“核苷酸序列”、“核酸”和“寡核苷酸”可互换地使用，通常是指具有任何长度的核苷酸的聚合形式，如脱氧核糖核苷酸或核糖核苷酸、或其类似物。多核苷酸可具有任何三维结构，并且可以执行已知或未知的任何功能。以下是多核 苷酸的非限制性实例：基因或基因片段的编码区或非编码区、根据连接分析定义的多个座位(一个座位)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、短干扰RNA(siRNA)、短发夹RNA(shRNA)、micro-RNA(miRNA)、核酶、cDNA、重组多核苷酸、分支多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针、和引物。多核苷酸可以包含一个或多个经修饰的核苷酸，如甲基化的核苷酸和核苷酸类似物。如果存在，可以在聚合物组装之前或之后进行核苷酸结构的修饰。核苷酸的序列可以被非核苷酸组分中断。多核苷酸可以在聚合后，如通过与标记的组分缀合来进一步修饰。
在本申请中，术语“K D”(同样地，“K D”或“KD”)通常指“亲和常数”或“平衡解离常数”，并指在滴定测量中在平衡时、或者通过将解离速率常数(k d)除以结合速率常数(k a)所获得的值。使用结合速率常数(k a)、解离速率常数(k d)和平衡解离常数(K D)表示结合蛋白(例如本申请所述的分离的抗原结合蛋白)对抗原(例如，PD-1蛋白)的结合亲和力。确定结合和解离速率常数的方法为本领域熟知。使用基于荧光的技术提供了高灵敏度以及在生理缓冲液中在平衡时检查样品的能力。例如，可以通过Octet(Sartorius公司)测定所述K D值，也可以使用其他实验途径和仪器例如BIAcore(生物分子相互作用分析系统，GE Healthcare Life Science)测定。另外，也可以使用Sapidyne Instruments(Boise，Idaho)公司的KinExA(动态排阻测定，Kinetic Exclusion Assay)测定所述K D值，或者使用其他表面等离子共振仪(SPR)测定所述K D值。在本申请中，可以通过BIAcore测定所述K D值。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。在某些情形中，“包含”也涵盖仅包括指定的组分的情况。在某些情况下，“包含”也涵盖“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
发明详述
本申请所述分离的抗原结合蛋白
一方面，本申请提供一种分离的抗原结合蛋白，在ELSA检测中，其可以以约11μg/mL或更低的EC 50(例如，所述EC 50不高于约11μg/mL、不高于约10μg/mL、不高于约9.5μg/mL、 不高于约9.0μg/mL、不高于约8.5μg/mL、不高于约8.0μg/mL、不高于约7.5μg/mL、不高于约7.0μg/mL、不高于6.5μg/mL、不高于约6.0μg/mL、不高于5.5μg/mL、不高于约5.0μg/mL、不高于约4.5μg/mL、不高于约4.0μg/mL、不高于约3.5μg/mL、不高于约3.0μg/mL、不高于约2.5μg/mL、不高于约2.0μg/mL、不高于约1.5μg/mL、不高于约1.0μg/mL、不高于约0.5μg/mL、不高于约0.4μg/mL、不高于约0.3μg/mL、不高于约0.2μg/mL或不高于约0.1μg/mL或以下)与PD-1结合。
在竞争ELISA检测中，本申请所述分离的抗原结合蛋白可以以约2μg/mL或更低的EC 50(例如，所述EC 50不高于约1.9μg/mL、不高于约1.8μg/mL、不高于约1.7μg/mL、不高于约1.5μg/mL、不高于约1.3μg/mL、不高于约1.1μg/mL、不高于约1.0μg/mL、不高于约0.9μg/mL、不高于0.8μg/mL、不高于约0.7μg/mL、不高于0.6μg/mL、不高于约0.5μg/mL、不高于约0.4μg/mL、不高于约0.3μg/mL、不高于约0.2μg/mL、或不高于约0.1μg/mL或以下)抑制人PD-L1与人PD-1结合。
在本申请中，所述分离的抗原结合蛋白可以与参比抗体结合相同或重叠的PD-1表位。例如，所述分离的抗原结合蛋白可以与所述参比抗体竞争结合PD-1。
在本申请中，所述分离的抗原结合蛋白对PD-1的结合亲和力可与所述参比抗体对PD-1的结合亲和力基本相同。例如，结合亲和力可以通过ELISA检测进行测定。例如，所述结合亲和力可由EC 50进行表征。例如，在ELISA检测结合亲和力实验中，所述分离的抗原结合蛋白对PD-1的EC 50约等于或低于所述参比抗体对PD-1的EC 50。
在本申请中，所述参比抗体可包含重链可变区VH，所述VH可包含HCDR1、HCDR2和HCDR3中的至少一个、两个或三个。
在本申请中，所述HCDRs的确切边界可根据不同系统不同地限定。例如，本申请所述的HCDRs可由Kabat系统限定(参见Kabat等人，Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987)和(1991))描述的系统)，这一限定方法不仅提供了可应用于抗原结合蛋白的任何可变区的明确残基编号系统，还提供了限定CDRs的精确残基边界。这些CDRs可以被称为Kabat CDRs。例如，本申请所述的HCDRs还可由Chothia系统限定(参见Chothia和同事的研究(Chothia和Lesk，J.Mol.Biol.196：901-917(1987)以及Chothia等人，Nature 342：877-883(1989)))。Chothia和同事发现尽管在氨基酸序列水平上具有大的多样性，但是Kabat CDRs内的某些亚部分采取几乎相同的肽主链构象。这些亚部分命名为L1、L2和L3或H1、H2和H3，其中“L”和“H”分别指轻链和重链区域。这些区域可以被称为Chothia CDRs，所述Chothia CDRs具有与Kabat CDRs重叠的边界。与Kabat CDRs 重叠的限定CDRs的其他边界已由Padlan(FASEB J.9：133-139(1995))和MacCallum(J Mol Biol 262(5)：732-45(1996))描述。另外，其他的CDR边界定义可能不严格地遵循上述系统之一，但仍将与Kabat CDRs重叠，尽管按照特定残基或残基组或甚至整个CDRs并不显著影响抗原结合的预测或实验发现，它们可以缩短或加长。在本申请中，HCDRs可根据IMGT定义规则确定。
在本申请中，所述参比抗体的HCDR3可包含SEQ ID NO：138所示的氨基酸序列。例如，所述参比抗体的HCDR3的序列可根据IMGT定义规则确定。
AADX 4DX 6X 7GFDY(SEQ ID NO：138)，其中，X 4可以是I或L，X 6可以是H，R或V，X 7可以是A，D，G，H，L，N，R，S，T或V。
在本申请中，所述参比抗体的HCDR3可包含SEQ ID NO：142所示的氨基酸序列。例如，所述参比抗体的HCDR3的序列可根据IMGT定义规则确定。
AADX 4DX 6X 7GFDY(SEQ ID NO：142)，其中，X 4可以是I或L，X 6可以是H或R，X 7可以是A，H或S。
在本申请中，所述参比抗体的HCDR3可包含SEQ ID NO：3、55、56、57、58、59、60、61、62、63、64、65、66和67中任一项所述的氨基酸序列。例如，所述参比抗体的HCDR3的序列可根据IMGT定义规则确定。
在本申请中，所述参比抗体的HCDR2可包含SEQ ID NO：137所示的氨基酸序列。例如，所述参比抗体的HCDR2的序列可根据IMGT定义规则确定。
X 1GWX 4X 5X 6TX 8(SEQ ID NO：137)，其中，X 1可以是I或L，X 4可以是A，N，R或S，X 5可以是A，F，W或Y，X 6可以是A，G或S，X 8可以是E，Q，S或T。
在本申请中，所述参比抗体的HCDR2可包含SEQ ID NO：141所示的氨基酸序列。例如，所述参比抗体的HCDR2的序列可根据IMGT定义规则确定。
IGWX 4X 5X 6TX 8(SEQ ID NO：141)，其中，X 4可以是A或N，X 5可以是F或Y，X 6可以是A或S，X 8可以是E，Q或T。
在本申请中，所述参比抗体的HCDR2可包含SEQ ID NO：2、42、43、44、45、46、47、48、49、50、51、52、53和54中任一项所述的氨基酸序列。例如，所述参比抗体的HCDR2的序列可根据IMGT定义规则确定。
在本申请中，所述参比抗体的HCDR1可包含SEQ ID NO：136所示的氨基酸序列。例如，所述参比抗体的HCDR1的序列可根据IMGT定义规则确定。
X 1X 2X 3X 4X 5IYA(SEQ ID NO：136)，其中，X 1可以是A，G，H，K，S或V，X 2可以 是A，G，H，L，N，P，R，S，T或V，X 3可以是D，E，F，G，I，K，Q，R，S或V，X 4可以是D，G，H，K，L，P，Q，R，S，T，V或Y，X 5可以是A，F，N，Q，R，S或T。
在本申请中，所述参比抗体的HCDR1可包含SEQ ID NO：140所示的氨基酸序列。例如，所述参比抗体的HCDR1的序列可根据IMGT定义规则确定。
X 1X 2X 3X 4X 5IYA(SEQ ID NO：140)，其中，X 1可以是G，H或K，X 2可以是A，G，L，N，S或V，X 3可以是D，R或V，X 4可以是G，H，P，Q，R或T，X 5可以是R，S或T。
在本申请中，所述参比抗体的HCDR1可包含SEQ ID NO：1、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40和41中任一项所述的氨基酸序列。例如，所述参比抗体的HCDR1的序列可根据IMGT定义规则确定。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：138所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：137所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：136所示的氨基酸序列。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：142所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：141所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：140所示的氨基酸序列。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：1所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：2所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A或与其具有相同HCDR1-3的抗体。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：1所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：42所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：55所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-1或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：4所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：43所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-2或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：5所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：44所示的氨基酸序列；且所述参比抗体的HCDR3可包含 SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-3或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：6所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：45所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-4或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：7所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-5或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：8所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：47所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-6或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-7或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：10所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：45所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-8或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：11所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：44所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-9或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：12所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：43所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-10或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：13所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：48所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-11或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：14所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：45所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-12或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：15所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：47所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-13或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：16所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：43所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-14或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：17所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：49所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-15或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：18所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-16或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：19所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-17或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：20所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：50所示的氨基酸序列；且所述参比抗体的HCDR3可包含 SEQ ID NO：56所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-18或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：21所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：57所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-19或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：22所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-20或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：51所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-21或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：58所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-22或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：23所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-23或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：24所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：59所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-24或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：25所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：60所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-25或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：26所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：61所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-26或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：27所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-27或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：28所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：52所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-28或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：29所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-29或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：25所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：52所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：62所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-30或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：50所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：63所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-31或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：30所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-32或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：31所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含 SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-33或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：32所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：53所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-35或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：33所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：52所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-36或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：34所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：64所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-37或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：35所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：57所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-38或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：54所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-39或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：65所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-40或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：36所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-41或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：37所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：52所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-42或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：59所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-43或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：38所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：54所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-44或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：39所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-45或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：40所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：55所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-46或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：41所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-47或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：66所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-48或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述参比抗体的HCDR3可包含 SEQ ID NO：67所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A-49或与其具有相同HCDR3(例如，与其具有相同HCDR1-3)的抗原结合蛋白(例如，单域抗体)。
例如，所述参比抗体的VH可包含框架区H-FR1，H-FR2，H-FR3和H-FR4。
在本申请中，所述H-FR1可包含SEQ ID NO：68、144-148、230和/或235所示的氨基酸序列。
在本申请中，所述H-FR2可包含SEQ ID NO：69、149-151和/或231所示的氨基酸序列。
在本申请中，所述H-FR3可包含SEQ ID NO：70、152-161和/或232所示的氨基酸序列。
在本申请中，所述H-FR4可包含SEQ ID NO：71、162和/或233所示的氨基酸序列。
在本申请中，所述参比抗体的H-FR1可包含SEQ ID NO：68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO：69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO：70所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO：71所示的氨基酸序列。例如，所述参比抗体可包括单域抗体MHPD1-A或与其具有相同H-FR1-4的抗体。
在本申请中，所述参比抗体可包含重链可变区，所述重链可变区可包含SEQ ID NO：139所示的氨基酸序列。
QVQLVESGGGSVQVGGSLTLSCAASX 26X 27X 28X 29X 30IYAMGWFRQAPGKEHEFVAGX 51GWX 54X 55X 56TX 58YYADSVKGRFGISRDNTKNTVALLMNSLKPEDTAIYYCAADX 100DX 102X 103GFDYWGQGTQVTVSS(SEQ ID NO：139)，其中，X 26可以是A，G，H，K，S或V，X 27可以是A，G，H，L，N，P，R，S，T或V，X 28可以是D，E，F，G，I，K，Q，R，S或V，X 29可以是D，G，H，K，L，P，Q，R，S，T，V或Y，X 30可以是A，F，N，Q，R，S或T，X 51可以是I或L，X 54可以是A，N，R或S，X 55可以是A，F，W或Y，X 56可以是A，G或S，X 58可以是E，Q，S或T，X 100可以是I或L，X 102可以是H，R或V，X 103可以是A，D，G，H，L，N，R，S，T或V。
在本申请中，所述参比抗体可包含重链可变区，所述重链可变区可包含SEQ ID NO：143所示的氨基酸序列。
QVQLVESGGGSVQVGGSLTLSCAASX 26X 27X 28X 29X 30IYAMGWFRQAPGKEHEFVAGIGWX 54X 55X 56TX 58YYADSVKGRFGISRDNTKNTVALLMNSLKPEDTAIYYCAADX 100DX 102X 103GFDYWGQGTQVTVSS(SEQ ID NO：143)，其中，X 26可以是G，H或K，X 27可以是A，G，L，N，S或V，X 28可以是D，R或V，X 29可以是G，H，P，Q，R或T，X 30可以是R，S或T，X 54可以是A或N，X 55可以是F或Y，X 56可以是A或S，X 58可以是E，Q或T，X 100可以是I或L，X 102可以是H或R，X 103可以是A，H或S。
在本申请中，所述参比抗体的重链可变区可包含SEQ ID NO：72-120和/或163-227中任一项所示的氨基酸序列。
