WO2023198015A1 - Antigen-binding molecule specifically binding to psma and cd28 and pharmaceutical use thereof - Google Patents

Antigen-binding molecule specifically binding to psma and cd28 and pharmaceutical use thereof Download PDF

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Publication number
WO2023198015A1
WO2023198015A1 PCT/CN2023/087463 CN2023087463W WO2023198015A1 WO 2023198015 A1 WO2023198015 A1 WO 2023198015A1 CN 2023087463 W CN2023087463 W CN 2023087463W WO 2023198015 A1 WO2023198015 A1 WO 2023198015A1
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amino acid
seq
acid sequence
psma
strand
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PCT/CN2023/087463
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French (fr)
Chinese (zh)
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石金平
杨昊
黎婷婷
应华
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Publication of WO2023198015A1 publication Critical patent/WO2023198015A1/en

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to PSMA/CD28 antigen-binding molecules and their applications.
  • PSMA belongs to Glutamate carboxypeptidase II (Glutamate carboxypeptidase II, GCPII) and consists of 750 amino acids.
  • the extracellular part of PSMA consists of three domains: protease-like domain, apical domain and C-terminus. All three domains are involved in substrate binding. Among them, the protease-like domain and the apical domain directly bind to the substrate, and the C-terminus allows PSMA to form a dimer to function.
  • the expression level of PSMA in tumor tissue is 100-1000 times higher than that in normal prostate. Especially in poorly differentiated, metastatic and hormone-refractory cancers, the expression of PSMA is significantly increased.
  • PSMA is also expressed in the endothelial cells of capillaries in the tumor periphery and intratumoral areas of some malignant tumors, but is not expressed in blood vessels of normal tissues.
  • PSMA is related to tumor angiogenesis (Silver D.A. (1997) Clinical Cancer Research 3: 81-85). Recently, PSMA has been shown to be expressed in endothelial cells of tumor-associated neovasculature in colon, breast, bladder, pancreatic, renal, and melanoma cancers (Chang, S.S. (2004) Curr Opin Investig Drugs 5: 611- 5). Therefore, PSMA can be used not only as a drug against prostate cancer, but may also be applicable to other types of tumors.
  • T cell activation relies on the first signal provided by TCR activated by APC cells or other cell surface MHC-peptide complexes.
  • costimulatory factors are required to provide a second signal to completely activate T cells.
  • CD28 is a member of the immunoglobulin superfamily. In humans, CD28 is mainly expressed on the surface of T cells, and is also expressed in small amounts on other cells such as bone marrow stromal cells, plasma cells, neutrophils, and eosinophils. CD28 molecules can further activate the downstream NFAT, NF- ⁇ B and AP-1 signaling pathways by binding to CD80/CD86 molecules expressed on the surface of activated APC cells, promoting the activation and proliferation of T cells.
  • providing bispecific antibodies targeting PSMA/CD28 is an effective means to treat tumors and other diseases.
  • the present disclosure provides an antibody that specifically binds CD28 and PSMA antigen-binding molecules and an antibody that specifically binds CD28.
  • the present disclosure provides an antigen-binding molecule comprising at least one specific binding
  • the antigen-binding module of CD28 and at least one antigen-binding module that specifically binds to PSMA includes the heavy chain variable region CD28-VH and the light chain variable region CD28-VL
  • the specificity The antigen-binding module that binds PSMA includes the heavy chain variable region PSMA-VH and the light chain variable region PSMA-VL.
  • the antigen-binding molecule as described above, wherein:
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28 in SEQ ID NO: 36, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 respectively -amino acid sequences of LCDR1, CD28-LCDR2 and CD28-LCDR3, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28-LCDR1, CD28-LCDR2 and CD28 in SEQ ID NO: 34, 72, 73, 74, 75, 76, 77, 78, 79 or 80 respectively -The amino acid sequence of LCDR3, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37, 129 or 132
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in the CD28-VL respectively comprise the amino acids of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, 138, 139, 141, 142 or 143 sequence, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, 46, 47, 48 or 49
  • the amino acid sequence, and CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in the CD28-VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32 or 52.
  • the antigen-binding molecule as described above, wherein:
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 36, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain the amino acids of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 80 respectively. amino acid sequence, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33, and the CD28-VL in the CD28-VL.
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 34, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32;
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28-VL in the CD28-VL.
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 52.
  • the CD28-HCDR1, CD28-HCDR2, CD28-HCDR3, CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 are defined according to Kabat, IMGT, Chothia, AbM or Contact numbering rules.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR1 comprising SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92 or 93 amino acid sequence of CD28-LCDR2 and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14 and CD28- comprising the amino acid sequence of SEQ ID NO: 15 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, comprising the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27 CD28-HCDR3 of the sequence, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28- comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42 amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43. ID NO: 12 amino acid sequence of CD28-LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 30 amino acid sequence of CD28-LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42.
  • the amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42.
  • the amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12 Amino acid sequence of CD28-LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23 and CD28- comprising the amino acid sequence of SEQ ID NO: 24 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • HCDR3 has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 62 and CD28- comprising the amino acid sequence of SEQ ID NO: 18 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27.
  • HCDR3 has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28- comprising the amino acid sequence of SEQ ID NO: 30 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 12 LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 22, such as SEQ ID NO: 23, 81, CD28-LCDR2 represented by 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and CD28-LCDR3 represented by SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and CD28-LCDR3 shown in SEQ ID NO: 18; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25 or 117, CD28-HCDR2 as shown in SEQ ID NO: 26 or 116, and CD28-HCDR1 as shown in SEQ ID NO: 27 HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 as set forth in SEQ ID NO: CD28-LCDR3 shown in 30; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28-HCDR2 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 CD28-HCDR3 as shown, and the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 11 or 43, and CD28-LCDR2 as shown in SEQ ID NO: 12 CD28-LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO: 28, CD28-LCDR2 as shown in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as shown in SEQ ID NO: 30 ;or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 116 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO: 28, CD28-LCDR2 as shown in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as shown in SEQ ID NO: 30 ;or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 117, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO: 28, CD28-LCDR2 as shown in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as shown in SEQ ID NO: 30 ;or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 117, CD28-HCDR2 as shown in SEQ ID NO: 116 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as set forth in SEQ ID NO: 30 .
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 -HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 10, CD28-LCDR2 as set forth in SEQ ID NO: 11, and CD28-LCDR3 as set forth in SEQ ID NO: 12; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 -HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 10, CD28-LCDR2 as set forth in SEQ ID NO: 43, and CD28-LCDR3 as set forth in SEQ ID NO: 12.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 22, CD28-LCDR2 as set forth in SEQ ID NO: 23, and CD28-LCDR3 as set forth in SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL is CD28-LCDR1 set forth in SEQ ID NO: 16, CD28-LCDR2 set forth in SEQ ID NO: 62, and CD28-LCDR3 set forth in SEQ ID NO: 18; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and
  • the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, and CD28-LCDR3 as set forth in SEQ ID NO: 30; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO:7, CD28-HCDR2 as shown in SEQ ID NO:8 and CD28-HCDR3 as shown in SEQ ID NO:9, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 43, and CD28-LCDR3 as shown in SEQ ID NO: 12.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO:22, CD28-LCDR2 as shown in SEQ ID NO:23, and CD28-LCDR3 as shown in SEQ ID NO:24.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and the CD28- VL is CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 62, and CD28-LCDR3 as shown in SEQ ID NO: 18.
  • the antigen-binding molecule as described in any one of the preceding items are defined according to the Kabat numbering rule.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 with the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and the FR region of the CD28-VL comprises one or more amino acid substitutions selected from the group consisting
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27
  • the sequence of CD28-HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 71V and 78A
  • the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 having the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 having the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and CD28-LCDR3 having the amino acid sequence of SEQ ID NO: 30, and said
  • the FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44V and 73L; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and SEQ ID NO: 9, 39, 40, 41 or a CD28-HCDR3 with an amino acid sequence of 42, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S; and the CD28- VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12
  • the CD28-LCDR3 of the column; and the FR region of the CD28-VL contains one or more amino acid substitutions selected from the group consisting of 43S and 73F.
  • the antigen-binding molecule as described in any one of the preceding items binds to human CD28 with a KD of less than 2 ⁇ 10 -8 M, 8 ⁇ 10 -9 M or 5 ⁇ 10 -9 M at 25°C. , the KD is measured by surface plasmon resonance method.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, and the FR region of CD28-VL includes the selected One or more amino acid substitutions from the group consisting of 41D, 42G, 43T, 44I and 71Y; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 62, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 62, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 18, and the FR region of the CD28-VL contains a protein selected from the group consisting of 43S and one or more amino acid substitutions in the group consisting of 70K; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27.
  • CD28-VL has: the amino acid sequence comprising SEQ ID NO: 28 CD28-LCDR1, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30, and the FR region of the CD28-VL comprises 41D, 42G, 43T One or more amino acid substitutions in the group consisting of , 44V and 73L; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 having the amino acid sequence of 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and said The FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 43S and 73F.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 166 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 168, and PSMA comprising the amino acid sequence of SEQ ID NO: 169 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 172 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 174, and PSMA comprising the amino acid sequence of SEQ ID NO: 175 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 158, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 159, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 161, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 162, and PSMA comprising the amino acid sequence of SEQ ID NO: 163 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 176, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 177, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 178 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 179, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 180, and PSMA comprising the amino acid sequence of SEQ ID NO: 181 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 213, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 214, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 215 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 216, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 217, and PSMA comprising the amino acid sequence of SEQ ID NO: 218 -LCDR3.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 172; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 174, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 175 .
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH has: PSMA-HCDR1 including the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 including the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR3 including the amino acid sequence of SEQ ID NO: 166; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 168, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 169 .
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 167, PSMA-LCDR2 as shown in SEQ ID NO: 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 173, PSMA-LCDR2 as set forth in SEQ ID NO: 174, and PSMA-LCDR3 as set forth in SEQ ID NO: 175; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 158, PSMA-HCDR2 as shown in SEQ ID NO: 159, and PSMA-HCDR3 as shown in SEQ ID NO: 160; and PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 161, PSMA-LCDR2 as set forth in SEQ ID NO: 162, and PSMA-LCDR3 as set forth in SEQ ID NO: 163; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 176, PSMA-HCDR2 as shown in SEQ ID NO: 177, and PSMA-HCDR3 as shown in SEQ ID NO: 178; and Said PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 179, PSMA-LCDR2 as set forth in SEQ ID NO: 180, and PSMA-LCDR3 as set forth in SEQ ID NO: 181; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 213, PSMA-HCDR2 as shown in SEQ ID NO: 214, and PSMA-HCDR3 as shown in SEQ ID NO: 215; and
  • the PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 216, PSMA-LCDR2 as shown in SEQ ID NO: 217, and PSMA-LCDR3 as shown in SEQ ID NO: 218.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and the PSMA- VL contains PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and
  • the PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO:167, PSMA-LCDR2 as shown in SEQ ID NO:168, and PSMA-LCDR3 as shown in SEQ ID NO:169.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 98 or a variant thereof, wherein the variant is in the FR region of SEQ ID NO: 98 and includes a sequence selected from 26A, 29L, 69L, 71V, 78A and 93S
  • One or more amino acid substitutions in the group consisting of and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 102 or a variant thereof, the variant being in the FR region of SEQ ID NO: 102 and comprising 41D selected from the group consisting of One or more amino acid substitutions from the group consisting of , 42G, 43T, 44I and 71Y; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof, wherein the variant contains a sequence selected from the group consisting of 28S, 69L, 71V, 73K and 94S in the FR region of SEQ ID NO: 69 One or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or a variant thereof, the variant being in the FR region of SEQ ID NO: 70 and including 43S and 70K One or more amino acid substitutions in the group consisting of; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 133 or a variant thereof, wherein the variant is one selected from the group consisting of 25P, 71V and 78A in the FR region of SEQ ID NO: 133 Or multiple amino acid substitutions, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 137 or a variant thereof, the variant is in the FR region of SEQ ID NO: 137.
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 53 or a variant thereof, wherein the variant contains a FR region selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S of SEQ ID NO: 53 consisting of one or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52 or a variant thereof, the variant being in the FR region of SEQ ID NO: 52 and comprising 43S selected from the group consisting of One or more amino acid substitutions from the group consisting of and 73F.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 35, 94, 96, 97 or 98
  • the CD28-VL comprises SEQ ID NO: 101, 36, 99, 100, 102, 103 , 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 amino acid sequence; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69
  • the CD28-VL comprises SEQ ID NO: 80, 34, 70, 71 , 72, 73, 74, 75, 76, 77, 78 or 79 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 37, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133
  • the CD28-VL comprises SEQ ID NO. : the amino acid sequence of 134, 38, 135, 136, 137, 138, 139, 140, 141, 142 or 143; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 31, 45, 46, 47, 48, 49 or 53
  • the CD28-VL comprises SEQ ID NO: 52, 32, 50 or 51 amino acid sequence.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98
  • the CD28-VL comprises SEQ ID NO: 101, 99, 100, 102, 103, 104, 105 , the amino acid sequence of 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 63, 64, 65, 66, 67 or 69
  • the CD28-VL comprises SEQ ID NO: 80, 70, 71, 72, 73 , 74, 75, 76, 77, 78 or 79 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133
  • the CD28-VL comprises SEQ ID NO: 134 , 135, 136, 137, 138, 139, 140, 141, 142 or 143 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 45, 46, 47, 48, 49 or 53
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52, 50 or 51.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102, or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL includes SEQ ID NO: 101, 99, 100, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 94
  • the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 96
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99 or 100, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 97
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99, 100 or 101, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80, 72, 73, 74, 75, 76, 77, 78 or 79, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 63
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or 71, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 64
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 65
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 66
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71, 72, 74, 75, 76 or 79, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 67
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 74, 75, 76 or 79, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135, 136, 137, 138 or 139, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 123
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135 or 136, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 124, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 135; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 125
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134 or 135, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 127
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 137, 138 or 139; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 128, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 137, 138, 139 or 142; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 129
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 138, 139, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 130
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 131
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52, 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 45
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 52, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 36;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 33
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 34;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 37
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 38;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 31, and the CD28-VL contains Contains the amino acid sequence of SEQ ID NO: 32.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 65
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 71
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 78; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 106; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 94
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134;
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 125, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 135; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 128, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 142; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 129
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 44, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 52; or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 46, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 52; or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 68, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 78; or
  • CD28-VH is shown in SEQ ID NO: 68
  • amino acid sequence of CD28-VL is shown in SEQ ID NO: 80; or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 106; or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 94, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 101; or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 125, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 135; or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 128, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 142; or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 129, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 138.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50, 51 or 52, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 45
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 52, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 63, 64, 65 or 66
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 63, 64, 65 or 66
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 71; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 64 or 65
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 66
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 71, 72, 74, 75, 76 or 79; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 67
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 74, 75, 76 or 79; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 73, 74, 75, 76, 78, 79 or 80; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 94
  • the CD28-VL comprises SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110 , 111, 112, 113, 114 or 115 amino acid sequence; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, The amino acid sequence of 112, 113, 114 or 115; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 96
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99 or 100; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 97
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99, 100 or 101; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 123
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135 or 136;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 124, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 135; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 125
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134 or 135, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135, 136, 137, 138 or 139; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 127
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 137, 138 or 139; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 128, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 137, 138, 139 or 142; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 129
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 138, 139, 140, 141, 142 or 143; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 130
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 131
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, and the CD28-VL contains Containing the amino acid sequence of SEQ ID NO: 101; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the antigen-binding molecule as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 95, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 101 shown.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 68, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 80 shown.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, 199, 332, 194, 195, 196 or 198
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201, 202, 333 or 200 ;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, 184 or 203
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206, 185 or 205; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, 182, 189 or 190
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191, 183, 192 or 193; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207 or 186
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208, 187, 209 or 210; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 211
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212;
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO:204, and the PSMA-VL includes the amino acid sequence of SEQ ID NO:206.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • amino acid sequence of the PSMA-VH is as shown in SEQ ID NO: 204
  • amino acid sequence of the PSMA-VL is as shown in SEQ ID NO: 206; or
  • amino acid sequence of the PSMA-VH is as shown in SEQ ID NO: 197
  • amino acid sequence of the PSMA-VL is as shown in SEQ ID NO: 201; or
  • amino acid sequence of PSMA-VH is shown in SEQ ID NO: 199
  • amino acid sequence of PSMA-VL is shown in SEQ ID NO: 202; or
  • amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 188
  • amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 191; or
  • the antigen-binding molecule of any one of the preceding items wherein:
  • amino acid sequence of PSMA-VH is shown in SEQ ID NO: 204
  • amino acid sequence of PSMA-VL is shown in SEQ ID NO: 206.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • amino acid sequence of PSMA-VH is shown in SEQ ID NO: 197
  • amino acid sequence of PSMA-VL is shown in SEQ ID NO: 201.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • amino acid sequence of PSMA-VH is shown in SEQ ID NO: 199
  • amino acid sequence of PSMA-VL is shown in SEQ ID NO: 202.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding module that specifically binds CD28 or the antigen-binding module that specifically binds PSMA comprises a Titin chain and Obscurin capable of forming a dimer chain.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding module that specifically binds CD28 comprises a Titin chain and an Obscurin chain capable of forming a dimer.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding module that specifically binds PSMA includes a Titin chain and an Obscurin chain capable of forming a dimer.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 334 to SEQ ID NO: 352, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of The amino acid sequence of the group consisting of SEQ ID NO: 353 to SEQ ID NO: 393.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 388.