在本申请中，所述参比抗体的HCDR3可包含SEQ ID NO：128所示的氨基酸序列。
在本申请中，所述参比抗体的HCDR2可包含SEQ ID NO：127所示的氨基酸序列。
在本申请中，所述参比抗体的HCDR1可包含SEQ ID NO：126所示的氨基酸序列。
例如，所述参比抗体的HCDR1可包含SEQ ID NO：126所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO：127所示的氨基酸序列；且所述参比抗体的HCDR3可包含SEQ ID NO：128所示的氨基酸序列。例如，所述参比抗体可包括帕博利珠单抗或与其具有相同HCDR1-3的抗体。
在本申请中，所述参比抗体可包括轻链可变区VL，所述VL可包含LCDR1、LCDR2和LCDR3。
在本申请中，所述参比抗体的LCDR3可包含SEQ ID NO：131所示的氨基酸序列。
在本申请中，所述参比抗体的LCDR2可包含SEQ ID NO：130所示的氨基酸序列。
在本申请中，所述参比抗体的LCDR1可包含SEQ ID NO：129所示的氨基酸序列。
例如，所述参比抗体的LCDR1可包含SEQ ID NO：129所示的氨基酸序列；所述参比抗体的LCDR2可包含SEQ ID NO：130所示的氨基酸序列；且所述参比抗体的LCDR3可包含SEQ ID NO：131所示的氨基酸序列。例如，所述参比抗体可包括帕博利珠单抗或与其具有相同LCDR1-3的抗体。
例如，所述参比抗体的HCDR1可包含SEQ ID NO:126所示的氨基酸序列；所述参比抗体的HCDR2可包含SEQ ID NO:127所示的氨基酸序列；所述参比抗体的HCDR3可包含SEQ ID NO:128所示的氨基酸序列；所述参比抗体的LCDR1可包括SEQ ID NO:129所示的氨基酸序列；所述参比抗体的LCDR2可包含SEQ ID NO:130所示的氨基酸序列；所述参比抗体的LCDR3可包含SEQ ID NO:131所示的氨基酸序列。例如，所述参比抗体可包括帕博利珠单抗或与其具有相同HCDR1-3和LCDR1-3的抗原结合蛋白。
例如，所述参比抗体的VH可包含SEQ ID NO:132所示的氨基酸序列。例如，所述参比抗体的VL可包含SEQ ID NO:133所示的氨基酸序列。例如，所述参比抗体可包括与帕博利珠单抗或与其具有相同VH和VL的抗原结合蛋白。
在本申请中，所述参比抗体可包含重链和轻链，例如，所述参比抗体的重链可包含SEQ ID NO:134所示的氨基酸序列。例如，所述参比抗体的轻链可包含SEQ ID NO:135所示的氨基酸序列。例如，所述参比抗体可包括与帕博利珠单抗或与其具有相同重链和轻链的抗原结合 蛋白。
在本申请中，所述抗原结合蛋白可包含重链可变区VH，所述VH可包含HCDR1、HCDR2和HCDR3中的至少一个、两个或三个。
在本申请中，所述抗原结合蛋白的HCDR3可包含SEQ ID NO：138所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR3的序列可根据IMGT定义规则确定。
AADX 4DX 6X 7GFDY(SEQ ID NO：138)，其中，X 4可以是I或L，X 6可以是H，R或V，X 7可以是A，D，G，H，L，N，R，S，T或V。
在本申请中，所述抗原结合蛋白的HCDR3可包含SEQ ID NO：142所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR3的序列可根据IMGT定义规则确定。
AADX 4DX 6X 7GFDY(SEQ ID NO：142)，其中，X 4可以是I或L，X 6可以是H或R，X7可以是A，H或S。
在本申请中，所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3、55、56、57、58、59、60、61、62、63、64、65、66和67中任一项所述的氨基酸序列。例如，所述抗原结合蛋白的HCDR3的序列可根据IMGT定义规则确定。
在本申请中，所述抗原结合蛋白的HCDR2可包含SEQ ID NO：137所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR2的序列可根据IMGT定义规则确定。
X 1GWX 4X 5X 6TX 8(SEQ ID NO：137)，其中，X 1可以是I或L，X 4可以是A，N，R或S，X 5可以是A，F，W或Y，X 6可以是A，G或S，X 8可以是E，Q，S或T。
在本申请中，所述抗原结合蛋白的HCDR2可包含SEQ ID NO：141所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR2的序列可根据IMGT定义规则确定。
IGWX 4X 5X 6TX 8(SEQ ID NO：141)，其中，X 4可以是A或N，X 5可以是F或Y，X 6可以是A或S，X 8可以是E，Q或T。
在本申请中，所述抗原结合蛋白的HCDR2可包含SEQ ID NO：2、42、43、44、45、46、47、48、49、50、51、52、53和54中任一项所述的氨基酸序列。例如，所述抗原结合蛋白的HCDR2的序列可根据IMGT定义规则确定。
在本申请中，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：136所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR1的序列可根据IMGT定义规则确定。
X 1X 2X 3X 4X 5IYA(SEQ ID NO：136)，其中，X 1可以是A，G，H，K，S或V，X 2可以是A，G，H，L，N，P，R，S，T或V，X 3可以是D，E，F，G，I，K，Q，R，S或V，X 4可以是D，G，H，K，L，P，Q，R，S，T，V或Y，X 5可以是A，F，N，Q，R，S或T。
在本申请中，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：140所示的氨基酸序列。例如，所述抗原结合蛋白的HCDR1的序列可根据IMGT定义规则确定。
X 1X 2X 3X 4X 5IYA(SEQ ID NO：140)，其中，X 1可以是G，H或K，X 2可以是A，G，L，N，S或V，X 3可以是D，R或V，X 4可以是G，H，P，Q，R或T，X 5可以是R，S或T。
在本申请中，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：1、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40和41中任一项所述的氨基酸序列。例如，所述抗原结合蛋白的HCDR1的序列可根据IMGT定义规则确定。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：138所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：137所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：136所示的氨基酸序列。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：142所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：141所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：140所示的氨基酸序列。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：1所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：2所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：1所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：42所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：55所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-1或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：4所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：43所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-2或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：5所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：44所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-3或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：6所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：45所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-4或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：7所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-5或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：8所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：47所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-6或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-7或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：10所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：45所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-8或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：11所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：44所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-9或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：12所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：43所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-10或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：13所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：48所示的氨基酸序列；且所述抗原结合蛋白的 HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-11或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：14所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：45所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-12或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：15所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：47所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-13或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：16所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：43所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-14或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：17所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：49所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-15或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：18所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-16或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：19所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-17或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：20所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：50所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：56所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-18或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：21所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：57所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-19或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：22所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-20或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：51所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-21或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：58所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-22或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：23所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-23或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：24所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：59所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-24或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：25所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：60所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-25或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：26所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的 HCDR3可包含SEQ ID NO：61所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-26或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：27所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-27或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：28所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：52所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-28或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：29所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-29或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：25所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：52所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：62所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-30或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：50所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：63所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-31或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：30所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-32或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：31所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-33或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：32所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：53所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-35或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：33所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：52所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-36或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：34所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：64所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-37或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：35所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：57所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-38或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：54所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-39或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：65所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-40或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：36所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-41或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：37所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：52所示的氨基酸序列；且所述抗原结合蛋白的 HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-42或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：59所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-43或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：38所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：54所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-44或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：39所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-45或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：40所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：55所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-46或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：41所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：3所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-47或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：66所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-48或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO：9所示的氨基酸序列；所述抗原结合蛋白的HCDR2可包含SEQ ID NO：46所示的氨基酸序列；且所述抗原结合蛋白的HCDR3可包含SEQ ID NO：67所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-49或与其具有相同HCDR1-3的抗体。
例如，所述抗原结合蛋白的VH可包含框架区H-FR1，H-FR2，H-FR3和H-FR4。
在本申请中，所述H-FR1可包含SEQ ID NO：230和/或235所示的氨基酸序列。在本申请中，所述H-FR2可包含SEQ ID NO：231所示的氨基酸序列。在本申请中，所述H-FR3可包含SEQ ID NO：232所示的氨基酸序列。在本申请中，所述H-FR4可包含SEQ ID NO：233所示的氨基酸序列。
在本申请中，所述H-FR1可包含SEQ ID NO：68、144-148、和/或230所示的氨基酸序列。
在本申请中，所述H-FR2可包含SEQ ID NO：69、和/或149-151所示的氨基酸序列。
在本申请中，所述H-FR3可包含SEQ ID NO：70、和/或152-161所示的氨基酸序列。
在本申请中，所述H-FR4可包含SEQ ID NO：71、和/或162所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的H-FR1可包含SEQ ID NO：68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO：69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO：70所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO：71所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A或与其具有相同H-FR1-4的抗体。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含SEQ ID NO：234所示的氨基酸序列。
QVQLVESGGGX 1VQX 2GGSLX 3LSCAX 4SX 5X 6X 7X 8X 9IYAX 10X 11WFRQAPGKX 12X 13EFVAGX 14GWX 15X 16X 17TX 18YYADSVKGRFX 19ISRDNX 20KNTX 21X 22LX 23MNSLX 24X 25EDTAX 26YYCAADX 27DX 28X 29GFDYWGQGTX 30VTVSS(SEQ ID NO：234)，其中，X 1可以是，L、或S，X 2可以是，P、或V，X 3可以是，R、或T，X 4可以是，A、或T，X 5可以是，A、G、H、K、S、或V，X 6可以是，A、G、H、L、N、P、R、S、T、或V，X 7可以是，D、E、F、G、I、K、Q、R、S、或V，X 8可以是，D、G、H、K、L、P、Q、R、S、T、V、或Y，X 9可以是，A、F、N、Q、R、S、或T，X 10可以是，A、或M，X 11可以是，G、或M，X 12可以是，E、或G，X 13可以是，H、或L，X 14可以是，I、或L，X 15可以是，A、N、R、或S，X 16可以是，A、F、W、或Y，X 17可以是，A、G、或S，X 18可以是，E、Q、S、或T，X 19可以是，G、或T，X 20可以是，S、或T，X 21可以是，L、或V，X 22可以是，A、或Y，X 23可以是，L、或Q，X 24可以是，K、或R，X 25可以是，A、或P，X 26可以是，I、或V，X 27可以是，I、或L，X 28可以是，H、R、或V，X 29可以是，A、D、G、H、L、N、R、S、T、或V，X 30可以是，L、或Q。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含SEQ ID NO： 236所示的氨基酸序列。
QVQLVESGGGLVQPGGSLRLSCAX 1SX 2X 3X 4X 5X 6IYAMGWFRQAPGKGLEFVAGIGWX 7X 8X 9TX 10YYADSVKGRFTISRDNTKNTLYLQMNSLRAEDTAVYYCAADX 11DX 12X 13GFDYWGQGTLVTVSS(SEQ ID NO：236)，其中，X 1可以是，A、或T，X 2可以是，G、H、或K，X 3可以是，A、G、L、N、S、或V，X 4可以是，D、R、或V，X 5可以是，G、H、P、Q、R、或T，X 6可以是，R、S、或T，X 7可以是，A、或N，X 8可以是，F、或Y，X 9可以是，A、或S，X 10可以是，E、Q、或T，X 11可以是，I、或L，X 12可以是，H、或R，X 13可以是，A、H、或S。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含SEQ ID NO：139所示的氨基酸序列。
QVQLVESGGGSVQVGGSLTLSCAASX 26X 27X 28X 29X 30IYAMGWFRQAPGKEHEFVAGX 51GWX 54X 55X 56TX 58YYADSVKGRFGISRDNTKNTVALLMNSLKPEDTAIYYCAADX 100DX 102X 103GFDYWGQGTQVTVSS(SEQ ID NO：139)，其中，X 26可以是A，G，H，K，S或V，X 27可以是A，G，H，L，N，P，R，S，T或V，X 28可以是D，E，F，G，I，K，Q，R，S或V，X 29可以是D，G，H，K，L，P，Q，R，S，T，V或Y，X 30可以是A，F，N，Q，R，S或T，X 51可以是I或L，X 54可以是A，N，R或S，X 55可以是A，F，W或Y，X 56可以是A，G或S，X 58可以是E，Q，S或T，X 100可以是I或L，X 102可以是H，R或V，X 103可以是A，D，G，H，L，N，R，S，T或V。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含SEQ ID NO：143所示的氨基酸序列。
QVQLVESGGGSVQVGGSLTLSCAASX 26X 27X 28X 29X 30IYAMGWFRQAPGKEHEFVAGIGWX 54X 55X 56TX 58YYADSVKGRFGISRDNTKNTVALLMNSLKPEDTAIYYCAADX 100DX 102X 103GFDYWGQGTQVTVSS(SEQ ID NO：143)，其中，X 26可以是G，H或K，X 27可以是A，G，L，N，S或V，X 28可以是D，R或V，X 29可以是G，H，P，Q，R或T，X 30可以是R，S或T，X 54可以是A或N，X 55可以是F或Y，X 56可以是A或S，X 58可以是E，Q或T，X 100可以是I或L，X 102可以是H或R，X 103可以是A，H或S。
在本申请中，所述重链可变区可包含SEQ ID NO：72-120和/或163-227中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:1所示的氨基酸序列；所述HCDR2 可包含SEQ ID NO:2所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:72所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:1所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:42所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:55所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-1或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:73所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-1或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-1具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:4所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:43所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-2或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:74所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-2或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-2 具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:44所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-3或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:75所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-3或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-3具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:6所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:45所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-4或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:76所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-4或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-4具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:6所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:45所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:147所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-4 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合 蛋白的重链可变区可包含SEQ ID NO:182所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-4 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-4 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:7所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-5或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:77所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-5或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:8所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:47所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-6或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:78所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-6或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-6具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:9所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO: 69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-7或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:79所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-7或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-7具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:9所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:148所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-7 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:185所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-7 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-7 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:10所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:45所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-8或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:80所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-8或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-8具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3 以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:10所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:45所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:147所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-8 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:186所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-8 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-8 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:11所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:44所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-9或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:81所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-9或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-9具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:12所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:43所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-10或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:82所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-10或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗 原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-10具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:13所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:48所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-11或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:83所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-11或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-11具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:14所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:45所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-12或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:84所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-12或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-12具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:15所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:47所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体 MHPD1-A-13或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:85所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-13或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-13具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:15所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:47所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:147所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-13 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:191所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-13 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-13 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:16所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:43所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-14或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:86所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-14或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-14具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:17所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:49所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸 序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-15或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:87所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-15或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-15具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:17所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:49所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:148所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-15 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:193所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-15 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-15 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-16或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:88所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-16或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-16具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:19所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-17或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:89所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-17或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-17具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:20所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:50所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:56所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-18或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:90所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-18或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-18具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:21所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:57所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-19或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:91所示的氨基酸序列。例如，所述抗原结合蛋白可包 括单域抗体MHPD1-A-19或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-19具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:22所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-20或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:92所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-20或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-20具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:9所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:51所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-21或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:93所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-21或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-21具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:9所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:58所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4 可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-22或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:94所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-22或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-22具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:23所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-23或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:95所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-23或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-23具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:24所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:59所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-24或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:96所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-24或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-24具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:24所示的氨基酸序列；所述HCDR2 可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:59所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:148所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-24 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:202所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-24 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-24 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:25所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:60所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-25或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:97所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-25或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-25具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:25所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:60所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:148所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-25 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:203所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-25 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述 MHPD1-A-25 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:26所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:61所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-26或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:98所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-26或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-26具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:27所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-27或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:99所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-27或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-27具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:28所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:52所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-28或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合 蛋白的重链可变区可包含SEQ ID NO:100所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-28或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-28具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:29所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-29或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:101所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-29或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-29具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:25所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:52所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:62所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-30或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:102所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-30或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-30具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:25所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:52所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:62所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:148所示的氨基酸序列；所述H-FR2可包含SEQ ID NO: 150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-30 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:208所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-30 