  • the antigen-binding molecule of any one of the preceding items wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 388.
  • the antigen-binding molecule of any one of the preceding items wherein the antigen-binding molecule comprises an IgG Fc region.
  • the antigen-binding molecule of any one of the preceding items wherein the antigen-binding molecule comprises an IgG1 Fc region.
  • the antigen-binding molecule of any one of the preceding items wherein the antigen-binding molecule comprises an Fc region comprising one or more amino acid substitutions capable of reducing the binding of the Fc region to Fc ⁇ receptors.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region is a human IgG1 Fc region, and the amino acids at positions 234 and 235 are A, and the numbering basis is EU index.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, so The Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule
  • the subunit includes an Fc region, the Fc region includes a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 has a convex structure according to the pestle and mortar technology, and the Fc2 has a pore structure according to the pestle and mortar technology.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, so
  • the Fc1 has a convex structure according to the pestle and mortar technology
  • the Fc2 has a pore structure according to the pestle and mortar technology
  • the amino acid of the Fc1 at position 366 is W
  • the amino acid of Fc2 at position 366 is S
  • the amino acid at position 368 is S.
  • the amino acid is A, and the amino acid at position 407 is V, and the numbering basis is the EU index.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, so
  • the Fc1 has a convex structure according to the pestle and mortar technology
  • the Fc2 has a pore structure according to the pestle and mortar technology, wherein the amino acid at the 354 position of the Fc1 is C and the amino acid at the 366 position is W; and the Fc2 is at 349
  • the amino acid at position 366 is S
  • the amino acid at position 368 is A
  • the amino acid at position 407 is V.
  • the numbering basis is the EU index.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein , the Fc1 includes the amino acid sequence of SEQ ID NO: 220; and the Fc2 includes the amino acid sequence of SEQ ID NO: 221 or 222.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein , the Fc1 includes the amino acid sequence of SEQ ID NO: 220; and the Fc2 includes the amino acid sequence of SEQ ID NO: 221.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds CD28 and an antigen-binding module that specifically binds PSMA.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds CD28 and an antigen-binding module that specifically binds PSMA, wherein:
  • the antigen-binding module that specifically binds to CD28 is a Fab
  • the antigen-binding module that specifically binds to PSMA is a replaced Fab that contains a Titin chain and an Obscurin chain capable of forming dimers.
  • the antigen-binding molecule as described in any one of the preceding items includes a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b) , a third chain having the structure shown in formula (c) and a fourth chain having the structure shown in formula (d),
  • linker 1 and the linker 2 are the same or different peptide linkers; the structures shown in formulas (a), (b), (c) and (d) are arranged from the N end to the C end. of.
  • the antigen-binding molecule as described in any one of the preceding items includes a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b) , a third chain having the structure shown in formula (c) and a fourth chain having the structure shown in formula (d),
  • the connector 1 and the connector 2 do not exist, that is, the connector 1 and the connector 2 are both bonds; as shown in formulas (a), (b), (c) and (d)
  • the structure is arranged from the N-terminus to the C-terminus.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204 sequence, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 33, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 34; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 65, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 71; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 78; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 31, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 32; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 50; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204 sequence, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 37
  • the CD28-VL includes the amino acid sequence of SEQ ID NO: 38
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence
  • said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 212.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 206.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 68, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 80; and the amino acid sequence of PSMA-VH is shown in SEQ ID NO: 204 is shown, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 206.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain and Obscurin chain can be selected from any Titin that can form dimers in Table 3-1 and Table 3-2 of the present disclosure. chain and Obscurin chain.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 388.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the linker 1 and the linker 2 are both peptide linkers known in the art, as long as the antigen-binding molecule can exhibit the desired antigen binding active.
  • the peptide linker can be a flexible peptide having 1-50 or 3-20 amino acid residues.
  • the peptide linker is 3-15 amino acid residues in length.
  • the peptide linkers each independently have the structure (GGGGS)n, where n is 1, 2, or 3.
  • the sequences of linker 1 and linker 2 are GGGGS (SEQ ID NO: 223).
  • the antigen-binding molecule as described in any one of the preceding items, wherein said CH1 is IgG The CH1 sequence.
  • the CH1 is CH1 of IgG1.
  • the CH1 comprises the amino acid sequence of SEQ ID NO: 219.
  • the antigen-binding molecule as described in any one of the preceding items wherein said CL is the light chain constant region of an antibody. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the CL is the light chain constant region of kappa or lamada. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 145.
  • a first strand as shown in SEQ ID NO:237, a second strand as shown in SEQ ID NO:239, a third strand as shown in SEQ ID NO:249 and a third strand as shown in SEQ ID NO:250 The fourth chain shown.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds CD28 and an antigen-binding module that specifically binds PSMA, and the antigen-binding module that specifically binds PSMA
  • the antigen-binding module is a Fab; the antigen-binding module that specifically binds to CD28 is a replaced Fab, which contains a Titin chain and an Obscurin chain capable of forming dimers.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule includes a first chain having a structure shown in formula (e), a second chain having a structure shown in formula (f) chain, a third chain having the structure shown in formula (g) and a fourth chain having the structure shown in formula (h),
  • linker 3 and the linker 4 are the same or different peptide linkers; the structures shown in formulas (e), (f), (g) and (h) are arranged from the N-terminus to the C-terminus.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule includes a first chain having a structure shown in formula (e), a second chain having a structure shown in formula (f) chain, one a third chain having a structure represented by formula (g) and a fourth chain having a structure represented by formula (h),
  • the connector 3 and the connector 4 do not exist, that is, the connector 3 and the connector 4 are both bonds; the structures shown in formulas (e), (f), (g) and (h) are from Arranged from N-terminus to C-terminus.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204 sequence, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 94, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 37, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 38; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL Comprising the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH comprising the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 129, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 138; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 206; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 208.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 201.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 206.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 101; and the amino acid sequence of PSMA-VH is shown in SEQ ID NO: 197 is shown, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 201.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 101; and the amino acid sequence of PSMA-VH is shown in SEQ ID NO: 204 is shown, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 206.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain and The Obscurin chain can be selected from any Titin chain and Obscurin chain that can form dimers in Table 3-1 and Table 3-2 in this disclosure.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 388.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the linker 3 and the linker 4 are both peptide linkers known in the art, as long as the antigen-binding molecule can exhibit the desired antigen binding active.
  • the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues.
  • the peptide linker is 3-15 amino acid residues in length.
  • the peptide linkers each independently have the structure (GGGGS)n, where n is 1, 2, or 3.
  • the sequences of linker 3 and linker 4 are GGGGS (SEQ ID NO: 223).
  • the antigen-binding molecule as described in any one of the preceding items, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 219.
  • the antigen-binding molecule as described in any one of the preceding items wherein said CL is the light chain constant region of an antibody. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the CL is the light chain constant region of kappa or lamada. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 145.
  • a first strand as shown in SEQ ID NO:261, a second strand as shown in SEQ ID NO:262, a third strand as shown in SEQ ID NO:272 and a third strand as shown in SEQ ID NO:273 The fourth chain shown.
  • a first strand as shown in SEQ ID NO: 261, a second strand as shown in SEQ ID NO: 262, a third strand as shown in SEQ ID NO: 274 and a third strand as shown in SEQ ID NO: 275 The fourth chain shown.
  • the present disclosure also provides an anti-CD28 antibody capable of specifically binding to CD28, the anti-CD28 antibody comprising a heavy chain variable region CD28-VH and a light chain variable region CD28-VL, wherein:
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28 in SEQ ID NO: 36, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 respectively -amino acid sequences of LCDR1, CD28-LCDR2 and CD28-LCDR3, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 34, 73, 74, 75, 76, 77, 78, 79 or 80 respectively the amino acid sequence, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37, 129 or 132
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in the CD28-VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, 139, 141, 142 or 143, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32, 46, 47, 48 or 49.
  • the anti-CD28 antibody as described above, wherein:
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 36, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 80, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33, and the CD28-VL in the CD28-VL.
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 34, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37
  • the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, or
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32;
  • CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28-VL in the CD28-VL.
  • CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 52.
  • the CD28-HCDR1, CD28-HCDR2, CD28-HCDR3, CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 are defined according to Kabat, IMGT, Chothia, AbM or Contact numbering rules.
  • the anti-CD28 antibody as described above, wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 24 CD28-LCDR3; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • HCDR3 has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, comprising the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62
  • the sequence of CD28-LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27 CD28-HCDR3 of the sequence, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28- comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42 amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43. ID NO: 12 amino acid sequence of CD28-LCDR3.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, A CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 30 amino acid sequence of CD28-LCDR3.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23 and CD28- comprising the amino acid sequence of SEQ ID NO: 24 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • HCDR3 has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 62 and CD28- comprising the amino acid sequence of SEQ ID NO: 18 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27.
  • HCDR3 has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28- comprising the amino acid sequence of SEQ ID NO: 30 LCDR3; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 12 LCDR3.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 22, such as SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 CD28-LCDR2 is shown and as CD28-LCDR3 shown in SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and CD28-LCDR3 shown in SEQ ID NO: 18; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25 or 117, CD28-HCDR2 as shown in SEQ ID NO: 26 or 116, and CD28-HCDR1 as shown in SEQ ID NO: 27 HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 as set forth in SEQ ID NO: CD28-LCDR3 shown in 30; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28-HCDR2 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 CD28-HCDR3 as shown, and the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 11 or 43, and CD28-LCDR2 as shown in SEQ ID NO: 12 CD28-LCDR3.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116 and amino acid sequence comprising SEQ ID NO: 27 CD28-HCDR3 with amino acid sequence
  • the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 CD28-LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO:30.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42.
  • the amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42.
  • the amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12 Amino acid sequence of CD28-LCDR3.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 22, CD28-LCDR2 as set forth in SEQ ID NO: 23, and CD28-LCDR3 as set forth in SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL is CD28-LCDR1 set forth in SEQ ID NO: 16, CD28-LCDR2 set forth in SEQ ID NO: 62, and CD28-LCDR3 set forth in SEQ ID NO: 18; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and
  • the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, and CD28-LCDR3 as set forth in SEQ ID NO: 30; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO:7, CD28-HCDR2 as shown in SEQ ID NO:8 and CD28-HCDR3 as shown in SEQ ID NO:9, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 43, and CD28-LCDR3 as shown in SEQ ID NO: 12.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and the CD28- VL includes CD28-LCDR1 as shown in SEQ ID NO:22, CD28-LCDR2 as shown in SEQ ID NO:23, and CD28-LCDR3 as shown in SEQ ID NO:24.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and the CD28- VL is CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 62, and CD28-LCDR3 as shown in SEQ ID NO: 18.
  • the anti-CD28 antibody of any one of the preceding items are defined according to the Kabat numbering rule.
  • the anti-CD28 antibody as described in any one of the preceding items wherein the CD28-VH and CD28-VL are humanized and both comprise the FR region of a human antibody.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 with the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and the FR region of the CD28-VL comprises one or more amino acid substitutions selected from the group consisting
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 with the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and the amino acid sequence of SEQ ID NO: 18 CD28-LCDR3, and the FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 43S and 70K; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27
  • the sequence of CD28-HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 71V and 78A
  • the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 having the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 having the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and CD28-LCDR3 having the amino acid sequence of SEQ ID NO: 30, and said
  • the FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44V and 73L; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42, and the FR region of the CD28-VH comprises a FR region selected from 1E, One or more amino acid substitutions in the group consisting of 69L, 71V, 73K, 78A and 94S; and the CD28-VL has: a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 11 or CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 12 and CD28-LCDR3 with the amino acid sequence of SEQ ID NO: 12; and the FR region of CD28-VL includes one or more amino acid substitutions selected from the group consisting of 43S and 73
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, and the FR region of CD28-VL includes the selected One or more amino acid substitutions from the group consisting of 41D, 42G, 43T, 44I and 71Y; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15.
  • the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 62, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 62, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 18, and the FR region of the CD28-VL contains a protein selected from the group consisting of 43S and one or more amino acid substitutions in the group consisting of 70K; or
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27.
  • CD28-VL has: the amino acid sequence comprising SEQ ID NO: 28 CD28-LCDR1, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30, and the FR region of the CD28-VL comprises 41D, 42G, 43T One or more amino acid substitutions in the group consisting of , 44V and 73L; or
  • the CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S; and the CD28-VL has: comprising SEQ CD28-LCDR1 with the amino acid sequence of ID NO: 10, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR3 with the amino acid sequence of SEQ ID NO: 12; and the FR region of CD28-VL includes One or more amino acid substitutions selected from the group consisting of 43S and 73F.
  • the anti-CD28 antibody as described in any one of the preceding items binds to human CD28 with a KD of less than 2 ⁇ 10 -8 M, 8 ⁇ 10 -9 M or 5 ⁇ 10 -9 M at 25°C. , the KD is measured by surface plasmon resonance method.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 98 or a variant thereof, the variant is included in the FR region of SEQ ID NO: 98 and the FR region of the CD28-VH includes a sequence selected from 26A , 29L, 69L, 71V, 78A and 93S, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 102 or a variant thereof, the variant being in SEQ ID NO.
  • the FR region of NO: 102 contains one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44I and 71Y; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof, wherein the variant contains a sequence selected from the group consisting of 28S, 69L, 71V, 73K and 94S in the FR region of SEQ ID NO: 69 One or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or a variant thereof, the variant being in the FR region of SEQ ID NO: 70 and including 43S and 70K One or more amino acid substitutions in the group consisting of; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 133 or a variant thereof, wherein the variant is one selected from the group consisting of 25P, 71V and 78A in the FR region of SEQ ID NO: 133 Or multiple amino acid substitutions, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 137 or a variant thereof, the variant is in the FR region of SEQ ID NO: 137.
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 53 or a variant thereof, wherein the variant contains a FR region selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S of SEQ ID NO: 53 consisting of one or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or a variant thereof, the variant being in the FR region of SEQ ID NO: 50 and comprising 43S selected from the group consisting of One or more amino acid substitutions in the group consisting of 73F; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 53 or a variant thereof, wherein the variant is a group selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S in the FR region of SEQ ID NO: 53.
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52 or a variant thereof, wherein the variant is composed of a FR region selected from 43S and 73F of SEQ ID NO: 52
  • One or more amino acids in the group are substituted.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 35, 94, 96, 97 or 98
  • the CD28-VL comprises SEQ ID NO: 101, 36, 99, 100, 102, 103 ,104,105
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69
  • the CD28-VL comprises SEQ ID NO: 80, 34, 70, 71 , 72, 73, 74, 75, 76, 77, 78 or 79 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 37, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133
  • the CD28-VL comprises SEQ ID NO. : the amino acid sequence of 134, 38, 135, 136, 137, 138, 139, 140, 141, 142 or 143; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 31, 45, 46, 47, 48, 49 or 53
  • the CD28-VL comprises SEQ ID NO: 52, 32, 50 or 51 amino acid sequence.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98
  • the CD28-VL comprises SEQ ID NO: 101, 99, 100, 102, 103, 104, 105 , the amino acid sequence of 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 63, 64, 65, 66, 67 or 69
  • the CD28-VL comprises SEQ ID NO: 80, 70, 71, 72, 73 , 74, 75, 76, 77, 78 or 79 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133
  • the CD28-VL comprises SEQ ID NO: 134 , 135, 136, 137, 138, 139, 140, 141, 142 or 143 amino acid sequence
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 45, 46, 47, 48 or 49
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52, 50, 51 or 53.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 35
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 36;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 33
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 34;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 37
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 38;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 31
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 32.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102, or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 94
  • the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
  • the CD28-VH includes the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 96
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99 or 100, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 97
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99, 100 or 101, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 63
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or 71
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 64
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 65
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 66
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71, 72, 74, 75, 76 or 79, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 67
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 74, 75, 76 or 79, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79 or 80, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 123
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135 or 136, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 124, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 135; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 125
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134 or 135, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135, 136, 137, 138 or 139, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 127
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 137, 138 or 139; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 128, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 137, 138, 139 or 142; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 129
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 138, 139, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 130
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 131
  • the CD28-VL comprises The amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50, 51 or 52, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 45
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 46
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 52, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 47
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 48
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51.
  • the anti-CD28 antibody of any one of the preceding items wherein:
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134;
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 95, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 101 shown.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 68, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 80 shown.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 126, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 134 shown.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 44, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 52 shown.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain constant region and a light chain constant region.
  • the anti-CD28 antibody of any one of the preceding items wherein the heavy chain constant region is a human IgGl constant region.
  • the anti-CD28 antibody as described in any one of the preceding items wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 144.