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-30 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:9所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:50所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:63所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-31或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:103所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-31或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-31具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:30所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-32或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:104所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-32或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-32具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3 以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:30所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:148所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-32 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:210所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-32 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-32 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:31所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-33或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:105所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-33或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-33具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:32所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:53所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-35或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:106所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-35或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗 原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-35具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:33所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:52所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-36或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:107所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-36或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-36具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:34所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:64所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-37或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:108所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-37或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-37具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:35所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:57所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体 MHPD1-A-38或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:109所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-38或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-38具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:9所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:54所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-39或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:110所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-39或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-39具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:9所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:54所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:148所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-39 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:217所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-39 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-39 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:9所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:65所示的氨基酸 序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-40或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:111所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-40或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-40具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:36所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-41或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:112所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-41或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-41具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:37所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:52所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-42或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:113所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-42或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-42具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:37所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:52所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:148所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-42 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:223所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-42 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-42 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:9所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:59所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-43或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:114所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-43或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-43具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:38所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:54所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-44或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:115所示的氨基酸序列。例如，所述抗原结合蛋白可包 括单域抗体MHPD1-A-44或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-44具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:38所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:54所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:148所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-44 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:222所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-44 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-44 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:39所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-45或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:116所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-45或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-45具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:39所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:148所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:150所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:161所示的氨基酸序列；所述H-FR4 可包含SEQ ID NO:162所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-45 H或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:223所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-45 H或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-45 H具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:40所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:55所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-46或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:117所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-46或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-46具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:41所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:3所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-47或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:118所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-47或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-47具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:9所示的氨基酸序列；所述HCDR2 可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:66所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-48或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:119所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-48或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-48具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述抗原结合蛋白可包含重链可变区，所述重链可变区可包含HCDR1-3以及H-FR1-4。例如，所述HCDR1可包含SEQ ID NO:9所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:46所示的氨基酸序列；所述HCDR3可包含SEQ ID NO:67所示的氨基酸序列；所述H-FR1可包含SEQ ID NO:68所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:69所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:70所示的氨基酸序列；所述H-FR4可包含SEQ ID NO:71所示的氨基酸序列；例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-49或与其具有相同HCDR1-3和H-FR1-4的抗原结合蛋白。例如，所述抗原结合蛋白的重链可变区可包含SEQ ID NO:120所示的氨基酸序列。例如，所述抗原结合蛋白可包括单域抗体MHPD1-A-49或与其具有相同HCDR3(例如，与其具有相同的HCDR1-3)的抗原结合蛋白(例如，单域抗体)。在某些情况下，所述抗原结合蛋白可包括与所述MHPD1-A-49具有相同VH的抗原结合蛋白(例如，单域抗体)。
在本申请中，所述分离的抗原结合蛋白还包含免疫球蛋白Fc区。例如，本申请提供了一种多肽，其包含本申请的抗原结合蛋白和免疫球蛋白的Fc区。
在本申请中，所述抗原结合蛋白的所述VH与所述Fc区直接或间接地连接。在本申请中，所述抗原结合蛋白的所述VH的C端与所述Fc区的N端直接或间接地连接。在本申请中，所述抗原结合蛋白的所述VH与所述Fc区在框内融合。
在本申请中，所述抗原结合蛋白的所述VH通过连接子与所述Fc区连接。例如，所述连接子包括肽连接子。例如，所述连接子包括柔性连接子。
在本申请中，所述分离的抗原结合蛋白从N端到C端依次包含所述抗原结合蛋白的所述VH，所述连接子和所述Fc区。
在本申请中，所述Fc区包括源自IgG1的Fc或源自IgG4的Fc。例如，所述Fc区包含 SEQ ID NO：121-125中任一项所示的氨基酸序列。
多肽和免疫缀合物
另一方面，本申请提供了一种或多种多肽，其可包含本申请的分离的抗原结合蛋白。例如，所述多肽可以包括融合蛋白。
另一方面，本申请提供了一种或多种免疫缀合物，所述免疫缀合物可包含本申请的分离的抗原结合蛋白。在某些实施方式中，所述免疫缀合物还可包含药学上可接受的治疗剂。
核酸、载体、细胞和药物组合物
另一方面，本申请还提供了分离的一种或多种核酸分子，所述一种或多种核酸分子可编码本申请所述分离的抗原结合蛋白。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述抗原结合蛋白，也可以编码其中的一部分(例如，HCDR1-3、重链可变区中的一种或多种)。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。例如，所述分离的核酸可以是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述分离的抗原结合蛋白的核酸，这些方法包括但不限于，采用逆转录PCR和PCR获得本申请所述分离的抗原结合蛋白的核酸分子。
另一方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷 酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。
所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体可为表达载体。例如，所述载体可为病毒载体。可以将病毒载体直接给予至患者(体内)或可以通过间接的形式，例如，在体外使用病毒处理细胞，然后将处理过的细胞给予至患者(离体)。病毒载体技术在本领域中是公知的，并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。常规的基于病毒的系统可以包括用于基因转移的逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关病毒载体以及单纯疱疹病毒载体。在某些情形中，可以用逆转录病毒、慢病毒和腺相关病毒的方法将基因转移整合进宿主基因组中，使插入的基因长期表达。慢病毒载体是能够转导或感染非分裂细胞并典型地产生较高病毒效价的逆转录病毒载体。慢病毒载体可包含长末端重复序列5’LTR和截短的3’LTR、RRE、rev应答元件(cPPT)、中央终止序列(CTS)和/或翻译后调控元件(WPRE)。本申请所述的载体可以被引入细胞。
另一方面，本申请提供了一种细胞。所述细胞可包含本申请所述的分离的抗原结合蛋白、所述的多肽、所述的免疫缀合物、一种或多种核酸分子和/或本申请所述的一种或多种载体。例如，每种或每个细胞可包含一个或一种本申请所述的核酸分子或载体。例如，每种或每个细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如原核细胞(例如，细菌细胞)、CHO细胞、NS/0细胞、HEK293T细胞、293F细胞或HEK293A细胞，或者其他真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。例如，所述细胞可以包括酵母细胞。例如，所述细胞可以包括大肠杆菌细胞。例如，所述细胞可以包括哺乳动物细胞。例如，所述细胞可以包括免疫细胞。
所述细胞可以包括免疫细胞。在某些情形中，所述细胞可以包括免疫细胞。例如，所述细胞可包括T细胞、B细胞、天然杀伤(NK)细胞、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
另一方面，本申请提供了一种药物组合物。所述药物组合物可包含本申请所述的分离的抗原结合蛋白、所述的多肽、所述的免疫缀合物、所述分离的核酸分子、所述的载体、所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。在本申请中，所述药学上可接受的佐剂可以 包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂。除非与本申请所述的细胞不相容，否则任何常规介质或试剂均可以考虑用于本申请的药物组合物中。在本申请中，所述药学上可接受的赋形剂可以包括在药物制剂中除主药以外的附加物，也可称为辅料。例如，所述赋形剂可以包括片剂中的粘合剂、填充剂、崩解剂、润滑剂。例如，所述赋形剂可以包括中药丸剂中的酒、醋、药汁等。例如，所述赋形剂可以包括半固体制剂软膏剂、霜剂中的基质部分。例如，所述赋形剂可以包括液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂。
检测方法、试剂盒、用途和方法
另一方面，本申请提供了一种用于检测PD-1的存在和/或含量的方法，其可包括施用所述分离的抗原结合蛋白或所述的多肽。
在本申请中，所述方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。
例如，所述方法可包括用于非诊断目的的检测PD-1的存在和/或含量的方法，其可包括下述步骤：
1)使样品与本申请的抗原结合蛋白接触；以及
2)检测样品结合的所述抗原结合蛋白的存在和/或含量来确定获自受试者的样品中PD-1的存在和/或表达水平。
另一方面，本申请提供了一种试剂盒，其可包含所述分离的抗原结合蛋白或所述的多肽。
在本申请中，所述试剂盒还可包含使用说明，所述使用说明记载用于检测PD-1的存在和/或含量的方法。例如，所述方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。
另一方面，本申请提供了一种所述的分离的抗原结合蛋白或所述的多肽在制备试剂盒中的用途，所述试剂盒可用于检测PD-1的存在和/或含量的方法。例如，所述方法可包括体外方法，离体方法，非诊断或非治疗目的的方法。
另一方面，本申请提供了一种所述分离的抗原结合蛋白和/或所述的多肽在制备药物中的用途，所述药物可用于预防和/或治疗疾病或病症。
另一方面，本申请提供了一种所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述的分离的核酸分子，所述的载体，所述的细胞和/或所述的药物组合物，其用于预防、缓解和/或治疗疾病或病症。
例如，所述疾病或病症可包括肿瘤。例如，所述肿瘤可包括实体瘤。例如，所述肿瘤可包括血液瘤。例如，所述肿瘤可包括与PD-L1的表达相关的肿瘤。例如，所述重链可包括与 PD-L1的表达上调相关的肿瘤。例如，所述肿瘤可选自下组：黑色素瘤，肺癌，头颈鳞状细胞癌，淋巴瘤，肝细胞癌，肾细胞癌，尿路上皮癌，结直肠癌和乳腺癌。
另一方面，本申请提供了一种预防和/或治疗疾病或病症的方法，其可包括向有需要的受试者施用有效量的所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述的分离的核酸分子，所述的载体，和/或所述的细胞。
例如，所述疾病或病症可包括肿瘤。例如，所述肿瘤可包括实体瘤。例如，所述肿瘤可包括血液瘤。例如，所述肿瘤可包括与PD-L1的表达相关的肿瘤。例如，所述重链可包括与PD-L1的表达上调相关的肿瘤。例如，所述肿瘤可选自下组：黑色素瘤，肺癌，头颈鳞状细胞癌，淋巴瘤，肝细胞癌，肾细胞癌，尿路上皮癌，结直肠癌和乳腺癌。
另一方面，本申请提供了一种抑制PD-1与PD-L1相互作用的方法，其包括向有需要的受试者施用有效量的所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述的分离的核酸分子，所述的载体，和/或所述的细胞。所述方法可以是离体或体外方法。在本申请中，所述方法可包括使生物样品与本申请所述的抗原结合蛋白和/或PD-L1在容许所述抗原结合蛋白和/或PD-L1结合PD-1的条件下接触，检测在所述抗原结合蛋白与PD-1之间是否形成复合物，和检测PD-1与PD-L1之间是否形成复合物。
另一方面，本申请提供了所述分离的抗原结合蛋白，所述的多肽，所述的免疫缀合物，所述的分离的核酸分子，所述的载体，所述的细胞和/或所述的药物组合物，其用于抑制PD-1与PD-L1的相互作用。
另一方面，本申请提供了一种所述的分离的抗原结合蛋白和/或所述的多肽在制备药物中的用途，所述药物用于抑制PD-1与PD-L1的相互作用。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的抗原结合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1羊驼免疫
采用重组人PD-1蛋白免疫一只健康的雌性成年羊驼(Alpaca)。首次免疫将1mg重组人PD-1蛋白与等体积弗氏完全佐剂乳化后皮下多点注射；加强免疫采用0.5mg重组人PD-1蛋白与等体积弗氏不完全佐剂乳化后皮下多点注射，共进行3次加强免疫。
实施例2羊驼免疫库构建
2.1免疫结束后，收集羊驼外周血，采用淋巴细胞分离液(Solarbio，Cat.P8900)分离得 到淋巴细胞；采用TRIzol TM Reagent(Thermo Fisher Scientific，Cat.15596018)提取总RNA。
2.2采用PrimeScript TM II 1st Strand cDNA Synthesis Kit(TAKARA,Code No.6210B)反转录得到cDNA，巢式PCR扩增VHH基因。
2.3采用胶纯化试剂盒(TAKARA,Code No.9761)回收VHH基因片段后采用限制性内切酶sfil(TAKARA,Code No.1244A)酶切消化，随后将其克隆至噬菌粒载体pComb 3XSS，将构建好的克隆产物转化至E.coli TG1电转化感受态细胞，得到PD-1单域抗体文库。
2.4采用平板梯度稀释法测定库容量为3.2×10 8，菌落PCR结果显示文库克隆率为98％。取适量文库菌液(菌体数量约3.2×10 9)接种于100mL LB培养基(含氨苄青霉素)中培养至对数中期，加入适量的辅助噬箘体M13KO7感染60分钟后，将培养基置换为200mL的2×YT(含氨苄青霉素和卡那霉素)，30℃剧烈震荡培养12小时，离心获取上清液，加入适量的20％PEG-NaCl溶液，使噬箘体沉淀，采用适量的PBS重悬沉淀，即得到噬箘体展示文库，可直接用于后续筛选。
实施例3纳米抗体筛选
采用固相亲和筛选法筛选PD-1纳米抗体，共进行三轮筛选：
3.1包被：采用重组人PD-1蛋白包被微孔板，第一轮包被浓度为100μg/mL，第二轮包被浓度为50μg/mL，第三轮包被浓度为20μg/mL，4℃过夜包被。
3.2封闭：第一轮和第三轮采用3％卵清白蛋白封闭，第二轮采用3％牛血清白蛋白封闭，封闭条件为37℃1小时.