  • the anti-CD28 antibody as described in any one of the preceding items wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO: 145.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein:
  • the heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence
  • the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%
  • An amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical; or
  • the heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence
  • the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%
  • An amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical; or
  • the heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence
  • the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%
  • An amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical; or
  • the heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence
  • the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, Amino acid sequences with 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein:
  • the heavy chain includes the amino acid sequence of SEQ ID NO: 146, and the light chain includes the amino acid sequence of SEQ ID NO: 147;
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 148, and the light chain comprises the amino acid sequence of SEQ ID NO: 149; or
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 150, and the light chain comprises the amino acid sequence of SEQ ID NO: 151; or
  • the heavy chain includes the amino acid sequence of SEQ ID NO:152, and the light chain includes the amino acid sequence of SEQ ID NO:153.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 150, and the light chain The chain contains the amino acid sequence of SEQ ID NO: 151.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 148, and the light chain The chain contains the amino acid sequence of SEQ ID NO: 149.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody is an antibody fragment, wherein the antibody fragment is Fab, Fab', F(ab')2, Fd, Fv , scFv, dsFv or dAb.
  • the anti-CD28 antibody of any one of the preceding items wherein the anti-CD28 antibody is a bispecific antibody.
  • the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody is a bispecific antibody, and the bispecific antibody specifically binds to CD28 and PSMA.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (e.g., a therapeutically effective amount) the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing, and a or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
  • the pharmaceutical composition further includes at least one (eg, one) second therapeutic agent.
  • the second therapeutic agent is any agent that is advantageously combined with a PSMA/CD28 bispecific antigen-binding molecule (including antineoplastic agents, radiotherapy, antibody drug conjugates, bispecific antibodies, and antineoplastic agent-conjugated bispecific antibodies, immune checkpoint inhibitors, or combinations thereof).
  • the second therapeutic agent is an antibody capable of specifically binding CD3; preferably, the second therapeutic agent is a bispecific antibody capable of specifically binding CD3; more preferably, the second therapeutic agent is a bispecific antibody capable of specifically binding CD3.
  • the therapeutic agent is a bispecific antibody that specifically binds to CD3 and tumor cell surface antigen (TAA); most preferably, the second therapeutic agent is a bispecific antibody that specifically binds to CD3 and PSMA.
  • the second therapeutic agent is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor targets PD-1 or CTLA4; preferably, the immune checkpoint inhibitor is an anti- PD-1 antibodies.
  • the present disclosure also provides an isolated nucleic acid encoding the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing.
  • the present disclosure also provides a vector comprising the aforementioned isolated nucleic acid.
  • the present disclosure also provides a host cell comprising the aforementioned vector.
  • the present disclosure also provides a method of treating a disease, the method comprising administering (e.g., a therapeutically effective amount) to a subject the antigen-binding molecule of any of the foregoing or the antigen-binding molecule of any of the foregoing.
  • administering e.g., a therapeutically effective amount
  • Anti-CD28 antibody or a composition as described in any one of the preceding items.
  • the present disclosure also provides the antigen-binding molecule of any of the foregoing or any of the foregoing.
  • the present disclosure also provides the antigen-binding molecule of any of the foregoing, the anti-CD28 antibody of any of the foregoing, or the composition of any of the foregoing for use as a medicament.
  • the medicament is used to treat disease.
  • the disease of any of the preceding items is a tumor.
  • the tumor of any one of the preceding items is prostate cancer, renal cancer, urothelial cancer, breast cancer, bladder cancer, primary liver cancer, pancreatic cancer, melanoma, glioblastoma (e.g.
  • Glioblastoma multiforme osteosarcoma, ovarian cancer, esophageal cancer, cervical squamous cell carcinoma, lung cancer (non-small cell lung cancer, small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma), colorectal cancer (including colon and rectal cancer), endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, gastroesophageal cancer (gastroesophageal adenocarcinoma), metastatic liver cancer, multiple myeloma, lymphoma , acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) or B-cell lymphoma.
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PSMA is expressed by cells of a tumor as described above or by vascular endothelial cells proximate the tumor.
  • the tumor as described above is a solid tumor.
  • the tumor as described above is a solid tumor
  • the solid tumor is prostate cancer, renal cancer, urothelial cancer, breast cancer, bladder cancer, primary liver cancer, pancreatic cancer, melanoma, glaucoma, Blastoma (such as glioblastoma multiforme), osteosarcoma, ovarian cancer, esophageal cancer, cervical squamous cell carcinoma, lung cancer (non-small cell lung cancer, small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma cancer), colorectal cancer (including colon cancer and rectal cancer), endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, gastroesophageal cancer (gastroesophageal adenocarcinoma), liver metastatic cancer.
  • prostate cancer renal cancer, urothelial cancer, breast cancer, bladder cancer, primary liver cancer, pancreatic cancer, melanoma, glaucoma, Blastoma (such
  • the tumor as described above is prostate cancer.
  • the tumor as described above is a non-solid tumor.
  • the tumor as described above is a non-solid tumor, the non-solid tumor being multiple myeloma, lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia Cellular leukemia (CLL) or B-cell lymphoma.
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia Cellular leukemia
  • B-cell lymphoma B-cell lymphoma
  • the prostate cancer as described above is refractory prostate cancer, prostatic intraepithelial neoplasia, androgen-independent prostate cancer, malignant prostate cancer, recurrent prostate cancer, or castration-resistant prostate cancer.
  • the aforementioned disease is a disease associated with PSMA; in some embodiments, the aforementioned disease is a disease expressing PSMA.
  • the method of treating a disease as described above further includes using a second therapeutic agent.
  • the second therapeutic agent includes an anti-tumor agent, radiation therapy, antibody drug conjugates, bispecific antibodies, bispecific antibodies conjugated to an anti-tumor agent, immune checkpoint inhibitors or combination thereof.
  • the second therapeutic agent is an antibody capable of specifically binding CD3; preferably, the second therapeutic agent is a bispecific antibody capable of specifically binding CD3; more preferably, The second therapeutic agent is a bispecific antibody that can specifically bind to CD3 and tumor cell surface antigen (TAA); most preferably, the second therapeutic agent is a bispecific antibody that can specifically bind to CD3 and PSMA.
  • the second therapeutic agent is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor targets PD-1 or CTLA4; preferably, the immune checkpoint inhibitor is an anti- PD-1 antibodies.
  • the second therapeutic agent of any one of the preceding and the antigen-binding molecule of any of this disclosure are administered simultaneously, sequentially, or separately.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding moiety that specifically binds PSMA and at least one antigen-binding moiety that specifically binds CD3.
  • Antigen binding module is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding moiety that specifically binds PSMA and at least one antigen-binding moiety that specifically binds CD3.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding moiety that specifically binds PSMA and at least one antigen-binding moiety that specifically binds CD3.
  • Antigen-binding module wherein the antigen-binding module that specifically binds to PSMA binds to a different epitope than the antigen-binding module that specifically binds to CD28 and PSMA in the antigen-binding molecule that specifically binds to CD28 and PSMA.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding moiety that specifically binds PSMA and at least one antigen-binding moiety that specifically binds CD3.
  • Antigen-binding module, the antigen-binding module that specifically binds to PSMA includes the heavy chain variable region PSMA-VH and the light chain variable region PSMA-VL, and the antigen-binding module that specifically binds to CD3 includes the heavy chain variable region CD3 -VH and light chain variable region CD3-VL.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 166 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 168, and PSMA comprising the amino acid sequence of SEQ ID NO: 169 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 172 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 174, and PSMA comprising the amino acid sequence of SEQ ID NO: 175 -LCDR3; or
  • the PSMA-VH has: comprising the amino acid sequence of SEQ ID NO: 158 PSMA-HCDR1, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 159, and PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 160; and said PSMA-VL has: comprising the amino acid sequence of SEQ ID NO: 161 PSMA-LCDR1, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 162, and PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 163; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 176, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 177, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 178 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 179, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 180, and PSMA comprising the amino acid sequence of SEQ ID NO: 181 -LCDR3; or
  • the PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 301, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 302, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 303 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 304, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 305, and PSMA comprising the amino acid sequence of SEQ ID NO: 306 -LCDR3.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH has: PSMA-HCDR1 including the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 including the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR3 including the amino acid sequence of SEQ ID NO: 172; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 174, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 175 .
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH has: PSMA-HCDR1 including the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 including the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR3 including the amino acid sequence of SEQ ID NO: 166; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 168, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 169
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH comprises PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and
  • the PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 167, as shown in SEQ ID NO: PSMA-LCDR2 as shown in 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 158, PSMA-HCDR2 as shown in SEQ ID NO: 159, and PSMA-HCDR3 as shown in SEQ ID NO: 160; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 161, PSMA-LCDR2 as shown in SEQ ID NO: 162, and PSMA-LCDR3 as shown in SEQ ID NO: 163; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 176, PSMA-HCDR2 as shown in SEQ ID NO: 177, and PSMA-HCDR3 as shown in SEQ ID NO: 178; and Said PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 179, PSMA-LCDR2 as set forth in SEQ ID NO: 180, and PSMA-LCDR3 as set forth in SEQ ID NO: 181; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 301, PSMA-HCDR2 as shown in SEQ ID NO: 302, and PSMA-HCDR3 as shown in SEQ ID NO: 303; and
  • the PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 304, PSMA-LCDR2 as shown in SEQ ID NO: 305, and PSMA-LCDR3 as shown in SEQ ID NO: 306.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and the PSMA -VL contains PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and the PSMA -VL contains PSMA-LCDR1 as shown in SEQ ID NO: 167, PSMA-LCDR2 as shown in SEQ ID NO: 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, 199, 332, 194, 195, 196 or 198
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201, 202, 333 or 200 ;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, 184 or 203
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206, 185 or 205; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, 182, 189 or 190
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191, 183, 192 or 193; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207 or 186
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208, 187, 209 or 210; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 313, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 314.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191;
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202; or
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, and said PSMA -VL contains the amino acid sequence of SEQ ID NO: 206.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, and said PSMA -VL contains the amino acid sequence of SEQ ID NO:201.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199, and said PSMA -VL contains the amino acid sequence of SEQ ID NO:202.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
  • the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 283, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 284, and CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 285 HCDR3; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 286, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 288; or
  • the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 307, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 308, and CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 309 HCDR3; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 310, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 311, and CD3 comprising the amino acid sequence of SEQ ID NO: 312 -LCDR3.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
  • the CD3-VH has: CD3-HCDR1 including the amino acid sequence of SEQ ID NO: 283, CD3-HCDR2 including the amino acid sequence of SEQ ID NO: 284, and CD3-HCDR3 including the amino acid sequence of SEQ ID NO: 285; And the CD3-VL has: CD3-LCDR1 including the amino acid sequence of SEQ ID NO: 286, CD3-LCDR2 including the amino acid sequence of SEQ ID NO: 287, and CD3-LCDR3 including the amino acid sequence of SEQ ID NO: 288 .
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
  • the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and
  • the CD3-VL includes CD3-LCDR1 as set forth in SEQ ID NO: 286, CD3-LCDR2 as set forth in SEQ ID NO: 287, and CD3-LCDR3 as set forth in SEQ ID NO: 288; or
  • the CD3-VH includes CD3-HCDR1 as set forth in SEQ ID NO: 307, CD3-HCDR2 as set forth in SEQ ID NO: 308, and CD3-HCDR3 as set forth in SEQ ID NO: 309; and
  • the CD3-VL includes CD3-LCDR1 as shown in SEQ ID NO:310, CD3-LCDR2 as shown in SEQ ID NO:311, and CD3-LCDR3 as shown in SEQ ID NO:312.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
  • the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and the CD3 -VL contains CD3-LCDR1 as shown in SEQ ID NO:286, CD3-LCDR2 as shown in SEQ ID NO:287, and CD3-LCDR3 as shown in SEQ ID NO:288.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, said CD3-VH comprising the amino acid sequence of SEQ ID NO: 289, and said CD3- VL contains the amino acid sequence of SEQ ID NO: 290; or
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 315
  • the CD3-VL comprises SEQ Amino acid sequence of ID NO:316.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and the PSMA - VL contains PSMA-LCDR1 as set forth in SEQ ID NO: 173, PSMA-LCDR2 as set forth in SEQ ID NO: 174, and PSMA-LCDR3 as set forth in SEQ ID NO: 175; and
  • the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and the CD3 -VL contains CD3-LCDR1 as shown in SEQ ID NO:286, CD3-LCDR2 as shown in SEQ ID NO:287, and CD3-LCDR3 as shown in SEQ ID NO:288.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and the PSMA -VL contains PSMA-LCDR1 as set forth in SEQ ID NO: 167, PSMA-LCDR2 as set forth in SEQ ID NO: 168, and PSMA-LCDR3 as set forth in SEQ ID NO: 169; and
  • the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and the CD3 -VL contains CD3-LCDR1 as shown in SEQ ID NO:286, CD3-LCDR2 as shown in SEQ ID NO:287, and CD3-LCDR3 as shown in SEQ ID NO:288.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 301, PSMA-HCDR2 as shown in SEQ ID NO: 302, and PSMA-HCDR3 as shown in SEQ ID NO: 303; and the PSMA -VL contains PSMA-LCDR1 as shown in SEQ ID NO:304, PSMA-LCDR2 as shown in SEQ ID NO:305, and PSMA-LCDR3 as shown in SEQ ID NO:306; and
  • the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 307, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 308, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 309; And the CD3-VL has: CD3-LCDR1 including the amino acid sequence of SEQ ID NO: 310, CD3-LCDR2 including the amino acid sequence of SEQ ID NO: 311, and CD3-LCDR3 including the amino acid sequence of SEQ ID NO: 312 .
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206;
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 201;
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO: 199, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 202; and
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO: 313, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 314; and
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 315
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO: 316.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an Fc region.
  • the Fc region is an IgG Fc region.
  • the Fc region is an IgGl Fc region.
  • the Fc region contains one or more amino acid substitutions that reduce binding of the Fc region to Fc ⁇ receptors.
  • the Fc region is a human IgGl Fc region, and the amino acids at positions 234 and 235 are A, numbered according to the EU index.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an Fc region comprising a first subunit Fcl capable of associating with each other and a second subunit Fc2, each of said Fc1 and Fc2 independently having one or more amino acid substitutions that reduce homodimerization of the Fc region.
  • the Fc1 has a convex structure according to the pestle and mortar technology
  • the Fc2 has a pore structure according to the pestle and mortar technology.
  • the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbering according to EU index.
  • the Fc1 amino acid at position 354 is C and the amino acid at position 366 is W; and the Fc2 amino acid at position 349 is C, the amino acid at position 366 is S, and the amino acid at position 368 is W is A, and the amino acid at position 407 is V, and the numbering basis is the EU index.
  • the Fcl comprises The amino acid sequence of SEQ ID NO: 220; the Fc2 includes the amino acid sequence of SEQ ID NO: 221.
  • the Fc1 comprises the amino acid sequence of SEQ ID NO:220; the Fc2 comprises the amino acid sequence of SEQ ID NO:222.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an antigen-binding moiety that specifically binds PSMA and an antigen-binding moiety that specifically binds CD3.
  • module, the antigen-binding module that specifically binds to PSMA and the antigen-binding module that specifically binds to CD3 are both scFv.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
  • linker 6, linker 7, linker 8 and/or linker 9 are the same or different peptide linkers.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197; and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289; and The CD3-V L includes the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199; and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289 Amino acid sequence; and said CD3-V L comprises the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
  • the structures shown in formulas (a) and (b) are arranged from the N end to the C end.
  • the linker 6 and the linker 8 are peptide linkers with the same sequence; the linker 7 and the linker 9 have different sequences. Peptide linker.
  • the second therapeutic agent as described in any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, and the linker 6, linker 7, linker 8, and linker 9 can be any Suitable peptide linkers are as long as the antigen-binding molecule can exhibit the desired antigen-binding activity.
  • the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues.
  • the peptide linkers each independently have the structure of L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, S, GS, GGS (SEQ ID NO: 399) or GGGS (SEQ ID NO: 400), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is key, G, GG, GGG (SEQ ID NO: 295) or GGGG (SEQ ID NO: 256), and the peptide linker is not a bond.
  • the peptide linker is 3-15 amino acid residues in length.
  • the second therapeutic agent as described in any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said linker 6 and linker 8 comprise the amino acid sequence of SEQ ID NO: 293 (GGGGGSGGGGSGGGGS).
  • the linker 7 includes the amino acid sequence of SEQ ID NO: 294 (SGGGGS).
  • the linker 9 includes the amino acid sequence of SEQ ID NO: 295 (GGG).
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said Fc1 comprises the amino acid sequence of SEQ ID NO: 220.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the Fc2 comprises the amino acid sequence of SEQ ID NO: 221.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the bispecific antibody that specifically binds PSMA and CD3 has: a bar comprising SEQ ID The first strand contains the amino acid sequence of SEQ ID NO: 296 and the second strand contains the amino acid sequence of SEQ ID NO: 220.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the bispecific antibody that specifically binds PSMA and CD3 has:
  • a first strand comprising the amino acid sequence of SEQ ID NO: 297 and a second strand comprising the amino acid sequence of SEQ ID NO: 220.
  • the second therapeutic agent of any one of the preceding items specifically binds PSMA and Bispecific antibodies to CD3, which comprise an antigen-binding module that specifically binds PSMA and an antigen-binding module that specifically binds CD3, and the antigen-binding module that specifically binds PSMA or the antigen-binding module that specifically binds CD3 includes an antigen-binding module that specifically binds to PSMA. Titin chain and Obscurin chain forming a dimer.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an antigen-binding moiety that specifically binds PSMA and an antigen-binding moiety that specifically binds CD3.
  • the antigen-binding module that specifically binds to PSMA is a Fab
  • the antigen-binding module that specifically binds to CD3 is a replaced Fab, which includes a Titin chain and an Obscurin chain capable of forming dimers.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 is a Fab; said specifically binds PSMA
  • the antigen-binding module is a replaced Fab containing a Titin chain and an Obscurin chain capable of forming dimers.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (c), a first chain having a structure represented by formula (c), d) a second chain having the structure shown in formula (e), a third chain having the structure shown in formula (e) and a fourth chain having the structure shown in formula (f),
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (c), a first chain having a structure represented by formula (c), d) a second chain having the structure shown in formula (e), a third chain having the structure shown in formula (e) and a fourth chain having the structure shown in formula (f),
  • the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204; and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 206; and the CD3-VH includes the amino acid sequence of SEQ ID NO: 289; and The CD3-VL includes the amino acid sequence of SEQ ID NO: 290.
  • the second therapeutic agent of any one of the preceding items specifically binds PSMA and A bispecific antibody to CD3, wherein the Titin chain and Obscurin chain are any Titin chain and Obscurin chain in Table 3-1 and Table 3-2 of the present disclosure that can form dimers.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 388.
  • the second therapeutic agent as described in any one of the preceding is a bispecific antibody that specifically binds PSMA and CD3, wherein the linker 10 and linker 11 can be any suitable peptide linkage as long as the antigen-binding molecule can exhibit the desired antigen-binding activity.
  • the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues.
  • the peptide linkers each independently have the structure of L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, S, GS, GGS (SEQ ID NO: 399) or GGGS (SEQ ID NO: 400), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is key, G, GG, GGG (SEQ ID NO: 295) or GGGG (SEQ ID NO: 256), and the peptide linker is not a bond.
  • the peptide linker is 3-15 amino acid residues in length.
  • the linker 10 and the linker 11 comprise the amino acid sequence set forth in SEQ ID NO:223.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the CH1 is the CH1 sequence of an IgG.
  • the CH1 is CH1 of IgG1.
  • the CH1 comprises the amino acid sequence of SEQ ID NO: 219.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the CL is the light chain constant region of kappa or lamada.
  • the CL comprises the amino acid sequence of SEQ ID NO: 145.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, having: a first chain comprising the amino acid sequence of SEQ ID NO: 298, a first chain comprising A second strand containing the amino acid sequence of SEQ ID NO: 275, a third strand containing the amino acid sequence of SEQ ID NO: 291 and a fourth strand containing the amino acid sequence of SEQ ID NO: 292.
  • the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, having an IgG-scFv structure.
  • the second therapeutic agent as described in any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising two first chains of the structure represented by formula (a) and two formulas ( b) Second strand of the structure shown:
  • the second therapeutic agent of any one of the preceding items specifically binds PSMA and A bispecific antibody to CD3, which has an IgG-scFv structure, which includes two first chains of the structure represented by formula (a) and two second chains of the structure represented by formula (b):
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 313; and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 314; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 315; and The CD3-VL includes the amino acid sequence of SEQ ID NO: 316.
  • the bispecific antibody that specifically binds PSMA and CD3 comprises two amino acid sequences of SEQ ID NO: 299 and two amino acid sequences of SEQ ID NO: 300.
  • the second therapeutic agent of any one of the preceding items is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor targets PD-1 or CTLA4.
  • the second therapeutic agent of any one of the preceding items is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
  • the second therapeutic agent of any one of the preceding items is an anti-PD-1 antibody, including any known therapeutically active anti-PD-1 antibody, including but not limited to camrelizumab (Camrelizumab), pembrolizumab (Keytruda), nivolumab (Opdivo) and cemiplimab (cemiplimab).
  • camrelizumab Camrelizumab
  • pembrolizumab Keytruda
  • nivolumab Opdivo
  • cemiplimab cemiplimab
  • the second therapeutic agent of any one of the preceding items is an anti-PD-1 antibody comprising a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, wherein:
  • the PD-1-VH has: PD-1-HCDR1 including the amino acid sequence of SEQ ID NO: 319, PD-1-HCDR2 including the amino acid sequence of SEQ ID NO: 320 and the amino acid sequence including SEQ ID NO: 321 PD-1-HCDR3, and the PD-1-VL has: PD-1-LCDR1 comprising the amino acid sequence of SEQ ID NO: 322, PD-1-LCDR2 comprising the amino acid sequence of SEQ ID NO: 323 and PD-1-LCDR3 of the amino acid sequence of SEQ ID NO: 324.
  • the second therapeutic agent of any one is an anti-PD-1 antibody comprising a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, wherein:
  • the PD-1-VH includes the amino acid sequence of SEQ ID NO: 325
  • the PD-1-VL includes the amino acid sequence of SEQ ID NO: 326.
  • the second therapeutic agent of any one of the preceding items is an anti-PD-1 antibody comprising a heavy chain of the amino acid sequence of SEQ ID NO: 328, and a light chain of the amino acid sequence of SEQ ID NO: 329 .
  • the antigen-binding molecules provided by the present disclosure have the characteristics of good therapeutic activity, safety, pharmacokinetic properties and druggability (such as stability).
  • Figure 1A Schematic structural diagram of Formula 1 of PSMA/CD28 bispecific antibody
  • FIG. 1B Schematic structural diagram of Formula 2 of PSMA/CD28 bispecific antibody
  • Figure 1C Schematic structural diagram of Formula 1 of PSMA/CD3 bispecific antibody
  • FIG. 1D Schematic structural diagram of Formula 5 of PSMA/CD3 bispecific antibody; where Ob represents Obscurin.
  • Figure 2 Fixed concentration of PSMA/CD3 bispecific antibody CC-1, killing results of different concentrations of PSMA/CD28 bispecific antibody on CHO-huPSMA cells.
  • Figure 3 Changes in the secretion of cytokine IFN- ⁇ in PBMCs under the combined action of PSMA/CD3 double antibodies, PSMA/CD28 double antibodies and CHO-huPSMA cells.
  • Figure 4 Changes in the secretion of cytokine IL-6 in PBMCs under the combined action of PSMA/CD3 double antibodies, PSMA/CD28 double antibodies and CHO-huPSMA cells.
  • CD28 cluster of differentiation 28, Tp44
  • CD28 refers to any CD28 protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., macaques) and rodents (e.g., mice and rats).
  • CD28 is expressed on T cells and provides costimulatory signals required for T cell activation and survival. In addition to T cell receptors (TCR), stimulation of T cells through CD28 can also provide effective signals for the production of various interleukins.
  • CD28 is the receptor for the CD80 (B7.1) and CD86 (B7.2) proteins and is the only B7 receptor constitutively expressed on naive T cells.
  • the amino acid sequence of human CD28 is shown in UniProt (www.uniprot.org) accession number P10747.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine.
  • Amino acid analogs have the same basic chemical structure as naturally occurring amino acids (i.e. alpha carbon bonded to hydrogen, carboxyl, amino and R groups) compounds such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium.
  • Such analogs have modified R groups (eg, norleucine) or modified peptide backbones but retain the same basic chemical structure as the naturally occurring amino acid.
  • Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of amino acids, but act in a manner similar to naturally occurring amino acids.
  • amino acid mutation includes amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to achieve the final construct, as long as the final construct possesses the desired properties, such as reduction or binding to Fc receptors.
  • Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and/or carboxyl terminus of the polypeptide chain.
  • Specific amino acid mutations may be amino acid substitutions.
  • the amino acid mutation is a non-conservative amino acid substitution, ie, one amino acid is replaced by another amino acid with different structural and/or chemical properties.
  • Amino acid substitutions include substitutions by non-naturally occurring amino acids or by derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) .
  • Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is expected that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be available. Various names may be used herein to refer to the same amino acid mutation.
  • the amino acid residue at a specific position can be represented by position + amino acid residue.
  • 366W means that the amino acid residue at position 366 is W.
  • T366W means that the amino acid residue at position 366 has been mutated from the original T to W.
  • antigen-binding molecule is used in the broadest sense and covers a variety of molecules that specifically bind to antigens, including but not limited to antibodies, other polypeptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of the two, as long as they exhibit the desired Antigen-binding activity.
  • the antigen-binding molecules herein comprise a variable region (VH) and a variable region (VL), which together constitute an antigen-binding domain.
  • VH variable region
  • VL variable region
  • the antigen-binding molecule herein is a bispecific antigen-binding molecule (eg, bispecific antibody).
  • antibody is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibodies fragments (or antigen-binding fragments, or antigen-binding portions) as long as they exhibit the desired antigen-binding activity.
  • natural IgG antibodies are heterotetrameric proteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as variable heavy domain, heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C terminus, each light chain has a variable region (VL), also called a variable light domain, or light chain variable domain, followed by a constant light domain (light chain constant region, CL ).
  • VH variable region
  • CH1, CH2, and CH3 constant domain
  • bispecific antibody refers to an antibody (including antibodies or antigen-binding fragments thereof, such as single-chain antibodies) that can specifically bind to two different antigens or at least two different epitopes of the same antigen.
  • Bispecific antibodies of various structures have been disclosed in the prior art. According to the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment type bispecific antibodies. According to the number of antigen-binding regions, they can be divided into bivalent antibodies. , trivalent, quadrivalent or more valent bispecific antibodies, which can be divided into symmetric structure bispecific antibodies according to whether the structure is symmetrical or not. and asymmetrically structured bispecific antibodies.
  • bispecific antibodies based on antibody fragments such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining 2 or more Fab fragments in one molecule, have lower immunogenicity, and Small molecular weight, high tumor tissue penetration, typical antibody structures of this type such as F(ab)2, scFv-Fab, (scFv)2-Fab; IgG-like bispecific antibodies (for example, with Fc fragment),
  • This type of antibody has a relatively large molecular weight.
  • the Fc fragment helps purify the antibody and improves its solubility and stability.
  • the Fc part may also bind to the receptor FcRn, increasing the serum half-life of the antibody.
  • Typical bispecific antibody structural model Such as KiH, CrossMAb, Triomab quadroma, Fc ⁇ Adp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 1Fab-IgG TDB, FynomAb, two-in-one/DAF, scFv-Fab-IgG , DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4 -Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol
  • variable region refers to the domain of an antigen-binding molecule that binds the antigen.
  • the heavy chain variable region in the antigen-binding module that specifically binds to PSMA is labeled PSMA-VH
  • the light chain variable region is labeled PSMA-VL
  • the heavy-chain variable region in the antigen-binding module that specifically binds to CD28 is labeled PSMA-VH.
  • CD28-VH the light chain variable region
  • CD28-VL each contain four conserved framework regions (FR) and three complementarity determining regions (CDR).
  • CDR complementarity determining region
  • framework or “FR” refers to the variable domain residues other than CDR residues.
  • VH contains 3 CDR areas: HCDR1, HCDR2 and HCDR3;
  • VL contains 3 CDR areas: LCDR1, LCDR2 and LCDR3.
  • the three CDR regions in PSMA-VH are labeled PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 respectively;
  • the three CDR regions in PSMA-VL are labeled PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 respectively.
  • CD28-VH The three CDR regions in CD28-VH are labeled CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 respectively; the three CDR regions in CD28-VL are labeled respectively CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3.
  • Each VH and VL from N end to C end are: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a single VH or VL may be sufficient to confer antigen binding specificity.
  • the amino acid sequence boundaries of CDRs can be determined by various well-known schemes, such as: “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health , Bethesda, MD), "Chothia” numbering rule, “ABM” numbering rule, "contact” numbering rule (see Martin, ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001) and ImMunoGenTics (IMGT) numbering Rules (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art of.
  • the numbering scheme for this disclosure is set forth in Table 1 below.
  • variable region and CDR sequences in the embodiments of the present disclosure are all subject to the "Kabat" numbering rule.
  • one numbering system such as Kabat
  • Kabat is used to define amino acid residues
  • the corresponding technical solutions in other numbering systems will be regarded as equivalent technical solutions.
  • antibody fragment refers to a molecule other than an intact antibody that contains portions of an intact antibody that retain the antigen-binding ability of the intact antibody.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • Fc region or “fragment crystallizable region” is used to define the C-terminal region of an antibody heavy chain, including native Fc regions and engineered Fc regions.
  • the Fc region contains two subunits that are the same or different.
  • the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus.
  • Suitable native sequence Fc regions for use in the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise stated, the numbering convention for the Fc region is the EU index.
  • Titin chain refers to a peptide segment in the Titin protein containing a Titin Ig-like 152 domain or a functional variant thereof with a length of 78-118 amino acids.
  • the Titin chain can combine with the Obscurin chain to form a dimerization complex. things.
  • the term "Obscurin chain” refers to a peptide segment of the Obscurin protein containing the Obscurin Ig-like 1 domain or a functional variant thereof with a length of 87-117 amino acids, or a segment of the Obscurin-like 1 protein with a length of 78-118 amino acids.
  • a peptide segment of an amino acid containing an Obscurin-like Ig-like 1 domain or a functional variant thereof, and the Obscurin chain can combine with the Titin chain to form a dimerization complex.
  • the Titin chain and Obscurin chain disclosed in the present disclosure can be used to replace CH1 and CL in Fab to form a substituted Fab (Fab-S). This substitution does not affect the binding of the antigen-binding molecule to the antigen.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a specific source or species and the remaining portion of the heavy and/or light chain is derived from a different source or species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. This can be accomplished, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions as well as the framework portions of the variable regions).
  • affinity refers to the overall strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • binding affinity refers to the internal binding affinity that reflects a 1:1 between the members of a binding pair (e.g., antibody to antigen) interaction.
  • the affinity of a molecule X for its ligand Y can generally be expressed by the equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.
  • KD refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as molar concentration (M).
  • M molar concentration
  • the term "monoclonal antibody” refers to a population of antibodies or members thereof that are substantially homogeneous, ie, the antibody molecules contained in the population are identical in amino acid sequence, except for natural mutations that may be present in minor amounts. In contrast, polyclonal antibody preparations typically contain multiple different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes. "Monoclonal” refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.
  • antigen refers to a molecule or portion of a molecule capable of being selectively recognized or bound by an antigen-binding molecule (eg, an antibody).
  • An antigen may have one or more epitopes capable of interacting with different antigen-binding molecules (eg, antibodies).
  • epitope refers to an area or region on an antigen that is capable of specifically binding to an antibody or antigen-binding fragment thereof.
  • Epitopes may be formed from contiguous amino acids (linear epitopes) or contain non-contiguous amino acids (conformational epitopes), for example due to the folding of the antigen (i.e. the tertiary folding of the antigen by its proteinaceous nature) such that the non-contiguous amino acids are spatially separated. near.
  • the difference between conformational epitopes and linear epitopes is that in the presence of denaturing solvents, the antibody's binding to the conformational epitope is lost.
  • An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
  • Screening for antibodies that bind a specific epitope can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443 -463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigen (see Prot.Sci.9 (2000) 487-496), and cross-blocking (see “Antibodies", Harlow and Lane (Cold Spring Harbor Press,Cold Spring Harb.,NY)).
  • the terms “capable of specifically binding”, “specifically binding” or “binding” refer to the ability of an antibody to bind to an antigen or epitope with higher affinity than to other antigens or epitopes.
  • antibodies bind an antigen or epitope with an equilibrium dissociation constant (KD) of about 1 ⁇ 10 ⁇ 7 M or less (eg, about 1 ⁇ 10 ⁇ 8 M or less).
  • KD equilibrium dissociation constant
  • the KD of the antibody binding to the antigen is 10% or less (eg, 1%) of the KD of the antibody binding to a non-specific antigen (eg, BSA, casein).
  • KD can be measured using known methods, such as by FACS or surface plasmon resonance assays.
  • antibodies that specifically bind to an antigen or its epitope may be cross-reactive to other related antigens, e.g., to antibodies from other species (homologous) such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus). , cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset) corresponding antigens are cross-reactive.
  • does not bind means that the antibody is unable to bind to an antigen or its epitope in the specific binding manner described above. For example, when the antibody binds the antigen or its epitope with an equilibrium dissociation constant (KD) of about 1 ⁇ 10 -6 M or greater.
  • KD equilibrium dissociation constant
  • antigen-binding module refers to a polypeptide molecule that specifically binds to a target antigen or an epitope thereof.
  • Specific antigen-binding modules include the antigen-binding domain of an antibody, for example, including a heavy chain variable region and a light chain variable region.