3.3结合与洗涤：第一轮筛选，除去封闭液后，向微孔板中加入100μL实施例2中构建的纳米抗体噬菌体展示文库，37℃孵育结合1小时后，除去未结合的文库，分别采用PBST和PBS洗涤微孔板(各5次)以去除不结合或结合力较低的噬菌体；第二轮PBST和PBS各洗涤10次，第三轮PBST和PBS各洗涤20次。
3.4特异性噬菌体的洗脱：向微孔板中加入100μL甘氨酸盐酸盐缓冲液(pH 2.2)以使特异性噬菌体解离，轻微震荡10分钟后，收集洗脱液。
3.5洗脱物滴度测定及扩增：将洗脱物感染处于对数生长期的E.coli TG1，取少量感染后的E.coli TG1梯度稀释，取适量梯度稀释液均匀涂布LB固体培养基(平板稀释法)，12-14小时后平板计数，计算噬箘体洗脱量；剩余的扩增获得特异性噬菌体富集文库，用于下一轮筛选。
3.6鉴定：从上述第二轮和第三轮测定洗脱物滴度的平板上各随机挑取93个克隆，扩增得到噬菌体，采用ELISA鉴定。方法为，包被重组人PD-1蛋白(包被浓度2μg/mL)，采用5％ 脱脂乳封闭后加入待鉴定噬菌体，37℃结合1小时，洗涤后加入Goat anti-Llama IgG(H+L)Secondary Antibody[HRP]，37℃结合1小时，洗涤后加入TMB底物液显色，采用酶标仪读取OD 450。设置阳性对照孔(每一轮的富集文库)、阴性对照(辅助噬箘体M13KO7)和空白孔(PBS)。待测孔OD值大于阴性对照3倍以上则判定为阳性。阳性孔所对应的克隆保菌、提质粒、测序，将DNA序列翻译成氨基酸序列后比对，把HCDR1、HCDR2、HCDR3序列均相同的克隆视为同一抗体株，而HCDR序列不同的克隆视为不同抗体株。
实施例4表达和纯化VHH
经实施例3筛选出的阳性克隆转化至Top10F’中，IPTG诱导VHH表达。离心收集细胞沉淀、超声破碎后收集上清，采用
Figure PCTCN2022074550-appb-000001
Metal Affinity Resins纯化并将缓冲液置换为磷酸缓冲液。通过测量OD280值，对纯化后的VHH进行定量，并保存于4℃备用。其中筛选出对抗原(人重组PD-1)有较高亲和力且可与人重组PD-L1竞争结合人重组PD-1的VHH的SDS PAGE如图1所示，得到的VHH命名为MHPD1-A。
实施例5 VHH与重组人PD-L1对抗原蛋白的竞争结合ELISA实验
在96孔板中加入重组人PD-1抗原(2μg/mL)，4℃包被过夜。采用1％酪蛋白封闭。将MHPD1-A样品采用0.1％BSA/PBS进行2倍梯度稀释，起始浓度20μg/mL。向抗原(重组人PD-1蛋白)包被的96孔板中加入4μg/mL重组人PD-L1-Fc及不同浓度的VHH，室温孵育1小时。不同孵育间采用PBST洗涤孔板。采用辣根过氧化物酶标记的抗人IgG二抗(1:5000，含1％酪蛋白)进行检测，采用Graphpad Prism软件拟合曲线并计算EC 50值，结果如表1所示。结果显示，MHPD1-A可有效抑制人重组PD-L1与人重组PD-1结合。
表1 VHH对抗原蛋白的竞争抑制结果
抗体名称 EC 50(μg/mL)
MHPD1-A 1.71
实施例6表达和纯化融合蛋白
合成包含编码MHPD1-A及人Fc段(VHH的C端与人Fc铰链区N端直接相连)的核酸序列的pTT5重组载体，采用以下步骤准备脂质体-DNA复合物：将15μg质粒DNA采用
Figure PCTCN2022074550-appb-000002
I稀释至总体积0.5mL；将30μL 293fectin TM试剂采用
Figure PCTCN2022074550-appb-000003
I稀释至总体积0.5mL；轻柔混合，室温孵育5分钟。将稀释的pTT5重组载体加入稀释的293fectin TM试剂中，轻柔混匀，室温孵育30分钟。将上述混合物加入含有1×10 6cells/mL细胞的摇瓶中， 置于37℃、含8％CO 2的细胞培养箱内以125rpm旋转的轨道振荡器上孵育。转染后第4天，收获上清液，采用Pierce TM的Protein A琼脂糖凝胶纯化，并将缓冲液置换为磷酸缓冲液，得到的融合蛋白命名为MHPD1-A Fc。
实施例7融合蛋白与人、猴及小鼠PD-1的种属交叉验证
96孔Greiner板分别包被2μg/mL的重组人PD-1-his蛋白、重组猴PD-1-his蛋白和重组小鼠PD-1-his蛋白于4℃过夜。第二天用1％酪蛋白封闭后，将5μg/mL和1.67μg/mL纳米抗体-Fc融合蛋白样品分别加入抗原包被板中，孵育2小时。然后用PBST清洗平板，加入抗人IgG-HRP(1％酪蛋白中1:5000稀释)孵育1小时，进行结合检测。如图2所示，MHPD1-A Fc可与重组人PD-1-his和重组猴PD-1-his结合，与重组小鼠PD-1-his无结合活性。
实施例8融合蛋白对重组人PD-1的亲和力检测
使用胺偶联试剂盒(GE Healthcare,Cat No.BR100050)，按照标准胺偶联步骤在25℃下将约250RU的重组人PD-1-his蛋白偶联于CM5芯片上。在25℃下以30μL/min的流速依次注入2倍系列稀释的MHPD1-A Fc样品(检测5个浓度)，结合时间为120s，解离时间为600s。使用
Figure PCTCN2022074550-appb-000004
分析软件(BIAevaluation)，采用1:1 Langmuir结合模型对曲线进行拟合，以确定其结合速率常数、解离速率常数及结合解离常数等动力学常数。
结果显示，MHPD1-A Fc与重组人PD-1有较高亲和力，MHPD1-A Fc与重组人PD1-his结合的动力学常数见表2。
表2.纳米抗体-Fc融合蛋白与hPD-1-his结合的动力学常数
分析物 ka(1/Ms) kd(1/s) Rmax(RU) KD Chi 2(RU 2)
MHPD1-A Fc 4.60E+05 1.95E-05 85.6 4.25E-11 1.57E-01
实施例9融合蛋白与重组人PD-L1对重组人PD-1的竞争结合ELISA实验
在96孔板中加入重组人PD-L1(C-Fc，0.2μg/孔)，4℃包被过夜。采用1％酪蛋白封闭。将MHPD1-A Fc采用0.1％BSA/PBS进行2倍梯度稀释，起始浓度10μg/mL。向抗原包被的96孔板中加入0.5μg/mL重组人PD-1(C-Fc&C-Avi&C-6×His)及不同浓度的MHPD1-A Fc，室温孵育1小时。不同孵育间采用PBST洗涤孔板。采用辣根过氧化物酶标记的链霉亲和素(1:10000)进行检测，采用Graphpad Prism软件拟合曲线并计算EC 50值，结果如表3所示。
结果显示，受试的MHPD1-A Fc可竞争性抑制重组人PD-L1与重组人PD-1结合，且活 性与阳性对照Nivolumab相当或优于阳性对照。
表3融合蛋白竞争性结合活性
抗体名称 EC 50(μg/mL)
MHPD1-A Fc 0.499
Nivolumab 0.593
实施例10融合蛋白对过表达人PD-1的HEK293细胞的结合活性
在含有10％FBS的DMEM培养基中于CO 2培养箱中(37℃，5％CO2)培养过表达人PD-1的HEK293细胞。收获细胞，并通过测试缓冲液洗涤细胞一次。进行细胞计数和活力检测，向各试管中加入3×10 5活细胞，并分别加入100μL不同浓度的MHPD1-A Fc(3000ng/mL、1000ng/mL、300ng/mL、100ng/mL、30ng/mL、10ng/mL、3ng/mL、1ng/mL和0.3ng/mL)，在4℃孵育1小时。用洗涤缓冲液洗涤细胞后，按照说明加入100μL PE-抗人IgG，在4℃下避光孵育1小时。用洗涤缓冲液洗涤细胞3次，将各样品重悬于200μL PBS中，采用流式细胞仪检测。使用FCS Express 6Plus和GraphPad Prism 5软件分析数据。
结果显示，MHPD1-A Fc可与过表达人PD-1的HEK293细胞结合，MHPD1-A Fc与过表达人PD-1的HEK293细胞结合的EC 50如表4所示。
表4融合蛋白与过表达人PD-1的HEK293细胞的结合活性
融合蛋白 EC 50(μg/mL)
MHPD1-A Fc 0.2448
实施例11融合蛋白与人PD-L1竞争结合过表达人PD-1的HEK293细胞
在CO 2培养箱中(37℃，5％CO 2)，用含有10％FBS和1mg/mL G418的的DMEM培养基培养过表达人PD-1的HEK293细胞。收获细胞，并通过测试缓冲液洗涤细胞一次。进行细胞计数和活力检测，向各试管中加入3×10 5活细胞(细胞活力≥95％)。将1.0μg/mL生物素化的人PD-L1蛋白和系列稀释的MHPD1-A Fc(浓度为0.03μg/mL至10μg/mL)分别在室温下混合并孵育30分钟，将混合物分别加入含有3×10 5活细胞的离心管中，在4℃孵育1小时。用洗涤缓冲液洗涤细胞后，按照说明加入100μL PE-SA，在4℃下孵育1小时。用洗涤缓冲液洗涤细胞3次，将各样品重悬于100μL PBS中，采用流式细胞仪检测。使用FCS Express 6 Plus和GraphPad Prism 5软件分析数据。
结果显示，MHPD1-A Fc可竞争性抑制重组人PD-L1与过表达hPD-1的HEK293细胞结 合，且MHPD1-A Fc的竞争性结合活性略优于阳性对照Nivolumab。MHPD1-A Fc与人PD-L1竞争结合HEK293/hPD1的EC 50值如表5所示。
表5融合蛋白与人PD-L1竞争结合过表达人PD-1的HEK293细胞
融合蛋白 EC 50(μg/mL)
MHPD1-A Fc 0.549
Nivolumab 0.580
实施例12融合蛋白与帕博利珠单抗(Pembrolizumab)对抗原蛋白的亲和动力学检测
采用
Figure PCTCN2022074550-appb-000005
8K(GE Healthcare)进行亲和动力学检测：使用胺偶联试剂盒(GE Healthcare,Cat No.BR100050)，按照标准胺偶联步骤在25℃下将重组人PD-1-his蛋白偶联于CM5芯片(GE Healthcare,Cat No.BR100530)上。一项实验中，在25℃下以30μL/min的流速依次注入Pembrolizumab(100nM)和MHPD1-A Fc(2μM)、或依次注入Pembrolizumab(100nM)和缓冲液、或依次注入缓冲液和MHPD1-A Fc(2μM)，每个样品结合时间为300s，亲和动力学曲线见图3A。另一实验中，在25℃下以30μL/min的流速依次注入MHPD1-A Fc(2μM)和Pembrolizumab(100nM)、或依次注入MHPD1-A Fc(2μM)和缓冲液、或依次注入缓冲液和Pembrolizumab(100nM)，每个样品结合时间为300s，亲和动力学曲线见图3B。综合上述检测结果，推测MHPD1-A Fc和Pembrolizumab与PD1-His蛋白结合的表位相同或者重叠。
实施例13构建VHH变体
通过对MHPD1-A的序列进行数据库序列分析，基于结构预测及经验对HCDR1-3的部分氨基酸位点进行突变，设计一系列VHH变体序列。根据所设计的VHH变体序列分别进行全基因合成(His标签)，对pcDNA3.4质粒进行NotI/XbaI酶切(NEB,Cat.R0189L、R0145L)、并与VHH变体基因序列重组，将重组质粒转化TOP10感受态并进行菌液涂布，37℃培养16-20h。挑选平板中的单菌落进行菌落PCR、电泳鉴定阳性克隆，选择阳性克隆测序，并对测序正确的克隆进行扩大培养抽提质粒。采用脂质体转染试剂对HEK293细胞进行瞬转，置于37℃、含5％CO2的细胞培养箱内，并在以125rpm旋转的轨道振荡器上孵育。待第5天离心收集上清，经Ni亲和层析柱纯化((Ni Smart Beads 6FF,GE#17531802)，并经透析将缓冲液置换为磷酸缓冲液，得到一系列VHH变体，分别命名为MHPD1-A-4、MHPD1-A-7、MHPD1-A-8、MHPD1-A-13、MHPD1-A-15、MHPD1-A-24、MHPD1-A-25、MHPD1-A-30、 MHPD1-A-32、MHPD1-A-39、MHPD1-A-42、MHPD1-A-44和MHPD1-A-45。
实施例14 VHH变体与重组人PD-L1对抗原蛋白的竞争结合ELISA实验
在96孔板中加入重组人PD-1蛋白(2μg/mL)，4℃包被过夜。采用1％酪蛋白封闭。将VHH变体采用0.1％BSA/PBS进行2倍梯度稀释，起始浓度20μg/mL。向抗原(重组人PD-1蛋白)包被的96孔板中加入4μg/mL重组人PD-L1-Fc及不同浓度的VHH变体，室温孵育1小时。不同孵育间采用PBST洗涤孔板。采用辣根过氧化物酶标记的抗人IgG二抗(1:5000，含1％酪蛋白)进行检测。经分析后，结果显示，VHH变体可有效抑制重组人PD-L1与重组人PD-1结合。
实施例15构建包含VHH变体的融合蛋白
选择亲和力较高的VHH变体，将其基因序列与Fc序列合成全长序列，对pcDNA3.4质粒进行NotI/XbaI酶切(NEB,Cat.R0189L、R0145L)、并与上述全长序列重组，将重组质粒转化TOP10感受态并进行菌液涂布，37℃培养16-20h。挑选平板中的单菌落进行菌落PCR、电泳鉴定阳性克隆，挑选阳性克隆测序并对测序正确的克隆扩大培养抽提质粒。采用脂质体转染试剂对HEK293细胞进行瞬转，置于37℃、含5％CO2的细胞培养箱内，并在以125rpm旋转的轨道振荡器上孵育。待第5天离心收集上清，经Protein A亲和层析柱纯化，并经透析将缓冲液置换为磷酸缓冲液，得到包含VHH变体的融合蛋白。
实施例16包含VHH变体的融合蛋白与重组人PD-L1对抗原蛋白的竞争结合ELISA实验
在96孔板中加入重组人PD-L1(C-Fc，0.2μg/孔)，4℃包被过夜。采用1％酪蛋白封闭。将VHH变体融合蛋白采用0.1％BSA/PBS进行2倍梯度稀释，起始浓度10μg/mL。向抗原包被的96孔板中加入0.5μg/mL重组人PD-1(C-Fc&C-Avi&C-6×His)及不同浓度的VHH变体融合蛋白，室温孵育1小时。不同孵育间采用PBST洗涤孔板。采用辣根过氧化物酶标记的链霉亲和素(1:10000)进行检测。经分析后，结果显示，VHH变体融合蛋白可有效抑制重组人PD-L1与重组人PD-1结合。
实施例17纳米抗体人源化及人源化的纳米抗体-Fc融合蛋白与抗原蛋白的结合活性检测
通过对原始驼源抗体MHPD1-A的序列进行数据库序列分析，明确参考的人源序列Germeline，进行人源化序列的设计，第一轮人源化后人源化程度从86.44％到90.68％不等， 将其构建成纳米抗体-Fc融合蛋白，在ExpiCHO-s中进行瞬转表达，根据人源化程度及亲和力结进行第二轮人源化。第二轮人源化后人源化程度均大于90％，将其构建成纳米抗体-Fc融合蛋白，在ExpiCHO-s中进行瞬转表达并验证其结合活性。
采用ELISA方法分别验证了两轮人源化后，不同人源化程度的纳米抗体-Fc融合蛋白与抗原蛋白(人PD-1)的结合活性。在96孔酶标板中，分别加入2μg/mL的重组人PD-1-his抗原蛋白，30μL/孔，4℃包被过夜。PBST洗板3次。加入5％PBS-Milk，室温孵育2h。PBST洗板3次。所有纳米抗体-Fc融合蛋白均以10μg/mL为初始浓度，进行3倍梯度稀释；30μL/孔加入到各孔中，室温孵育1h。PBST洗板3次。1：8000稀释二抗Goat-Anti-Human-IgG-Fc-HRP(购自Sigma)，30μL/孔加入到各孔中，室温孵育50min。PBST洗板3次。加TMB(购自SurModics)到各孔中，30μL/孔，室温，避光显色1min-5min。加2M盐酸，30μL/孔，终止反应。采用酶标仪，读取OD450吸光值。采用Graphpad Prism软件拟合曲线并计算EC 50值，如表6所示。结果显示，经人源化后的纳米抗体-Fc融合蛋白仍可与抗原蛋白有效结合，且部分人源化纳米抗体-Fc融合蛋白与抗原蛋白的亲和力与母本抗体相当或优于母本抗体。
表6人源化纳米抗体-Fc融合蛋白与抗原蛋白人PD-1结合活性
Figure PCTCN2022074550-appb-000006
实施例18人源化纳米抗体的亲和力成熟及纳米抗体-Fc融合蛋白与抗原的结合活性检测(第一轮)
将母本抗体MHPD1-A-H19的VHH克隆至乙方噬菌体展示载体；设计一系列引物分别对其HCDR进行单点或者连续三点突变进行亲和力成熟噬菌体展示文库的构建。采用免疫管 筛选(即固相筛选)，使用抗原蛋白hPD-1-His包被免疫管，加入亲和力成熟噬菌体展示文库进行孵育、洗涤和洗脱，经历2-3轮的淘选，以富集高亲和力的特异性单克隆抗体。经过亲和力成熟筛选后，在ELISA水平筛选对hPD-1-His抗原亲和力较高的候选抗体，并根据VHH水平的亲和力和序列，从中选择了14个候选分子进行纳米抗体-Fc融合蛋白构建。
采用ELISA方法验证经亲和力成熟后的纳米抗体-Fc融合蛋白与抗原蛋白(hPD-1-his)的结合活性：在96孔酶标板中，分别加入2μg/mL的重组人PD-1-his抗原蛋白，30μL/孔，4℃包被过夜。PBST洗板3次。5％PBS-Milk，室温孵育2h。PBST洗板3次。所有纳米抗体-Fc融合蛋白均以10μg/mL为初始浓度，进行3倍梯度稀释；30μL/孔加入到各孔中，室温孵育1h。PBST洗板3次。1：8000稀释二抗Goat-Anti-Human-IgG-Fc-HRP，30μL/孔加入到各孔中，室温孵育50min。PBST洗板9次。加TMB到各孔中，30μL/孔，室温，避光显色1min-5min。加2M盐酸，30μL/孔，终止反应。采用酶标仪，读取OD450吸光值。采用Graphpad Prism软件拟合曲线并计算EC 50值(见表7)。结果显示，经亲和力成熟后，多个纳米抗体-Fc融合蛋白与抗原蛋白人PD-1的结合活性优于母本MHPD1-A-H19 Fc。
表7亲和力成熟纳米抗体-Fc融合蛋白与抗原蛋白人PD-1结合活性
Figure PCTCN2022074550-appb-000007
实施例19亲和力成熟纳米抗体-Fc融合蛋白与重组人PD-L1对重组人PD-1的竞争结合ELISA实验
在96孔板中加入重组人PD-L1-mFc(8μg/mL)，4℃包被过夜。PBST洗板3次。5％PBS-Milk，室温孵育1小时。所有纳米抗体-Fc融合蛋白均以10μg/mL为初始浓度，进行3倍梯度稀释；30μL/孔加入到各孔中，室温孵育1小时。向抗原包被的96孔板中加入0.5μg/mL重组人PD-1-Fc-Biotin，30μL/孔，室温孵育1小时。不同孵育间采用PBST洗涤孔板。采用辣根过氧化物酶标记的链霉亲和素(1:10000)进行检测，采用Graphpad Prism软件拟合曲线 并计算IC 50值，如表8所示。结果显示，经亲和力成熟后，有多个纳米抗体-Fc融合蛋白对抗原蛋白人PD-1与配体人PD-L1结合的竞争抑制活性较亲和力成熟前提高，且优于阳性对照。
表8亲和力成熟纳米抗体-Fc融合蛋白与重组人PD-L1对人重组PD-1竞争抑制活性
抗体代号 IC 50(nM) 抗体代号 IC 50(nM)
MHPD1-A-H19 Fc 1.310 MHPD1-A-8 H Fc 0.323
MHPD1-A-1 H Fc 0.572 MHPD1-A-9 H Fc 0.256
MHPD1-A-2 H Fc 0.419 MHPD1-A-10 H Fc 0.287
MHPD1-A-3 H Fc 0.275 MHPD1-A-11 H Fc 0.365
MHPD1-A-4 H Fc 0.281 MHPD1-A-12 H Fc 0.293
MHPD1-A-5 H Fc 0.306 MHPD1-A-13 H Fc 0.395
MHPD1-A-6 H Fc 0.407 MHPD1-A-14 H Fc 0.852
MHPD1-A-7 H Fc 0.316 Pembrolizumab 0.410
Nivolumab 0.402    
实施例20纳米抗体-Fc融合蛋白生物活性检测
利用Jurkat-NFAT-PD1转基因细胞作为效应细胞，采用Hep3B-OS8-PDL1作为靶细胞，通过荧光素酶检测系统，检测纳米抗体-Fc融合蛋白阻断PD1/PDL1结合的生物活性。取Hep3B-OS8-PDL1细胞重悬于加入10％FBS的PRMI1640培养基中，接种于96孔细胞培养板中，置于细胞培养箱内培养过夜；第二天，移除细胞培养板中的培养基，每孔加入50uL预先配制的含有不同浓度纳米抗体-Fc融合蛋白的培养基及50uL Jurkat-NFAT-PD1细胞悬液，置于二氧化碳培养箱(37℃，5％CO 2)中孵育6小时。将细胞培养板取出置于室温平衡5-10分钟，每孔加入100uL荧光素酶报告基因检测试剂(One-Glo TM Luciferase assay system,Promega)，孵育至少3分钟后，采用酶标仪测定荧光信号(见表9)。结果显示，经亲和力成熟后的抗体A-3 H Fc、A-4 H Fc、A-7 H Fc可有效阻断PD-1、PD-L1结合对下游报告基因荧光信号的抑制，且生物活性与阳性对照Pembrolizumab相当或优于阳性对照。
表9亲和力成熟纳米抗体-Fc融合蛋白生物活性
抗体代号 EC 50(nM)
MHPD1-A-3 H Fc 0.078
MHPD1-A-4 H Fc 0.090
MHPD1-A-7 H Fc 0.070
Pembrolizumab 0.091
实施例21纳米抗体的亲和力成熟及纳米抗体-Fc融合蛋白与人PD-1结合、与人PD-L1对人PD-1竞争结合活性检测(第二轮)
将母本MHPD1-A-7 H的VHH克隆至乙方噬菌体展示载体；设计一系列引物分别对其HCDR进行单点或者连续三点突变进行亲和力成熟噬菌体展示文库的构建。采用免疫管筛选(即固相筛选)，使用抗原蛋白hPD-1-His包被免疫管，加入亲和力成熟噬菌体展示文库进行孵育、洗涤和洗脱，经历2-3轮的淘选，以富集高亲和力的特异性单克隆抗体。经过亲和力成熟筛选后，在ELISA水平筛选对hPD-1-His抗原亲和力较高的候选抗体，并根据VHH水平的亲和力和序列，从中选择了29个候选分子进行纳米抗体-Fc融合蛋白构建。
采用ELISA方法验证经亲和力成熟后的纳米抗体-Fc融合蛋白与抗原蛋白(hPD-1-his)的结合活性：在96孔酶标板中，分别加入2μg/mL的重组人PD-1-his抗原蛋白，30μL/孔，4℃包被过夜。PBST洗板3次。5％PBS-Milk，室温孵育2h。PBST洗板3次。所有纳米抗体-Fc融合蛋白均以10μg/mL为初始浓度，进行3倍梯度稀释；30μL/孔加入到各孔中，室温孵育1h。PBST洗板3次。1：8000稀释二抗Goat-Anti-Human-IgG-Fc-HRP，30μL/孔加入到各孔中，室温孵育50min。PBST洗板9次。加TMB到各孔中，30μL/孔，室温，避光显色1min-5min。加2M盐酸，30μL/孔，终止反应。采用酶标仪，读取OD450吸光值。采用Graphpad Prism软件拟合曲线并计算EC 50值，如表10所示，多个纳米抗体-Fc融合蛋白与抗原蛋白人PD-1的结合活性与母本MHPD1-A-7 H Fc相当。
表10亲和力成熟纳米抗体-Fc融合蛋白与抗原蛋白结合活性
抗体代号 EC 50(nM) 抗体代号 EC 50(nM)
MHPD1-A-7 H Fc 0.149 MHPD1-A-30 H Fc 0.166
MHPD1-A-15 H Fc 0.134 MHPD1-A-31 H Fc 0.219
MHPD1-A-16 H Fc 0.187 MHPD1-A-32 H Fc 0.156
MHPD1-A-17 H Fc 0.196 MHPD1-A-33 H Fc 0.628
MHPD1-A-18 H Fc 0.178 MHPD1-A-35 H Fc 0.824
MHPD1-A-19 H Fc 0.270 MHPD1-A-37 H Fc 0.141
MHPD1-A-20 H Fc 0.175 MHPD1-A-38 H Fc 0.421
MHPD1-A-21 H Fc 0.179 MHPD1-A-39 H Fc 0.158
MHPD1-A-22 H Fc 0.242 MHPD1-A-42 H Fc 0.193
MHPD1-A-23 H Fc 0.152 MHPD1-A-43 H Fc 0.387
MHPD1-A-24 H Fc 0.177 MHPD1-A-44 H Fc 0.198
MHPD1-A-25 H Fc 0.211 MHPD1-A-45 H Fc 0.146
MHPD1-A-27 H Fc 0.540 MHPD1-A-46 H Fc 0.252
MHPD1-A-28 H Fc 0.242 MHPD1-A-47 H Fc 0.238
MHPD1-A-29 H Fc 2.181 MHPD1-A-49 H Fc 0.171
选取部分与人PD-1亲和力较高的融合蛋白进行与重组PD-L1对人PD-1竞争结合活性检测。在96孔板中加入重组人PD-L1-mFc(8μg/mL)，4℃包被过夜。PBST洗板3次。5％PBS-Milk，室温孵育1小时。所有纳米抗体-Fc融合蛋白均以10μg/mL为初始浓度，进行3倍梯度稀释；30μL/孔加入到各孔中，室温孵育1小时。向抗原包被的96孔板中加入0.5μg/mL重组人PD-1-Fc-Biotin，30μL/孔，室温孵育1小时。不同孵育间采用PBST洗涤孔板。采用辣根过氧化物酶标记的链霉亲和素(1:10000)进行检测，采用Graphpad Prism软件拟合曲线并计算IC 50值，如表11所示。结果显示，经亲和力成熟后，MHPD1-A-15 H Fc对抗原蛋白与配体h-PD-L1结合的竞争抑制活性较亲和力成熟前提高，且有多个融合蛋白的竞争抑制活性优于阳性对照。
表11亲和力成熟纳米抗体-Fc融合蛋白与重组人PD-L1对人重组PD-1竞争抑制活性
抗体代号 IC 50(nM) 抗体代号 IC 50(nM)
MHPD1-A-7 H Fc 0.778 MHPD1-A-32 H Fc 1.718