  • antigen-binding module that specifically binds CD28 refers to a module that is capable of binding CD28 or an epitope thereof with sufficient affinity such that molecules containing the module can be used as diagnostic and/or therapeutic agents targeting CD28.
  • an antigen-binding module that specifically binds CD28 has the following equilibrium dissociation constant (KD): ⁇ approximately 2 ⁇ 10 ⁇ 8 M, as measured by surface plasmon resonance assay.
  • Antigen binding moieties include antibody fragments as defined herein, such as Fab, substituted Fab or scFv.
  • linker refers to a linking unit that joins two polypeptide fragments.
  • linkers appearing in the same structural formula may be the same or different.
  • the linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids.
  • the linkers used herein may be the same or different.
  • Tm is the solution denaturation temperature (intrinsic fluorescence). When proteins are denatured (heated or denatured), the tertiary structure is opened and the aromatic amino acid microenvironment changes, resulting in a change in the emission fluorescence spectrum.
  • Tm1 refers to the temperature at which the fluorescence changes to half of its maximum value.
  • Tonset is the denaturation starting temperature. It means the temperature at which the protein begins to denature, that is, the temperature at which the fluorescence value begins to change.
  • Tagg is the aggregation onset temperature. By static light scattering, aggregates are detected at two wavelengths, 266 nm and 473 nm, and the temperature at which the sample begins to aggregate is monitored. Tagg 266 refers to the aggregation onset temperature monitored at 266nm.
  • nucleic acid is used interchangeably herein with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring and non-naturally occurring, have similar binding properties to the reference nucleic acid, and are Metabolized in a manner similar to the reference nucleotide.
  • nucleic acid molecules that has been separated from components of its natural environment.
  • Isolated nucleic acid encoding said antigen-binding molecule refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such one or more nucleic acids in a single vector or separate vectors molecule, and such one or more nucleic acid molecules present at one or more locations in a host cell.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences thereof as well as sequences explicitly indicated.
  • degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a degenerate base and/or Deoxyinosine residue substitution.
  • polypeptide and "protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are the corresponding artificial chemical mimetics of naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless stated otherwise, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • sequence identity refers to the extent (percentage) that the amino acids/nucleic acids of two sequences are identical at equivalent positions when the two sequences are optimally aligned. During the alignment process, gaps may be allowed to be introduced when necessary to achieve maximum percent sequence identity, but any conservative substitutions are not considered to form part of the sequence identity. To determine percent sequence identity, alignment can be accomplished by techniques known to those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximal alignment over the full length of the sequences being compared.
  • fusion means that the components (eg, the antigen-binding module and the Fc domain) are covalently linked, either directly or via a linker.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked.
  • plasmid refers to a circular double-stranded DNA circle into which additional DNA segments can be ligated.
  • viral vector such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome.
  • AAV adeno-associated viral vector
  • Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
  • vectors eg, non-episomal mammalian vectors
  • expression vector or "expression construct” refers to a vector suitable for transformation of a host cell and containing the direction and/or control of the expression of one or more heterologous coding regions operably linked thereto (together with the host cell).
  • Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, RNA splicing of the coding region operably linked thereto.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cells, but may contain mutations.
  • mutant progeny that possess the same functional or biological activity as cells screened or selected in primary transformed cells.
  • Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell line plant cells, and fungal cells.
  • Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.
  • Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Cokra Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans), Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp., Saccharomycescerevisiae, Saccharomyces genus, Hansenula polymorpha, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans ), Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium
  • Pichia pastoris any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Luck Chrysosporium lucknowense, any species of Fusarium, Yarrowia lipolytica and Neurospora crassa.
  • the host cells of this patent do not include subjects that are not authorized under the patent law.
  • composition means a mixture containing one or more antigen-binding molecules or antibodies described herein together with other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients.
  • pharmaceutically acceptable carrier refers to an ingredient of a pharmaceutical formulation that is distinct from the active ingredient and is not toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • subject or “individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles.
  • patient or “subject” are used interchangeably herein.
  • cyno or “cynomolgus” refers to the crab-eating monkey (Macaca fascicularis).
  • the individual or subject is a human.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, means the administration of an exogenous drug, therapeutic, diagnostic or composition to an animal, human , contact with subjects, cells, tissues, organs or biological fluids.
  • sample refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject.
  • exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph fluid, urine, saliva, cyst fluid, tears, excreta, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites , fluids from the pleura, pericardium, peritoneum, peritoneal cavity and other body cavities, fluids collected from bronchial lavage, synovial fluids, fluid solutions in contact with subjects or biological sources, For example, cell and organ culture media (including cell or organ conditioned media), lavage fluid, etc., tissue biopsy samples, fine needle aspiration, surgically resected tissue, organ culture or cell culture.
  • biological fluids such as blood, serum and serosal fluids, plasma, lymph fluid, urine, saliva, cyst fluid, tears, excreta, sputum, mucos
  • Treatment refers to a clinical intervention attempted to be applied to the individual being treated, and may be administered for preventive purposes, or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and regressing or improving prognosis .
  • molecules of the present disclosure are used to delay the development of a disease or slow the progression of a disease.
  • recurrence refers to the return of cancer or disease following clinical assessment of disease resolution.
  • diagnosis of distant cancer metastasis or local recurrence may be considered recurrence.
  • refractory or “resistant” refers to a cancer or disease that does not respond to treatment.
  • an “effective amount” is generally one sufficient to reduce the severity and/or frequency of symptoms, eliminate those symptoms and/or underlying causes, prevent the occurrence of symptoms and/or their underlying causes, and/or ameliorate or ameliorate impairments caused by or associated with the disease state. amount.
  • the effective amount is a therapeutically effective amount or a prophylactically effective amount.
  • a "therapeutically effective amount” is one sufficient to treat a disease state or symptom, particularly a condition or symptom associated with that disease state, or to otherwise prevent, hinder, delay or reverse the disease state or any other adverse effect in any way related to the disease. The ideal amount of symptomatic progression.
  • a “prophylactically effective amount” is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. . Complete therapeutic or prophylactic effect does not necessarily occur after administration of one dose but may occur after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.
  • “Therapeutically effective amount” and “prophylactically effective amount” may vary depending on a variety of factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual.
  • Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.
  • Immune checkpoint means a group of molecules on the cell surface of CD4 T cells and CD8 T cells. These molecules can effectively act as “brakes” to downregulate or inhibit anti-tumor immune responses. Immune checkpoint molecules include, but are not limited to, programmed death 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), B7H1, B7H4, OX-40, CD137, CD40, and LAG-3, which directly inhibit Immune Cells.
  • PD-1 programmed death 1
  • CTL-4 cytotoxic T lymphocyte antigen 4
  • B7H1, B7H4, OX-40 CD137, CD40, and LAG-3
  • Immune checkpoint inhibitors for use in the present disclosure are substances that inhibit the function of immune checkpoint molecules.
  • the immune checkpoint inhibitor is not particularly limited as long as the inhibitor is a substance that can inhibit the function (signal) of the immune checkpoint molecule.
  • Immune checkpoint inhibitors that can be used in the methods of the present disclosure include, but are not limited to, PD-1, PD-L2, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, Inhibitors of CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5) and/or TGFR ⁇ . Inhibition of inhibitory molecules can be effected by inhibition at the DNA, RNA or protein level.
  • inhibitory nucleic acids eg, dsRNA, siRNA, or shRNA
  • the inhibitor of an inhibitory signal is a polypeptide that binds to an inhibitory molecule, e.g., a soluble ligand or an antibody.
  • immune checkpoint inhibitors used in this disclosure can This includes, but is not particularly limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, and anti-PD-1 antibodies and anti-PD-L1 antibodies may be preferred.
  • Antigen binding molecules of the present disclosure are provided.
  • the present disclosure provides antigen-binding molecules that have many advantageous properties, such as good in vitro killing activity, therapeutic activity, safety, pharmacokinetic properties and druggability (such as yield, purity and stability, etc.).
  • Antigen-binding molecules of the present disclosure include bispecific antigen-binding molecules (eg, bispecific antibodies) and anti-CD28 antibodies that specifically bind to CD28 and PSMA.
  • the antigen-binding molecules of the present disclosure have any of the following properties:
  • the anti-CD28 antibody binds human CD28 with an EC50 of less than 1 nM, as measured by ELISA.
  • the antigen-binding molecule activates IL-2-expressing Jurkat cells with an EC50 of less than 0.1 ⁇ g/mL in the presence of a first activation signal.
  • combination with PSMA/CD3 bispecific antibody or anti-PD-1 antibody has better in vivo therapeutic activity.
  • PSMA/CD3 bispecific antibody Combined with PSMA/CD3 bispecific antibody, it can significantly increase the killing effect of PSMA/CD3 bispecific antibody on tumor cells.
  • the present disclosure provides an antigen-binding molecule comprising at least one antigen-binding module that specifically binds to CD28 and at least one antigen-binding module that specifically binds to PSMA, the antigen-binding module that specifically binds to CD28 comprises CD28-VH and CD28 -VL, the antigen-binding module that specifically binds PSMA includes PSMA-VH and PSMA-VL.
  • the present disclosure also provides an anti-CD28 antibody capable of specifically binding to CD28, the antibody comprising CD28-VH and CD28-VL.
  • the Examples herein disclose antibody series 81, 94, 97, and 129. The antibodies of this article are described below using antibodies 97 and 94 as examples.
  • An antigen-binding molecule or anti-CD28 antibody that specifically binds CD28 and PSMA wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 22, as shown in SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 CD28-LCDR2 as shown and CD28-LCDR3 as shown in SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 16, such as SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and CD28-LCDR3 as shown in SEQ ID NO: 18.
  • An antigen-binding molecule or anti-CD28 antibody that specifically binds CD28 and PSMA wherein:
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and
  • the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 22, CD28-LCDR2 as set forth in SEQ ID NO: 23, and CD28-LCDR3 as set forth in SEQ ID NO: 24; or
  • the CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and
  • the CD28-VL is CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 62, and CD28-LCDR3 as shown in SEQ ID NO: 18.
  • the antigen-binding molecule or anti-CD28 antibody as described above, the CD28-VH and/or the CD28-VL is murine or humanized. In some embodiments, the CD28-VH and/or the CD28-VL are humanized.
  • the FR1, FR2, FR3, and FR4 of the humanized CD28-VH are at least 60%, 70%, 80%, or 90% identical to the FR1, FR2, FR3, and FR4 of SEQ ID NO:98 Sequence identity; FR1, FR2, FR3 and FR4 of the humanized CD28-VL have at least 60%, 70%, 80% or 90% identity with the FR1, FR2, FR3 and FR4 of SEQ ID NO: 102 Sequence identity.
  • the humanized CD28-VH has FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is unsubstituted or has FR4 derived from IGHJ6*01 , one or more amino acid substitutions in the group consisting of 26A, 29L, 69L, 71V, 78A and 93S; and/or the humanized CD28-VL has FR1, FR2, FR3 derived from IGKV1-33*01 and FR4 derived from IGKJ4*01 and which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44I and 71Y.
  • the amino acid positions in the variable regions described above are defined according to Kabat numbering rules.
  • the humanized CD28-VH comprises the amino acid sequence of SEQ ID NO: 98 or a variant thereof.
  • the variant is one or more amino acid substitutions in the FR region of SEQ ID NO: 98 selected from the group consisting of 26A, 29L, 69L, 71V, 78A, and 93S.
  • the humanized CD28-VL comprises the amino acid sequence of SEQ ID NO: 102, or a variant thereof.
  • the variant is one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44I, and 71Y in the FR region of SEQ ID NO: 102.
  • the FR1, FR2, FR3, and FR4 of the humanized CD28-VH are at least 60%, 70%, 80%, or 90% identical to the FR1, FR2, FR3, and FR4 of SEQ ID NO: 69 Sequence identity; FR1, FR2, FR3 and FR4 of the humanized CD28-VL have at least 60%, 70%, 80% or 90% identity with the FR1, FR2, FR3 and FR4 of SEQ ID NO:80 Sequence identity.
  • the humanized CD28-VH has FR1 derived from IGHV1-3*01, FR2, FR3 and FR4 derived from IGHJ1*01 and which are unsubstituted or have one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and/or the
  • the humanized CD28-VL has FR1, FR2, FR3 derived from IGKV1-12*01 and FR4 derived from IGKJ4*01, and is unsubstituted or has a group selected from the group consisting of 43S and 70K One or more amino acid substitutions.
  • the amino acid positions in the variable regions described above are defined according to Kabat numbering rules.
  • the humanized CD28-VH comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof.
  • the variant is one or more amino acid substitutions selected from the group consisting of 28S, 69L, 71V, 73K, and 94S in the FR region of SEQ ID NO: 69.
  • the humanized CD28-VL comprises the amino acid sequence of SEQ ID NO: 80, or a variant thereof.
  • the variant is one or more amino acid substitutions in the FR region of SEQ ID NO: 80.
  • the antigen-binding molecule or anti-CD28 antibody of any one of the above wherein the amino acid sequence of CD28-VH is at least 90% identical to SEQ ID NO: 95, 35, 94, 96, 97 or 98 , 95%, 96%, 97%, 98% or 99% sequence identity, and the amino acid sequence of CD28-VL is identical to SEQ ID NO: 101, 36, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 have at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the CD28-VH has an amino acid sequence as set forth in SEQ ID NO: 95, 35, 94, 96, 97 or 98
  • the CD28-VL has an amino acid sequence as SEQ ID NO: 101, 36 , 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115.
  • the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 94, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115, or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 95
  • the amino acid sequence of CD28-VL is shown in SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109 , 110, 111, 112, 113, 114 or 115, or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 96, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 99 or 100, or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 97
  • the amino acid sequence of CD28-VL is shown in SEQ ID NO: 99, 100 or 101.
  • the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 95, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO:101 shown.
  • the antigen-binding molecule or anti-CD28 antibody of any one of the preceding items wherein the amino acid sequence of CD28-VH is consistent with SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69 Tool There is at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and the amino acid sequence of CD28-VL is identical to SEQ ID NO: 80, 34, 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79 have a sequence identity of at least 90%, 95%, 96%, 97%, 98% or 99%.
  • the CD28-VH has an amino acid sequence set forth in SEQ ID NO: 68, 33, 63, 64, 65, 66, 67, or 69
  • the CD28-VL has an amino acid sequence set forth in SEQ ID NO. :80, 34, 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79.
  • the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 63, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO:70 or 71 shown, or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 64, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 70, 71 or 72, or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 65
  • the amino acid sequence of CD28-VL is shown in SEQ ID NO: 70, 71 or 72, or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 66
  • the amino acid sequence of CD28-VL is shown in SEQ ID NO: 70, 71, 72, 74, 75, 76 or 79, or
  • CD28-VH The amino acid sequence of CD28-VH is shown in SEQ ID NO: 67
  • amino acid sequence of CD28-VL is shown in SEQ ID NO: 72, 74, 75, 76 or 79, or
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 68
  • the amino acid sequence of CD28-VL is shown in SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79 or 80 .
  • the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 68, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO:80 is shown.
  • the antigen-binding molecule as described above, wherein:
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 167, PSMA-LCDR2 as shown in SEQ ID NO: 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169; or
  • the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175.
  • the antigen-binding molecule of any one of the preceding items, said PSMA-VH and/or said PSMA-VL is murine or humanized. In some embodiments, the PSMA-VH and/or the PSMA-VL are humanized. In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
  • amino acid sequence of PSMA-VH is shown in SEQ ID NO: 197, 199, 332, 194, 195, 196 or 198, and the amino acid sequence of PSMA-VL is SEQ ID NO: 201, 202, 333 or 200 shown; or
  • amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 204, 184 or 203
  • amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 206, 185 or 205.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199
  • the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202.
  • variable regions and CDRs described above are defined according to the Kabat numbering rule.
  • the antibody series 81 and 129 disclosed according to the embodiments herein have a similar technical solution scope to the antibody series 97 and 94 described above.
  • the bispecific antigen-binding molecules of the present disclosure are not limited to a specific molecular structure as long as they have the desired antigen-binding function.
  • the bispecific antigen-binding molecules herein may be bivalent (1+1), trivalent (2+1), or tetravalent (2+2).
  • the antigen-binding module in the antigen-binding molecule can be any antibody fragment with antigen-binding activity, which is fused through a peptide linker.
  • the peptide linkers of the present disclosure eg, linkers 1 to 11
  • the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues.
  • the peptide linkers each independently have the structure of L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, A, GS, GGS (SEQ ID NO: 399), GGGS (SEQ ID NO: 400), SGGGGS (SEQ ID NO: 294), GGGTKLTVLGGG (SEQ ID NO: 401), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is bond, G, GG, GGG (SEQ ID NO:295) or GGGG (SEQ ID NO:256), and the peptide linker is not a bond.
  • the peptide linker is 3-15 amino acid residues in length.
  • the peptide linkers each independently have the structure (GGGGS)n, where n is 1, 2, or 3.
  • the amino acid sequences of linker 1, linker 2, linker 3 and linker 4 are as shown in SEQ ID NO: 223.
  • the antigen-binding molecule of the present disclosure has a first chain with a structure represented by formula (a), a second chain with a structure represented by formula (b), and a second chain with a structure represented by formula (c-1).
  • antigen binding molecules include:
  • the antigen-binding molecule includes a first chain having a structure shown in formula (e-1), a second chain having a structure shown in formula (f-1), and a first chain having a structure shown in formula (g)
  • antigen binding molecules include:
  • amino acid sequence variants of the antigen-binding molecules provided herein are contemplated.
  • Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions, and/or substitutions of residues within the amino acid sequence of the antigen-binding molecule. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, so long as the final construct possesses the desired characteristics, such as antigen-binding properties.
  • antigen-binding molecule variants having one or more amino acid substitutions are provided. Substitutions can be made in CDR and FR. Conservative substitutions are shown in Table 2 under the heading "Preferred substitutions”. More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions" and are described further below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest and the product screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • amino acids can be grouped as follows:
  • a non-conservative substitution would be the substitution of a member of one class for a member of another class.
  • substitution variant involves the substitution of one or more CDR residues of a parent antibody (eg, a humanized or human antibody).
  • a parent antibody eg, a humanized or human antibody
  • the resulting variants selected for further study will have alterations (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will be substantially Retains certain biological properties of the parent antibody.
  • One exemplary substitution variant is an affinity matured antibody, which may be conveniently produced, for example, using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).
  • Changes can be made to the CDRs, for example to improve antibody affinity.
  • affinity maturation diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. middle. Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity.
  • CDR orientation in which several CDR residues (eg 4-6 residues at a time) are randomized.
  • CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.
  • substitutions, insertions, or deletions may occur within one or more CDRs as long as such changes do not substantially reduce the ability of the antibody to bind the antigen.
  • conservative changes eg, conservative substitutions, as provided herein
  • each CDR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.
  • alanine scanning mutagenesis One method that can be used to identify residues or regions in an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis.”
  • a residue or group of residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., Ala or polyalanine
  • Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution.
  • the contact points between the antibody and the antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include: fusion of 1 residue at the amino and/or carboxyl terminus or polypeptides of 100 or more residues in length; and intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include antibodies with an N-terminal methionyl residue.
  • Other insertional variants of antibody molecules include fusions with enzymes (or polypeptides that extend the serum half-life of the antibody) fused to the N- or C-terminus of the antibody.
  • one of the antigen-binding module that specifically binds PSMA and the antigen-binding module that specifically binds CD28 is a replaced Fab
  • the replaced Fab comprises Heavy chain variable region, light chain variable region, Titin chain and Obscurin chain.
  • the original CH1 and CL of the Fab are replaced by Titin chains and Obscurin chains.
  • the sequences of Titin chain and Obscurin chain are shown in Table 3-1 and Table 3-2.
  • the Fc region of an antigen-binding molecule of the present disclosure includes one or more amino acid substitutions that reduce its binding to an Fc receptor, e.g., its binding to an Fc ⁇ receptor, and reduce or Eliminate effector functions.
  • the native IgG Fc region specifically the IgG1 Fc region or the IgG4 Fc region, may cause the antigen-binding molecules of the present disclosure to target cells expressing Fc receptors rather than cells expressing the antigen.
  • the engineered Fc region of the present disclosure exhibits reduced binding affinity for Fc receptors and/or reduced effector function.
  • the engineered Fc region has a reduced binding affinity for the Fc receptor by more than 50%, 80%, 90%, or 95% compared to the native Fc region.
  • the Fc receptor is an Fc ⁇ receptor.
  • the Fc receptor is a human Fc ⁇ receptor, such as Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIB, Fc ⁇ RIIIa.
  • the engineered Fc region also has reduced binding affinity for complement, such as Clq, compared to the native Fc region.
  • the engineered Fc region has no reduced binding affinity for the neonatal Fc receptor (FcRn) compared to the native Fc region.
  • the engineered Fc region has reduced effector functions, which may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced Antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced interaction with NK cells binding, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced of T cells.
  • CDC complement-dependent cytotoxicity
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • ADCP reduced antibody-dependent cellular phagocytosis
  • cytokine secretion reduced immune complex-mediated antigen uptake by antigen-presenting cells
  • reduced interaction with NK cells binding reduced binding to macrophages
  • monocytes reduced binding to monocytes
  • substitutions of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function.
  • the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index.
  • amino acid residue substitutions at positions such as 228 may reduce effector function.
  • Antigen-binding molecules may also contain disulfide bond modifications, such as 354C in the first subunit and 349C in the second subunit.
  • disulfide bond modifications such as 354C in the first subunit and 349C in the second subunit.
  • mutations 252Y, 254T, and 256E can be introduced.
  • the Fc region of the present disclosure includes modifications according to the knob-into-hole (KIH) technique, which involves introducing a knob at the interface of the first subunit and a knob at the interface of the second subunit.
  • KH knob-into-hole
  • a hole structure is introduced at the interface of the base. This allows the protruding structure to be positioned in the pore structure, promoting the formation of heterodimers and inhibiting the production of homodimers.
  • the bulge structure is built by replacing small amino acid side chains from the interface of the first subunit with larger side chains, such as tyrosine or tryptophan.
  • the pore structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller amino acid side chains, such as alanine or threonine.
  • the bulge and pore structures are prepared by altering the nucleic acid encoding the polypeptide with optional amino acid substitutions as shown in the table below:
  • the C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; it can also be a truncated C-terminus, for example, one or two C-terminal amino acid residues have been removed from the truncated C-terminus.
  • the C-terminus of the heavy chain is a shortened C-terminus ending in PG.
  • a composition of intact antibodies may include a population of antibodies with all K447 residues and/or G446+K447 residues removed.
  • the composition of intact antibodies can include a population of antibodies without removal of the K447 residue and/or G446+K447 residues.
  • the composition of intact antibodies has a population of antibodies with and without a K447 residue and/or an antibody mixture of G446+K447 residues.
  • Antigen-binding molecules can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the antigen-binding molecules are provided.
  • nucleic acids In the case of native antibodies, native antibody fragments or bispecific antibodies with homodimeric heavy chains, two nucleic acids are required, one for the light chain or fragments thereof and one for the heavy chain or fragments thereof.
  • nucleic acids encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (eg, the light chain and/or heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.
  • nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heterologous monomeric Fc region polypeptide, and one one for the second light chain, and one for the second heavy chain comprising a second heterologous monomeric Fc region polypeptide.
  • These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors, usually these nucleic acids are located on two or three expression vectors, that is, one vector can contain more than one of these nucleic acids.
  • the present disclosure provides an isolated nucleic acid encoding an antibody as described above. Such nucleic acids can independently encode any of the aforementioned polypeptide chains.
  • the present disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids.
  • the present disclosure provides host cells comprising such nucleic acids.
  • a method of preparing an antigen-binding molecule is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally The antibody is recovered from the host cell (or host cell culture medium).
  • nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • nucleic acids can be easily can be isolated and sequenced (for example, by using oligonucleotide probes capable of binding specifically to the genes encoding the antibody heavy and light chains), or produced by recombinant methods or obtained by chemical synthesis.
  • Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein.
  • antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required for the antibody. After expression, the antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of vectors with Antibodies with partially or fully human glycosylation patterns.
  • Suitable host cells for expression of (glycosylated) antibodies may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells.
  • baculovirus strains have been identified that can be used in combination with insect cells, especially for transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, such as US5959177, US 6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, such as mammalian cell lines adapted to growth in suspension.
  • Suitable mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); mouse Sertoli ( sertoli) cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat (buffalo rat) liver cells ( BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse breast tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells.
  • COS-7 monkey kidney CV1 line
  • TM4 cells mouse Sertoli ( sertoli) cells
  • CV1 monkey kidney cells
  • VEO-76 African green monkey kidney cells
  • HELA human cervical cancer cells
  • BRL3A canine kidney cells
  • MDCK buffalo rat (buffalo rat) liver cells
  • W138 human liver cells
  • Hep G2
  • Suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as Y0, NSO, and Sp2/0.
  • CHO Chinese hamster ovary
  • myeloma cell lines such as Y0, NSO, and Sp2/0.
  • the present disclosure provides antigen-binding molecules that can be used to detect the presence of PSMA and/or CD3 in a biological sample.
  • detection encompasses either quantitative or qualitative detection.
  • a biological sample includes cells or tissue, such as tumor tissue.
  • antigen binding molecules for use in diagnostic or detection methods are provided.
  • a method of detecting the presence of PSMA and/or CD3 in a biological sample is provided.
  • the method includes contacting a biological sample with an antigen-binding molecule under appropriate conditions and detecting whether a complex is formed between the detection reagent and the antigen.
  • antigen binding molecules are used to select subjects suitable for treatment, for example PSMA and/or CD3 are biomarkers used to select patients.
  • Exemplary conditions that can be diagnosed using the antigen-binding molecules of the present disclosure are, for example, tumors or cancers.
  • labeled antigen binding molecules include, but are not limited to, direct Labels or moieties that detect directly (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties that detect indirectly (e.g., moieties that detect indirectly via enzymatic reactions or molecular interactions, such as enzymes or ligands) body).
  • direct Labels or moieties that detect directly such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels
  • moieties that detect indirectly e.g., moieties that detect indirectly via enzymatic reactions or molecular interactions, such as enzymes or ligands
  • compositions comprising the antigen-binding molecules are provided, eg, for use in any of the following methods of treatment.
  • a pharmaceutical composition includes any of the antigen-binding molecules provided herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition includes any of the antigen-binding molecules provided herein and at least one additional therapeutic agent.
  • compositions of antigen-binding molecules of the present disclosure are prepared by mixing such antigen-binding molecules with the desired purity with one or more optional pharmaceutically acceptable carriers, the pharmaceutical compositions In the form of lyophilized compositions or aqueous solutions.
  • Formulations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile membrane.
  • antigen-binding molecules Any of the antigen-binding molecules provided herein can be used in therapeutic methods.
  • the present disclosure provides use of an antigen-binding molecule in the manufacture or preparation of a medicament.
  • the medicament is used to treat proliferative diseases or tumors.
  • the drug is present in an effective amount for the above-mentioned diseases.
  • the effective amount is a unit daily dose or a unit weekly dose.
  • the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional treatments). agent).
  • a "subject" according to any of the above embodiments may be a human.
  • a pharmaceutical composition comprising the antigen-binding molecule, eg, for use in any of the above pharmaceutical uses or methods of treatment.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • the antigen-binding molecules of the present disclosure can be used alone or in combination with other agents for treatment.
  • the antigen-binding molecules of the present disclosure can be co-administered with at least one additional therapeutic agent.
  • the additional therapeutic agent is a bispecific antibody or an anti-PD-1 antibody that specifically binds PSMA and CD3.
  • the antigen-binding molecules of the present disclosure may be administered by any suitable means, including parenterally, intrapulmonary, and intranasal, and, if local treatment is desired, intralesional administration.
  • Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.
  • Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the antigen-binding molecules of the present disclosure will be formulated, administered, and administered in a manner consistent with GOOD MEDICAL PRACTICE. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and others known to the medical practitioner factor.
  • the antigen-binding molecules need not be, but are optionally, formulated with one or more agents currently used to prevent or treat the disorder. Such other reagents
  • the effective amount depends on the amount of antigen-binding molecule present in the pharmaceutical composition, the type of condition or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and by any route empirically/clinically determined to be appropriate.
  • the antigen-binding molecules of the present disclosure when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the nature of the therapeutic molecule Type, severity and duration of disease, whether administration is for prophylactic or therapeutic purposes, previous treatments, patient's clinical history and response to therapeutic molecules, and the judgment of the attending physician.
  • the therapeutic molecules are appropriately administered to the patient in one session or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg of the antigen-binding molecule may be an initial candidate dose for administration to the patient, whether, for example, by one or more divided administrations or by continuous infusion .
  • a typical daily dose may range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the exemplary unit daily dose is 50 ⁇ g-5g.
  • an article of manufacture contains materials useful in treating, preventing, and/or diagnosing the conditions described above.
  • the article includes a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like.
  • Containers can be formed from a variety of materials such as glass or plastic.
  • At least one active agent in the composition is an antigen-binding molecule of the present disclosure.
  • the label or package insert indicates use of the composition to treat the selected condition.
  • an article of manufacture may comprise: (a) a first container having a composition therein, wherein the composition comprises an antigen-binding molecule of the present disclosure; and (b) a second container having a composition therein, wherein the composition The agent contains additional cytotoxic agents or other therapeutic agents.
  • the article of manufacture in this embodiment of the present disclosure may further comprise a package insert indicating that the composition may be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer. It may further include other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • the article of manufacture is prepared in the form of a kit.
  • Titin chain/Obscurin chain of the present disclosure can be derived from any suitable polypeptide, including polypeptides derived from WO2021139758 (incorporated herein by reference in its entirety) and CN202110527339.7 and the patents (incorporated herein by reference in their entirety) which are priority documents. .
  • DI bispecific antibodies against hNGF and hRANKL are constructed: DI-2 to DI-20, which include the first heavy chain, the second heavy chain, the first light chain and the second Light chain:
  • the first heavy chain: from N-terminus to C-terminus is: [VH1-I]-[linker 1]-[Obscurin chain]-[Fc2],
  • the first light chain from N-terminal to C-terminal: [VL1-I]-[Linker 2]-[Titin chain],
  • Second heavy chain from N-terminus to C-terminus: [VH2-D]-[CH1]-[Fc1], and
  • Second light chain from N-terminus to C-terminus: [VL2-D]-[CL];
  • VH1-I and VL1-I are respectively the heavy chain variable region and light chain variable region of I0 in WO2021139758
  • VH2-D and VL2-D are respectively the heavy chain variable region and light chain variable region of D0 in WO2021139758. district.
  • the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the DI bispecific antibody are shown in the table below.
  • Test Example 4 of WO2021139758 was used to detect the binding activity of the DI-2 to DI-20 bispecific antibodies and their antigens.
  • Thermal stability studies of antibodies were performed. Research method: Use PBS to dilute the concentration of the antibody to 5 mg/mL, and use a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit) to measure its thermal stability. The experimental results showed that the antigen-binding activity of the modified bispecific antibody did not change significantly; and, compared with DI-2, the Tm1 of DI-4 to DI-8, DI-10 to DI-16, and DI-20 (°C) and Tonset (°C) are significantly improved, and the thermal stability of bispecific antibodies is better.
  • PL bispecific antibodies against hPDL1 and hCTLA4 were constructed: PL-1 to PL-19, which comprise the first heavy chain, the second heavy chain, the first light chain and the second light chain as follows:
  • the first heavy chain from N end to C end: [VH1-P]-[Linker 1]-[Obscurin chain]-[Fc1],
  • the first light chain from N-terminal to C-terminal: [VL1-P]-[Linker 2]-[Titin chain],
  • Second heavy chain from N-terminus to C-terminus: [VH2-L]-[CH1]-[Fc2], and
  • Second light chain from N-terminus to C-terminus: [VL2-L]-[CL];
  • VH1-P and VL1-P are the heavy chain variable region and light chain variable region of the h1831K antibody in WO2020177733A1, respectively.
  • the amino acid sequences of VH2-L and VL2-L are as follows.
  • the binding activity of the PL bispecific antibody was detected with reference to the ELISA method in Test Example 4 of WO2021139758, in which hPDL1 and hCTLA4 antigens were purchased from: Sino biology. Thermal stability studies of antibodies were performed. Method: Use PBS to dilute the concentration of the antibody to 1.4-3mg/mL, and use a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit) to measure its thermal stability.
  • the experimental results show that the PL bispecific antibody still has good binding activity to the antigen; and, compared with PL-1, the Tm1 (°C), Tagg 266 (°C), Tonset (°C) of PL-2 to PL-19 There is a significant improvement, and the thermal stability of bispecific antibodies is better.
  • HJ bispecific antibodies against hIL5 and hTSLP were constructed: HJ-3 to HJ11, which comprise the first heavy chain, the second heavy chain, the first light chain and the second light chain as follows:
  • the first heavy chain: from N-terminal to C-terminal is: [VH1-H]-[Linker 1]-[Titin chain]-[Fc1],
  • the first light chain from N end to C end: [VL1-H]-[Linker 2]-[Obscurin chain],
  • Second heavy chain from N terminus to C terminus: [VH2-J]-[CH1]-[Fc2], and
  • Second light chain from N-terminus to C-terminus: [VL2-J]-[CL];
  • VH1-H and VL1-H are respectively the heavy chain variable region and light chain variable region of H0 in WO2021139758
  • VH2-J and VL2-J are respectively the heavy chain variable region and light chain variable region of J1 in WO2021139758. district.
  • the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the HJ bispecific antibody in this example are shown in the table below.
  • the antigen-binding activity of the HJ bispecific antibody was detected with reference to the method in Test Example 4 in WO2021139758.