MHPD1-A-15 H Fc 0.517 MHPD1-A-37 H Fc 1.544
MHPD1-A-20 H Fc 1.211 MHPD1-A-39 H Fc 0.850
MHPD1-A-21 H Fc 1.609 MHPD1-A-45 H Fc 0.944
MHPD1-A-23 H Fc 1.202 MHPD1-A-49 H Fc 1.262
MHPD1-A-30 H Fc 1.078 Pembrolizumab 1.248

Claims (80)

  1. 分离的抗原结合蛋白，其与参比抗体结合相同或重叠的PD-1表位。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其与所述参比抗体竞争结合PD-1。
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其对PD-1的结合亲和力与所述参比抗体对PD-1的结合亲和力基本相同。
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其具有下述性质中的一种或多种：
    1)在ELISA检测中，以约11μg/mL或更低的EC50与PD-1结合；
    2)在竞争ELISA检测中，以约2μg/mL或更低的EC50抑制人PD-L1与人PD-1结合。
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其能够结合源自灵长类动物的PD-1。
  6. 根据权利要求5所述的分离的抗原结合蛋白，其中所述灵长类动物包括人和/或猴。
  7. 根据权利要求1-6中任一项所述的分离的抗原结合蛋白，其中所述参比抗体包含HCDR3，所述HCDR3包含SEQ ID NO:128所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的分离的抗原结合蛋白，其中所述参比抗体包含HCDR2，所述HCDR2包含SEQ ID NO:127所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的分离的抗原结合蛋白，其中所述参比抗体包含HCDR1，所述HCDR1包含SEQ ID NO:126所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的分离的抗原结合蛋白，其中所述参比抗体包含重链可变区VH，所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:128所示的氨基酸序列；所述HCDR2包含SEQ ID NO:127所示的氨基酸序列；且所述HCDR1包含SEQ ID NO:126所示的氨基酸序列。
  11. 根据权利要求10所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:132所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的分离的抗原结合蛋白，其中所述参比抗体包含LCDR3，所述LCDR3包含SEQ ID NO:131所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的分离的抗原结合蛋白，其中所述参比抗体包含LCDR2，所述LCDR2包含SEQ ID NO:130所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的分离的抗原结合蛋白，其中所述参比抗体包含LCDR1，所述LCDR1包含SEQ ID NO:129所示的氨基酸序列。
  15. 根据权利要求1-14中任一项所述的分离的抗原结合蛋白，其中所述参比抗体包含轻链可变区VL，所述VL包含所述LCDR1、LCDR2和LCDR3，所述LCDR3包含SEQ ID NO:131所示的氨基酸序列；所述LCDR2包含SEQ ID NO:130所示的氨基酸序列；且所述 LCDR1包含SEQ ID NO:129所示的氨基酸序列。
  16. 根据权利要求15所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NO:133所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的分离的抗原结合蛋白，其中所述参比抗体包括帕博利珠单抗或其抗原结合片段。
  18. 根据权利要求1-17中任一项所述的分离的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:138所示的氨基酸序列。
  19. 根据权利要求18所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:142所示的氨基酸序列。
  20. 根据权利要求18-19中任一项所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:3、55、56、57、58、59、60、61、62、63、64、65、66和67中任一项所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的分离的抗原结合蛋白，其包含HCDR2，所述HCDR2包含SEQ ID NO：137所示的氨基酸序列。
  22. 根据权利要求21所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO：141
    所示的氨基酸序列。
  23. 根据权利要求21-22中任一项所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:2、42、43、44、45、46、47、48、49、50、51、52、53和54中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的分离的抗原结合蛋白，其包含HCDR1，所述HCDR1包含SEQ ID NO：136所示的氨基酸序列。
  25. 根据权利要求24所述的分离的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO：140所示的氨基酸序列。
  26. 根据权利要求24-25中任一项所述的分离的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO:1、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40和41中任一项所示的氨基酸序列。
  27. 根据权利要求1-26中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO：138所示的氨基酸序列；所述HCDR2包含SEQ ID NO：137所示的氨基酸序列；且所述HCDR1包含SEQ ID NO：136所示的氨基酸序列。
  28. 根据权利要求27所述的分离的抗原结合蛋白，其中所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO：142所示的氨基酸序列；所述HCDR2包含SEQ ID NO：141所示的氨基酸序列；且所述HCDR1包含SEQ ID NO：140所示的氨基酸序列。
  29. 根据权利要求27-28中任一项所述的分离的抗原结合蛋白，其中所述VH包含所述HCDR1、HCDR2和HCDR3，所述HCDR3包含SEQ ID NO:3、55、56、57、58、59、60、61、62、63、64、65、66和67中任一项所示的氨基酸序列；所述HCDR2包含SEQ ID NO:2、42、43、44、45、46、47、48、49、50、51、52、53和54中任一项所示的氨基酸序列；且所述HCDR1包含SEQ ID NO:1、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40和41中任一项所示的氨基酸序列。
  30. 根据权利要求27-29中任一项所述的分离的抗原结合蛋白，其中所述HCDR1、HCDR2和HCDR3包含选自下述的任意一组氨基酸序列：
    1)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，和HCDR3：SEQ ID NO:3；
    2)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:42，和HCDR3：SEQ ID NO:55；
    3)HCDR1：SEQ ID NO:4，HCDR2：SEQ ID NO:43，和HCDR3：SEQ ID NO:3；
    4)HCDR1：SEQ ID NO:5，HCDR2：SEQ ID NO:44，和HCDR3：SEQ ID NO:3；
    5)HCDR1：SEQ ID NO:6，HCDR2：SEQ ID NO:45，和HCDR3：SEQ ID NO:3；
    6)HCDR1：SEQ ID NO:7，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    7)HCDR1：SEQ ID NO:8，HCDR2：SEQ ID NO:47，和HCDR3：SEQ ID NO:3；
    8)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    9)HCDR1：SEQ ID NO:10，HCDR2：SEQ ID NO:45，和HCDR3：SEQ ID NO:3；
    10)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:44，和HCDR3：SEQ ID NO:3；
    11)HCDR1：SEQ ID NO:12，HCDR2：SEQ ID NO:43，和HCDR3：SEQ ID NO:3；
    12)HCDR1：SEQ ID NO:13，HCDR2：SEQ ID NO:48，和HCDR3：SEQ ID NO:3；
    13)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:45，和HCDR3：SEQ ID NO:3；
    14)HCDR1：SEQ ID NO:15，HCDR2：SEQ ID NO:47，和HCDR3：SEQ ID NO:3；
    15)HCDR1：SEQ ID NO:16，HCDR2：SEQ ID NO:43，和HCDR3：SEQ ID NO:3；
    16)HCDR1：SEQ ID NO:17，HCDR2：SEQ ID NO:49，和HCDR3：SEQ ID NO:3；
    17)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    18)HCDR1：SEQ ID NO:19，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    19)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:56；
    20)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:57；
    21)HCDR1：SEQ ID NO:22，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    22)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:51，和HCDR3：SEQ ID NO:3；
    23)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:58；
    24)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    25)HCDR1：SEQ ID NO:24，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:59；
    26)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:60；
    27)HCDR1：SEQ ID NO:26，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:61；
    28)HCDR1：SEQ ID NO:27，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    29)HCDR1：SEQ ID NO:28，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:3；