  • the method is: prepare the HJ bispecific antibody dilution solution with a buffer solution of 10mM acetic acid pH 5.5 and 9% sucrose, and then concentrate the bispecific antibody through ultrafiltration concentration to obtain different concentrations. HJ bispecific antibody solution (see Table 13-2 for the concentration of HJ bispecific antibody), and then place the concentrated solution in a 40°C incubator for incubation.
  • CHO-hPSMA Construct CHO-hPSMA, CHO-APC-hPSMA, CHO-hCD28, HEK293-hCD28, CHO-APC-hPSMA-PDL1 and Jurkat-PD1 cell lines.
  • the relevant protein sequences are as follows:
  • HEK293T cells co-transfect HEK293T cells (ATCC, CRL-11268) with three plasmids: pVSV-G, pCMV-dR8.91 and pCDH-hPSMA for packaging.
  • Virus 48 hours after transfection, virus-infected HEK293 (ATCC, CRL-1573), CHO-K1 (ATCC, CCL-61) or CHO-APC cells (Promega, JA9441) were collected. After two weeks of pressure screening, the cells were subdivided. After cloning, CHO-hPSMA, CHO-APC-hPSMA, CHO-hCD28 and HEK293-hCD28 cell lines with high expression of PSMA or CD28 were obtained through FACS detection.
  • the gene encoding human PDL1 or human PD1 full-length was cloned into the mammalian cell expression vector pCDH, and HEK293T cells were co-transfected with three plasmids: pVSV-G, pCMV-dR8.91 and pCDH-hPSMA ( CRL-11268) packaged virus, 48 hours after transfection, collect the virus to infect CHO-APC-hPSMA or Jurkat (ATCC, TIB-152) cells as mentioned above, and perform cell sorting through pressure screening to obtain high expression of PDL1 or PD1 CHO-APC-hPSMA-PDL1 and Jurkat-PD1 cell lines.
  • This disclosure uses hybridoma technology to prepare monoclonal antibodies against human CD28.
  • the resulting antibodies specifically bind to human CD28 with higher affinity, and can cross-bind with cynomolgus CD28 and bind to human CD28 and cynomolgus monkeys on the cell surface.
  • CD28 has good binding activity.
  • the initial immunization with protein is 50 ⁇ g, and the booster immunization is 25 ⁇ g each time.
  • Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No. 77161) is an adjuvant for cross-immunization.
  • Cellular immunity was immunized according to 5E6 each time. After the initial immunization and seven boosting immunizations, mice with high serum antibody titers were selected for spleen cell fusion.
  • the immunogen sequence is as follows:
  • hybridoma culture supernatant was detected according to the hybridoma cell growth density. Hybridomas with good binding activity to human CD28 on the cell surface and good activation function were screened out. Monoclonal hybridoma cells were collected, RNA was extracted with NucleoZol (MN) (according to the kit instructions), and reverse transcription was performed (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and then sequenced to obtain the amino acid sequence of the CDR and variable region of the antibody in the hybridoma cell line.
  • MN NucleoZol
  • variable region sequence of the mouse anti-CD28 antibody was combined with the heavy chain and light chain constant regions shown in SEQ ID NO: 144 and SEQ ID NO: 145 to obtain a chimeric antibody.
  • Chi81 represents a chimeric antibody comprising the m81 murine heavy chain variable region, the light chain variable region, and the heavy chain and light chain constant regions shown in SEQ ID NO: 144 and SEQ ID NO: 145. Chi94, Chi97 and Chi129 and so on.
  • FR1, FR2, and FR3 of IGKV1-12*01 and FR4 of IGKJ4*01 were selected as the light chain framework region template; FR1, FR2, FR3 of IGHV1-46*01, and IGHJ1*01 were selected.
  • FR4 serves as the heavy chain framework region template.
  • the amino acid residues at positions 43, 54 and/or 73 of the light chain variable region of the humanized antibody are substituted; and/or the amino acid residues at positions 1, 54 and/or 73 of the heavy chain variable region of the humanized antibody are substituted.
  • the amino acid residues at positions 69, 71, 73, 78, 94, 100a, 100b and/or 100c are substituted.
  • R71V means that according to the Kabat numbering system, R at position 71 is mutated to V; 100a means that according to the kabat numbering rules 100A bit, and so on.
  • the CDRs of the humanized antibody of m81 are as follows:
  • variable region of humanized antibody h81 is as follows: Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are fr.
  • the humanized antibody of m94 selects FR1, FR2, FR3 of IGKV1-12*01, and FR4 of IGKJ4*01 as the light chain framework region template; selects FR1, FR2, FR3 of IGHV1-3*01 and FR4 of IGHJ1*01 As a heavy chain framework region template.
  • the amino acid residues at positions 43, 50, 51, 53, 54, 55 and/or 70 of the light chain variable region of the humanized antibody are substituted; and/or the heavy amino acid residues of the humanized antibody are substituted.
  • the amino acid residues at positions 1, 28, 66, 69, 71, 73 and/or 94 in the variable region of the chain are substituted.
  • R94S means that R at position 94 is mutated to S according to the Kabat numbering system.
  • the CDR region sequence of the humanized antibody of m94 is as follows:
  • the sequence of the humanized antibody h94 variable region is as follows: Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are fr.
  • FR1, FR2, FR3 of IGKV1-33*01 and FR4 of IGKJ4*01 were selected as the light chain framework region template; FR1, FR2, FR3 and IGHJ6* of IGHV1-46*01 were selected.
  • FR4 of 01 serves as the heavy chain framework region template.
  • the amino acid residues at positions 41, 42, 43, 44, 50, 51, 52, 53, 54, 55, 56 and/or 71 on the light chain variable region of the humanized antibody are substituted; and/or substitution of amino acid residues at positions 1, 26, 29, 69, 71, 78 and/or 93 of the heavy chain variable region of the humanized antibody.
  • F71Y means that F at position 71 is mutated back to Y according to the Kabat numbering system.
  • the CDR regions of the m97 humanized antibody are as follows:
  • variable region of humanized antibody h97 is as follows: Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are fr.
  • the humanized antibody of mouse antibody m129 selects FR1, FR2, FR3 of IGKV1-33*01, and FR4 of IGKJ4*01 as the light chain framework region template; selects FR1, FR2, FR3 and IGHJ6* of IGHV1-46*01 FR4 of 01 serves as the heavy chain framework region template.
  • the amino acid residues at positions 41, 42, 43, 44, 50, 53, 54, 55 and/or 73 of the light chain variable region of the humanized antibody are substituted; and/or human The amino acid residues at positions 1, 25, 30, 34, 52, 71 and/or 78 of the heavy chain variable region of the humanized antibody are substituted.
  • the CDRs of the humanized antibody to m129 are as follows:
  • variable region sequence of humanized antibody 129 is as follows:
  • the heavy chain variable region and light chain variable region of the anti-CD28 humanized antibody were recombined with the heavy chain constant region hIgG1:CH1-Fc and the light chain constant region CL, respectively, to obtain a full-length humanized antibody.
  • the humanized antibodies against CD28 of the present disclosure are as follows:
  • the full-length sequence of the anti-CD28 humanized antibody is as follows:
  • Control antibodies for anti-CD28 antibodies used in this disclosure are as follows:
  • REGN-hIgG1 was constructed with reference to patent US20190389951A1, and its sequence is as follows:
  • CD28 super agonist TGN1412 was prepared with reference to patent US07585960B2. The specific sequence is as follows:
  • the present disclosure prepares monoclonal antibodies against human PSMA through hybridoma technology.
  • the obtained antibody specifically binds to human PSMA with high affinity and can cross-react with cynomolgus monkey PSMA; the obtained antibody has good binding activity to human PSMA and cynomolgus monkey PSMA on the cell surface.
  • MN NucleoZol
  • the cDNA obtained by reverse transcription was PCR amplified and sequenced using mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503).
  • the amino acid sequences of the CDR and variable regions of the screened monoclonal hybridoma cell lines are as follows:

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Abstract

The present disclosure relates to an antigen-binding molecule specifically binding to PSMA and CD28 and pharmaceutical use thereof. The antigen-binding molecule can be used to treat tumors.

Description

特异性结合PSMA和CD28的抗原结合分子及其医药用途Antigen-binding molecules that specifically bind PSMA and CD28 and their medical uses
本披露要求2022年04月11日提交的中国专利申请202210371696.3的优先权。This disclosure claims priority to Chinese patent application 202210371696.3 filed on April 11, 2022.
技术领域Technical field
本披露属于生物技术领域，更具体地，本披露涉及PSMA/CD28抗原结合分子及其应用。The present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to PSMA/CD28 antigen-binding molecules and their applications.
背景技术Background technique
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.
PSMA属于谷氨酸羧肽酶II(Glutamate carboxypeptidase II，GCPII)，由750个氨基酸组成。PSMA的胞外部分由3个结构域组成：蛋白酶样结构域(protease-like domain)、顶极结构域(apical domain)和C-末端。三个结构域都参与底物的结合，其中，蛋白酶样结构域和顶极结构域直接与底物结合，C-末端使PSMA形成二聚体发挥作用。在前列腺癌患者中，PSMA在肿瘤组织上的表达水平相较于正常前列腺提高了100-1000倍,特别是在低分化的、转移的和激素难以治疗的癌中，PSMA的表达明显增加。在一些恶性肿瘤的肿瘤外周和肿瘤内区域中的毛细血管的内皮细胞中也表达PSMA，但在正常组织的血管中不表达。另外，PSMA与肿瘤血管生成相关(Silver D.A.(1997)Clinical Cancer Research 3：81-85)。最近，已经证明PSMA在结肠癌、乳腺癌、膀胱癌、胰腺癌、肾癌和黑色素瘤中的肿瘤相关新血管系统的内皮细胞中表达(Chang，S.S.(2004)Curr Opin Investig Drugs 5：611-5)。因此，PSMA不仅能作为针对前列腺癌的药物，也可能适用于其他类型的肿瘤。PSMA belongs to Glutamate carboxypeptidase II (Glutamate carboxypeptidase II, GCPII) and consists of 750 amino acids. The extracellular part of PSMA consists of three domains: protease-like domain, apical domain and C-terminus. All three domains are involved in substrate binding. Among them, the protease-like domain and the apical domain directly bind to the substrate, and the C-terminus allows PSMA to form a dimer to function. In patients with prostate cancer, the expression level of PSMA in tumor tissue is 100-1000 times higher than that in normal prostate. Especially in poorly differentiated, metastatic and hormone-refractory cancers, the expression of PSMA is significantly increased. PSMA is also expressed in the endothelial cells of capillaries in the tumor periphery and intratumoral areas of some malignant tumors, but is not expressed in blood vessels of normal tissues. In addition, PSMA is related to tumor angiogenesis (Silver D.A. (1997) Clinical Cancer Research 3: 81-85). Recently, PSMA has been shown to be expressed in endothelial cells of tumor-associated neovasculature in colon, breast, bladder, pancreatic, renal, and melanoma cancers (Chang, S.S. (2004) Curr Opin Investig Drugs 5: 611- 5). Therefore, PSMA can be used not only as a drug against prostate cancer, but may also be applicable to other types of tumors.
一般情况下，T细胞的激活依赖于被APC细胞或其他细胞表面MHC-肽复合物激活的TCR提供的第一信号，同时需要共刺激因子提供第二信号才可彻底激活T细胞。CD28作为T细胞表面主要共刺激之一，为免疫球蛋白超家族成员之一。在人类中，CD28主要表达于T细胞表面，也少量表达于骨髓基质细胞、浆细胞、中性粒细胞和嗜酸性粒细胞等其他细胞中。CD28分子可通过结合表达于激活APC细胞表面的CD80/CD86分子进一步激活下游NFAT、NF-κB和AP-1信号通路，促进T细胞的激活和增殖。In general, T cell activation relies on the first signal provided by TCR activated by APC cells or other cell surface MHC-peptide complexes. At the same time, costimulatory factors are required to provide a second signal to completely activate T cells. As one of the major costimulators on the surface of T cells, CD28 is a member of the immunoglobulin superfamily. In humans, CD28 is mainly expressed on the surface of T cells, and is also expressed in small amounts on other cells such as bone marrow stromal cells, plasma cells, neutrophils, and eosinophils. CD28 molecules can further activate the downstream NFAT, NF-κB and AP-1 signaling pathways by binding to CD80/CD86 molecules expressed on the surface of activated APC cells, promoting the activation and proliferation of T cells.
因此，提供靶向PSMA/CD28的双特异性抗体是治疗肿瘤等疾病的一个有效手段。Therefore, providing bispecific antibodies targeting PSMA/CD28 is an effective means to treat tumors and other diseases.
发明内容Contents of the invention
本披露提供了一种特异性结合CD28和PSMA抗原结合分子和特异性结合CD28的抗体。The present disclosure provides an antibody that specifically binds CD28 and PSMA antigen-binding molecules and an antibody that specifically binds CD28.
在一个方面，本披露提供了一种抗原结合分子，其包含至少一个特异性结合 CD28的抗原结合模块和至少一个特异性结合PSMA的抗原结合模块，所述特异性结合CD28的抗原结合模块包含重链可变区CD28-VH和轻链可变区CD28-VL，所述特异性结合PSMA的抗原结合模块包含重链可变区PSMA-VH和轻链可变区PSMA-VL。In one aspect, the present disclosure provides an antigen-binding molecule comprising at least one specific binding The antigen-binding module of CD28 and at least one antigen-binding module that specifically binds to PSMA, the antigen-binding module that specifically binds to CD28 includes the heavy chain variable region CD28-VH and the light chain variable region CD28-VL, and the specificity The antigen-binding module that binds PSMA includes the heavy chain variable region PSMA-VH and the light chain variable region PSMA-VL.
在一些实施方式中，如前所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule as described above, wherein:
(i)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：35中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：36、103、104、105、106、107、108、109、110、111、112、113、114或115中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或(i) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28 in SEQ ID NO: 36, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 respectively -amino acid sequences of LCDR1, CD28-LCDR2 and CD28-LCDR3, or
(ii)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：33中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：34、72、73、74、75、76、77、78、79或80中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或(ii) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28-LCDR1, CD28-LCDR2 and CD28 in SEQ ID NO: 34, 72, 73, 74, 75, 76, 77, 78, 79 or 80 respectively -The amino acid sequence of LCDR3, or
(iii)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：37、129或132中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：38、138、139、141、142或143中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或(iii) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37, 129 or 132, and CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in the CD28-VL respectively comprise the amino acids of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, 138, 139, 141, 142 or 143 sequence, or
(iv)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：31、46、47、48或49中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：32或52中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列。(iv) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, 46, 47, 48 or 49 The amino acid sequence, and CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in the CD28-VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32 or 52.
在一些实施方式中，如前所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule as described above, wherein:
(i)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：35中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：36中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或(i) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 36, or
(ii)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：33中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：80中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨 基酸序列，或(ii) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain the amino acids of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 80 respectively. amino acid sequence, or
所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：33中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：34中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33, and the CD28-VL in the CD28-VL. CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 34, or
(iii)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：37中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：38中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或(iii) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, or
(iv)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：31中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：32中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列；或(iv) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32; or
所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：31中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：52中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列。CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28-VL in the CD28-VL. CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 52.