    30)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    31)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:62；
    32)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:63；
    33)HCDR1：SEQ ID NO:30，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    34)HCDR1：SEQ ID NO:31，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    35)HCDR1：SEQ ID NO:32，HCDR2：SEQ ID NO:53，和HCDR3：SEQ ID NO:3；
    36)HCDR1：SEQ ID NO:33，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:3；
    37)HCDR1：SEQ ID NO:34，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:64；
    38)HCDR1：SEQ ID NO:35，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:57；
    39)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:54，和HCDR3：SEQ ID NO:3；
    40)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:65；
    41)HCDR1：SEQ ID NO:36，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    42)HCDR1：SEQ ID NO:37，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:3；
    43)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:59；
    44)HCDR1：SEQ ID NO:38，HCDR2：SEQ ID NO:54，和HCDR3：SEQ ID NO:3；
    45)HCDR1：SEQ ID NO:39，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    46)HCDR1：SEQ ID NO:40，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:55；
    47)HCDR1：SEQ ID NO:41，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:3；
    48)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:66；
    49)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:46，和HCDR3：SEQ ID NO:67。
  31. 根据权利要求1-30中任一项所述的分离的抗原结合蛋白，其包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接地相连，且所述H-FR1包含SEQ ID NO:68、144-148、230和/或235所示的氨基酸序列。
  32. 根据权利要求1-31中任一项所述的分离的抗原结合蛋白，其包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:69、149-151和/或231所示的氨基酸序列。
  33. 根据权利要求1-32中任一项所述的分离的抗原结合蛋白，其包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:70、152-161和/或232所示的氨基酸序列。
  34. 根据权利要求1-33中任一项所述的分离的抗原结合蛋白，其包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端直接或间接地相连，且所述H-FR4包含SEQ ID NO:71、162和/或233所示的氨基酸序列。
  35. 根据权利要求1-34中任一项所述的分离的抗原结合蛋白，其包含H-FR1，H-FR2，H-FR3和H-FR4，所述H-FR1包含SEQ ID NO:68、144-148、230和/或235所示的氨基酸序列；所述H-FR2包含SEQ ID NO:69、149-151和/或231所示的氨基酸序列；所述H-FR3包含SEQ ID NO:70、152-161和/或232所示的氨基酸序列；且所述H-FR4包含SEQ ID NO:71、162和/或233所示的氨基酸序列。
  36. 根据权利要求1-35中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，所述VH包含SEQ ID NO：139和/或234所示的氨基酸序列。
  37. 根据权利要求36所述的分离的抗原结合蛋白，其包含重链可变区VH，所述VH包含SEQ ID NO：143和/或236所示的氨基酸序列。
  38. 根据权利要求36-37中任一项所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:中72-120和/或163-227任一项所示的氨基酸序列。
  39. 根据权利要求1-38中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
  40. 根据权利要求39所述的分离的抗原结合蛋白，其包括单域抗体或其抗原结合片段。
  41. 根据权利要求39-40中任一项所述的分离的抗原结合蛋白，其中所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和/或dAb。
  42. 根据权利要求39-41中任一项所述的分离的抗原结合蛋白，其中所述抗体包括嵌合抗体、人源化抗体和/或全人源抗体。
  43. 根据权利要求1-42中任一项所述的分离的抗原结合蛋白，其包含SEQ ID NO:72-120和/或163-227中任一项所示的氨基酸序列。
  44. 根据权利要求1-43中任一项所述的分离的抗原结合蛋白，其还包含免疫球蛋白Fc区。
  45. 根据权利要求44所述的分离的抗原结合蛋白，其中所述抗原结合蛋白的所述VH与所述Fc区直接或间接地连接。
  46. 根据权利要求44-45中任一项所述的分离的抗原结合蛋白，其中所述抗原结合蛋白的所述VH的C端与所述Fc区的N端直接或间接地连接。
  47. 根据权利要求44-46中任一项所述的分离的抗原结合蛋白，其中所述抗原结合蛋白的所述VH与所述Fc区在框内融合。
  48. 根据权利要求44-47中任一项所述的分离的抗原结合蛋白，其中所述抗原结合蛋白的所述VH通过连接子与所述Fc区连接。
  49. 根据权利要求48所述的分离的抗原结合蛋白，其中所述连接子包括肽连接子。
  50. 根据权利要求48-49中任一项所述的分离的抗原结合蛋白，其中所述连接子包括柔性连接子。
  51. 根据权利要求48-50中任一项所述的分离的抗原结合蛋白，从N端到C端依次包含所述抗原结合蛋白的所述VH，所述连接子和所述Fc区。
  52. 根据权利要求44-51中任一项所述的分离的抗原结合蛋白，其中所述Fc区包括源自IgG1的Fc或源自IgG4的Fc。
  53. 根据权利要求44-52中任一项所述的分离的抗原结合蛋白，其中所述Fc区包含SEQ ID NO：121-125和/或228-229中任一项所示的氨基酸序列。
  54. 多肽，其包含权利要求1-53中任一项所述的分离的抗原结合蛋白。
  55. 免疫缀合物，其包含权利要求1-53中任一项所述的分离的抗原结合蛋白或权利要求54所述的多肽。
  56. 分离的核酸分子，其编码权利要求1-53中任一项所述的分离的抗原结合蛋白，或者权利要求54所述的多肽。
  57. 载体，其包含权利要求56所述的分离的核酸分子。
  58. 细胞，其包含和/或表达权利要求1-53中任一项所述的分离的抗原结合蛋白，权利要求54所述的多肽，权利要求55所述的免疫缀合物，权利要求56所述的分离的核酸分子和/或权利要求57所述的载体。
  59. 制备权利要求1-53中任一项所述的分离的抗原结合蛋白和/或权利要求54所述的多肽的方法，所述方法包括在使得所述的分离的抗原结合蛋白和/或所述的多肽表达的条件下，培 养权利要求58所述的细胞。
  60. 药物组合物，其包含权利要求1-53中任一项所述的分离的抗原结合蛋白，权利要求54所述的多肽，权利要求55所述的免疫缀合物，权利要求56所述的分离的核酸分子，权利要求57所述的载体，权利要求58所述的细胞，以及任选地药学上可接受的佐剂和/或赋形剂。
  61. 一种用于检测PD-1的存在和/或含量的方法，其包括：
    施用权利要求1-53中任一项所述的分离的抗原结合蛋白或权利要求54所述的多肽。
  62. 试剂盒，其包含权利要求1-53中任一项所述的分离的抗原结合蛋白或权利要求54所述的多肽。
  63. 根据权利要求62所述的试剂盒，其包含使用说明，所述使用说明记载用于检测PD-1的存在和/或含量的方法。
  64. 权利要求1-53中任一项所述的分离的抗原结合蛋白或权利要求54所述的多肽在制备试剂盒中的用途，所述试剂盒用于检测PD-1的存在和/或含量的方法。
  65. 权利要求1-53中任一项所述的分离的抗原结合蛋白和/或权利要求54所述的多肽在制备药物中的用途，所述药物用于预防和/或治疗疾病或病症。
  66. 权利要求1-53中任一项所述的分离的抗原结合蛋白，权利要求54所述的多肽，权利要求55所述的免疫缀合物，权利要求56所述的分离的核酸分子，权利要求57所述的载体，权利要求58所述的细胞和/或权利要求60中任一项所述的药物组合物，其用于预防、缓解和/或治疗疾病或病症。
  67. 根据权利要求65-66中任一项所述的用途，其中所述疾病或病症包括肿瘤。
  68. 根据权利要求67所述的用途，其中所述肿瘤包括实体瘤。
  69. 根据权利要求67-68中任一项所述的用途，其中所述肿瘤包括血液瘤。
  70. 根据权利要求67-69中任一项所述的用途，其中所述肿瘤包括与PD-L1的表达相关的肿瘤。
  71. 根据权利要求67-70中任一项所述的用途，其中所述肿瘤选自下组：黑色素瘤，肺癌，头颈鳞状细胞癌，淋巴瘤，肝细胞癌，肾细胞癌，尿路上皮癌，结直肠癌和乳腺癌。
  72. 一种预防和/或治疗疾病或病症的方法，其包括向有需要的受试者施用有效量的权利要求1-53中任一项所述的分离的抗原结合蛋白，权利要求54所述的多肽，权利要求55所述的免疫缀合物，权利要求56所述的分离的核酸分子，权利要求57所述的载体，和/或权利要求58所述的细胞。
  73. 根据权利要求72所述的方法，其中所述疾病或病症包括肿瘤。
  74. 根据权利要求73所述的方法，其中所述肿瘤包括实体瘤。
  75. 根据权利要求73-74中任一项所述的方法，其中所述肿瘤包括血液瘤。
  76. 根据权利要求73-75中任一项所述的方法，其中所述肿瘤包括与PD-L1的表达相关的肿瘤。
  77. 根据权利要求73-76中任一项所述的方法，其中所述肿瘤选自下组：黑色素瘤，肺癌，头颈鳞状细胞癌，淋巴瘤，肝细胞癌，肾细胞癌，尿路上皮癌，结直肠癌和乳腺癌。
  78. 抑制PD-1与PD-L1相互作用的方法，其包括向有需要的受试者施用有效量的权利要求1-53中任一项所述的分离的抗原结合蛋白，权利要求54所述的多肽，权利要求55所述的免疫缀合物，权利要求56所述的分离的核酸分子，权利要求57所述的载体，和/或权利要求58所述的细胞。
  79. 权利要求1-53中任一项所述的分离的抗原结合蛋白，权利要求54所述的多肽，权利要求55所述的免疫缀合物，权利要求56所述的分离的核酸分子，权利要求57所述的载体，权利要求58所述的细胞和/或权利要求60中任一项所述的药物组合物，其用于抑制PD-1与PD-L1的相互作用。
  80. 权利要求1-53中任一项所述的分离的抗原结合蛋白和/或权利要求54所述的多肽在制备药物中的用途，所述药物用于抑制PD-1与PD-L1的相互作用。
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