在一些实施方案中，所述CD28-HCDR1、CD28-HCDR2、CD28-HCDR3、CD28-LCDR1、CD28-LCDR2和CD28-LCDR3根据Kabat、IMGT、Chothia、AbM或Contact编号规则定义。In some embodiments, the CD28-HCDR1, CD28-HCDR2, CD28-HCDR3, CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 are defined according to Kabat, IMGT, Chothia, AbM or Contact numbering rules.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH具有：包含SEQ ID NO：19的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：20的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：21的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：22的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：23、81、82、83、84、85、86、87、88、89、90、91、92或93的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：24的氨基酸序列的CD28-LCDR3；或(i) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR1 comprising SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92 or 93 amino acid sequence of CD28-LCDR2 and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24; or
(ii)所述CD28-VH具有：包含SEQ ID NO：13的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：14的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：15的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：16的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：17、54、55、56、57、58、59、60、61或62的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：18的氨基酸序列的 CD28-LCDR3；或(ii) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14 and CD28- comprising the amino acid sequence of SEQ ID NO: 15 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, comprising the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 The sequence of CD28-LCDR2 and the amino acid sequence comprising SEQ ID NO: 18 CD28-LCDR3; or
(iii)所述CD28-VH具有：包含SEQ ID NO：25或117的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26或116的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或(iii) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27 CD28-HCDR3 of the sequence, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28- comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
(iv)所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9、39、40、41或42的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：11或43的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3。(iv) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42 amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43. ID NO: 12 amino acid sequence of CD28-LCDR3.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述CD28-VH具有：包含SEQ ID NO：25的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and The CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
所述CD28-VH具有：包含SEQ ID NO：25的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：116的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and The CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
所述CD28-VH具有：包含SEQ ID NO：117的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and The CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
所述CD28-VH具有：包含SEQ ID NO：117的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：116的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3。 The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and The CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 30 amino acid sequence of CD28-LCDR3.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9、39、40、41或42的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：11的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3；或The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42. The amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12 The amino acid sequence of CD28-LCDR3; or
所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9、39、40、41或42的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：43的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3。The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42. The amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12 Amino acid sequence of CD28-LCDR3.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH具有：包含SEQ ID NO：19的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：20的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：21的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：22的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：23的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：24的氨基酸序列的CD28-LCDR3；或(i) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23 and CD28- comprising the amino acid sequence of SEQ ID NO: 24 LCDR3; or
(ii)所述CD28-VH具有：包含SEQ ID NO：13的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：14的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：15的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：16的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：62的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：18的氨基酸序列的CD28-LCDR3；或(ii) The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15. HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 62 and CD28- comprising the amino acid sequence of SEQ ID NO: 18 LCDR3; or
(iii)所述CD28-VH具有：包含SEQ ID NO：25的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或(iii) The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27. HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28- comprising the amino acid sequence of SEQ ID NO: 30 LCDR3; or
(iv)所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：43的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3。(iv) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 12 LCDR3.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH包含如SEQ ID NO：19所示的CD28-HCDR1、如SEQ ID NO：20所示的CD28-HCDR2和如SEQ ID NO：21所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：22所示的CD28-LCDR1、如SEQ ID NO：23、81、 82、83、84、85、86、87、88、89、90、91、92或93所示的CD28-LCDR2和如SEQ ID NO：24所示的的CD28-LCDR3；或(i) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and The CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 22, such as SEQ ID NO: 23, 81, CD28-LCDR2 represented by 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and CD28-LCDR3 represented by SEQ ID NO: 24; or
(ii)所述CD28-VH包含如SEQ ID NO：13所示的CD28-HCDR1、如SEQ ID NO：14所示的CD28-HCDR2和如SEQ ID NO：15所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：16所示的CD28-LCDR1、如SEQ ID NO：17、54、55、56、57、58、59、60、61或62所示的CD28-LCDR2和如SEQ ID NO：18所示的CD28-LCDR3；或(ii) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and The CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and CD28-LCDR3 shown in SEQ ID NO: 18; or
(iii)所述CD28-VH包含如SEQ ID NO：25或117所示的CD28-HCDR1、如SEQ ID NO：26或116所示的CD28-HCDR2和如SEQ ID NO：27所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：28所示的CD28-LCDR1、如SEQ ID NO：29、118、119、120、121或122所示的CD28-LCDR2和如SEQ ID NO：30所示的CD28-LCDR3；或(iii) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25 or 117, CD28-HCDR2 as shown in SEQ ID NO: 26 or 116, and CD28-HCDR1 as shown in SEQ ID NO: 27 HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 as set forth in SEQ ID NO: CD28-LCDR3 shown in 30; or
(iv)所述CD28-VH包含如SEQ ID NO：7所示的CD28-HCDR1、如SEQ ID NO：8所示的CD28-HCDR2和如SEQ ID NO：9、39、40、41或42所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：10所示的CD28-LCDR1、如SEQ ID NO：11或43所示的CD28-LCDR2和如SEQ ID NO：12所示的CD28-LCDR3。(iv) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28-HCDR2 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 CD28-HCDR3 as shown, and the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 11 or 43, and CD28-LCDR2 as shown in SEQ ID NO: 12 CD28-LCDR3.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述CD28-VH包含如SEQ ID NO：25所示的CD28-HCDR1、如SEQ ID NO：26所示的CD28-HCDR2和如SEQ ID NO：27所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：28所示的CD28-LCDR1、如SEQ ID NO：29、118、119、120、121或122所示的CD28-LCDR2和如SEQ ID NO：30所示的CD28-LCDR3；或The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO: 28, CD28-LCDR2 as shown in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as shown in SEQ ID NO: 30 ;or
所述CD28-VH包含如SEQ ID NO：25所示的CD28-HCDR1、如SEQ ID NO：116所示的CD28-HCDR2和如SEQ ID NO：27所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：28所示的CD28-LCDR1、如SEQ ID NO：29、118、119、120、121或122所示的CD28-LCDR2和如SEQ ID NO：30所示的CD28-LCDR3；或The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 116 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO: 28, CD28-LCDR2 as shown in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as shown in SEQ ID NO: 30 ;or
所述CD28-VH包含如SEQ ID NO：117所示的CD28-HCDR1、如SEQ ID NO：26所示的CD28-HCDR2和如SEQ ID NO：27所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：28所示的CD28-LCDR1、如SEQ ID NO：29、118、119、120、121或122所示的CD28-LCDR2和如SEQ ID NO：30所示的CD28-LCDR3；或The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 117, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO: 28, CD28-LCDR2 as shown in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as shown in SEQ ID NO: 30 ;or
所述CD28-VH包含如SEQ ID NO：117所示的CD28-HCDR1、如SEQ ID NO：116所示的CD28-HCDR2和如SEQ ID NO：27所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：28所示的CD28-LCDR1、如SEQ ID NO：29、118、119、120、121或122所示的CD28-LCDR2和如SEQ ID NO：30所示的CD28-LCDR3。 The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 117, CD28-HCDR2 as shown in SEQ ID NO: 116 and CD28-HCDR3 as shown in SEQ ID NO: 27, and the CD28- VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR3 as set forth in SEQ ID NO: 30 .
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述CD28-VH包含如SEQ ID NO：7所示的CD28-HCDR1、如SEQ ID NO：8所示的CD28-HCDR2和如SEQ ID NO：9、39、40、41或42所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：10所示的CD28-LCDR1、如SEQ ID NO：11所示的CD28-LCDR2和如SEQ ID NO：12所示的CD28-LCDR3；或The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 -HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 10, CD28-LCDR2 as set forth in SEQ ID NO: 11, and CD28-LCDR3 as set forth in SEQ ID NO: 12; or
所述CD28-VH包含如SEQ ID NO：7所示的CD28-HCDR1、如SEQ ID NO：8所示的CD28-HCDR2和如SEQ ID NO：9、39、40、41或42所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：10所示的CD28-LCDR1、如SEQ ID NO：43所示的CD28-LCDR2和如SEQ ID NO：12所示的CD28-LCDR3。The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 -HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 10, CD28-LCDR2 as set forth in SEQ ID NO: 43, and CD28-LCDR3 as set forth in SEQ ID NO: 12.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH包含如SEQ ID NO：19所示的CD28-HCDR1、如SEQ ID NO：20所示的CD28-HCDR2和如SEQ ID NO：21所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：22所示的CD28-LCDR1、如SEQ ID NO：23所示的CD28-LCDR2和如SEQ ID NO：24所示的CD28-LCDR3；或(i) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and The CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 22, CD28-LCDR2 as set forth in SEQ ID NO: 23, and CD28-LCDR3 as set forth in SEQ ID NO: 24; or
(ii)所述CD28-VH包含如SEQ ID NO：13所示的CD28-HCDR1、如SEQ ID NO：14所示的CD28-HCDR2和如SEQ ID NO：15所示的CD28-HCDR3，和所述CD28-VL如SEQ ID NO：16所示的CD28-LCDR1、如SEQ ID NO：62所示的CD28-LCDR2和如SEQ ID NO：18所示的CD28-LCDR3；或(ii) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and The CD28-VL is CD28-LCDR1 set forth in SEQ ID NO: 16, CD28-LCDR2 set forth in SEQ ID NO: 62, and CD28-LCDR3 set forth in SEQ ID NO: 18; or
(iii)所述CD28-VH包含如SEQ ID NO：25所示的CD28-HCDR1、如SEQ ID NO：26所示的CD28-HCDR2和如SEQ ID NO：27所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：28所示的CD28-LCDR1、如SEQ ID NO：29所示的CD28-LCDR2和如SEQ ID NO：30所示的CD28-LCDR3；或(iii) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and The CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, and CD28-LCDR3 as set forth in SEQ ID NO: 30; or
(iv)所述CD28-VH包含如SEQ ID NO：7所示的CD28-HCDR1、如SEQ ID NO：8所示的CD28-HCDR2和如SEQ ID NO：9所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：10所示的CD28-LCDR1、如SEQ ID NO：43所示的CD28-LCDR2和如SEQ ID NO：12所示的CD28-LCDR3。(iv) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO:7, CD28-HCDR2 as shown in SEQ ID NO:8 and CD28-HCDR3 as shown in SEQ ID NO:9, and The CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 43, and CD28-LCDR3 as shown in SEQ ID NO: 12.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述CD28-VH包含如SEQ ID NO：19所示的CD28-HCDR1、如SEQ ID NO：20所示的CD28-HCDR2和如SEQ ID NO：21所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：22所示的CD28-LCDR1、如SEQ ID NO：23所示的CD28-LCDR2和如SEQ ID NO：24所示的CD28-LCDR3。The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and the CD28- VL contains CD28-LCDR1 as shown in SEQ ID NO:22, CD28-LCDR2 as shown in SEQ ID NO:23, and CD28-LCDR3 as shown in SEQ ID NO:24.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述CD28-VH包含如SEQ ID NO：13所示的CD28-HCDR1、如SEQ ID NO：14所示的CD28-HCDR2和如SEQ ID NO：15所示的CD28-HCDR3，和所述CD28-VL如SEQ ID NO：16所示的CD28-LCDR1、如SEQ ID NO：62所示的CD28-LCDR2和如SEQ ID NO：18所示的CD28-LCDR3。 The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and the CD28- VL is CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 62, and CD28-LCDR3 as shown in SEQ ID NO: 18.
在一些实施方式中，如前任一项所述的抗原结合分子，所述CD28-HCDR1、CD28-HCDR2、CD28-HCDR3、CD28-LCDR1、CD28-LCDR2和CD28-LCDR3是根据Kabat编号规则定义的。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the CD28-HCDR1, CD28-HCDR2, CD28-HCDR3, CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 are defined according to the Kabat numbering rule.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的CD28-VH和CD28-VL是人源化的，均包含人抗体的FR区。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the CD28-VH and CD28-VL are humanized, both comprise the FR region of a human antibody.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH具有：包含SEQ ID NO：19的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：20的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：21的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、26A、29L、69L、71V、78A和93S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：22的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：23、81、82、83、84、85、86、87、88、89、90、91、92或93的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：24的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自41D、42G、43T、44I和71Y组成的组中的一个或多个氨基酸取代；或(ii)所述CD28-VH具有：包含SEQ ID NO：13的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：14的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：15的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、28S、69L、71V、73K和94S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：16的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：17、54、55、56、57、58、59、60、61或62的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：18的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自43S和70K组成的组中的一个或多个氨基酸取代；或(i) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 with the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and the FR region of the CD28-VL comprises one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44I and 71Y; or ( ii) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14 and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 15 , and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID NO: 16 CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 18 CD28-LCDR3, and the FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 43S and 70K; or
(iii)所述CD28-VH具有：包含SEQ ID NO：25或117的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26或116的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、25P、71V和78A组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自41D、42G、43T、44V和73L组成的组中的一个或多个氨基酸取代；或(iii) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27 The sequence of CD28-HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 71V and 78A; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 having the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 having the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and CD28-LCDR3 having the amino acid sequence of SEQ ID NO: 30, and said The FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44V and 73L; or
(iv)所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9、39、40、41或42的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、69L、71V、73K、78A和94S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：11或43的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序 列的CD28-LCDR3；且所述CD28-VL的FR区包含选自43S和73F组成的组中的一个或多个氨基酸取代。(iv) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and SEQ ID NO: 9, 39, 40, 41 or a CD28-HCDR3 with an amino acid sequence of 42, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S; and the CD28- VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12 The CD28-LCDR3 of the column; and the FR region of the CD28-VL contains one or more amino acid substitutions selected from the group consisting of 43S and 73F.
在一些实施方式中，如前任一项所述的抗原结合分子，其在25℃条件下以小于2×10-8M、8×10-9M或5×10-9M的KD结合人CD28，所述KD是通过表面等离子体共振法测量的。In some embodiments, the antigen-binding molecule as described in any one of the preceding items binds to human CD28 with a KD of less than 2×10 -8 M, 8×10 -9 M or 5×10 -9 M at 25°C. , the KD is measured by surface plasmon resonance method.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH具有：包含SEQ ID NO：19的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：20的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：21的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、26A、29L、69L、71V、78A和93S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：22的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：23的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：24的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自41D、42G、43T、44I和71Y组成的组中的一个或多个氨基酸取代；或(i) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, and the FR region of CD28-VL includes the selected One or more amino acid substitutions from the group consisting of 41D, 42G, 43T, 44I and 71Y; or
(ii)所述CD28-VH具有：包含SEQ ID NO：13的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：14的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：15的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、28S、69L、71V、73K和94S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：16的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：62的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：18的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自43S和70K组成的组中的一个或多个氨基酸取代；或(ii) The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15. HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 62, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 62, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 18, and the FR region of the CD28-VL contains a protein selected from the group consisting of 43S and one or more amino acid substitutions in the group consisting of 70K; or
(iii)所述CD28-VH具有：包含SEQ ID NO：25的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、25P、71V和78A组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自41D、42G、43T、44V和73L组成的组中的一个或多个氨基酸取代；或(iii) The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27. HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 71V and 78A; and the CD28-VL has: the amino acid sequence comprising SEQ ID NO: 28 CD28-LCDR1, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30, and the FR region of the CD28-VL comprises 41D, 42G, 43T One or more amino acid substitutions in the group consisting of , 44V and 73L; or
(iv)所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、69L、71V、73K、78A和94S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：43的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3；且所述 CD28-VL的FR区包含选自43S和73F组成的组中的一个或多个氨基酸取代。(iv) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 having the amino acid sequence of 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and said The FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 43S and 73F.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
i)所述PSMA-VH具有：包含SEQ ID NO：164的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：165的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：166的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：167的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：168的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：169的氨基酸序列的PSMA-LCDR3；或i) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 166 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 168, and PSMA comprising the amino acid sequence of SEQ ID NO: 169 -LCDR3; or
ii)所述PSMA-VH具有：包含SEQ ID NO：170的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：171的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：172的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：173的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：174的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：175的氨基酸序列的PSMA-LCDR3；或ii) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 172 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 174, and PSMA comprising the amino acid sequence of SEQ ID NO: 175 -LCDR3; or
iii)所述PSMA-VH具有：包含SEQ ID NO：158的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：159的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：160的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：161的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：162的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：163的氨基酸序列的PSMA-LCDR3；或iii) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 158, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 159, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 161, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 162, and PSMA comprising the amino acid sequence of SEQ ID NO: 163 -LCDR3; or
iv)所述PSMA-VH具有：包含SEQ ID NO：176的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：177的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：178的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：179的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：180的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：181的氨基酸序列的PSMA-LCDR3；或iv) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 176, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 177, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 178 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 179, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 180, and PSMA comprising the amino acid sequence of SEQ ID NO: 181 -LCDR3; or
v)所述PSMA-VH具有：包含SEQ ID NO：213的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：214的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：215的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：216的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：217的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：218的氨基酸序列的PSMA-LCDR3。v) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 213, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 214, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 215 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 216, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 217, and PSMA comprising the amino acid sequence of SEQ ID NO: 218 -LCDR3.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述PSMA-VH具有：包含SEQ ID NO：170的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：171的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：172的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：173的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：174的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：175的氨基酸序列的PSMA-LCDR3。 The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 172; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 174, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 175 .
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述PSMA-VH具有：包含SEQ ID NO：164的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：165的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：166的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：167的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：168的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：169的氨基酸序列的PSMA-LCDR3。The PSMA-VH has: PSMA-HCDR1 including the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 including the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR3 including the amino acid sequence of SEQ ID NO: 166; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 168, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 169 .
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
i)所述PSMA-VH包含如SEQ ID NO：164所示的PSMA-HCDR1，如SEQ ID NO：165所示的PSMA-HCDR2，和如SEQ ID NO：166所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：167所示的PSMA-LCDR1，如SEQ ID NO：168所示的PSMA-LCDR2，和如SEQ ID NO：169所示的PSMA-LCDR3；或i) the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 167, PSMA-LCDR2 as shown in SEQ ID NO: 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169; or
ii)所述PSMA-VH包含如SEQ ID NO：170所示的PSMA-HCDR1，如SEQ ID NO：171所示PSMA-HCDR2，和如SEQ ID NO：172所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：173所示的PSMA-LCDR1，如SEQ ID NO：174所示的PSMA-LCDR2，和如SEQ ID NO：175所示的PSMA-LCDR3；或ii) the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 173, PSMA-LCDR2 as set forth in SEQ ID NO: 174, and PSMA-LCDR3 as set forth in SEQ ID NO: 175; or
iii)所述PSMA-VH包含如SEQ ID NO：158所示的PSMA-HCDR1，如SEQ ID NO：159所示PSMA-HCDR2，和如SEQ ID NO：160所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：161所示的PSMA-LCDR1，如SEQ ID NO：162所示的PSMA-LCDR2，和如SEQ ID NO：163所示的PSMA-LCDR3；或iii) the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 158, PSMA-HCDR2 as shown in SEQ ID NO: 159, and PSMA-HCDR3 as shown in SEQ ID NO: 160; and PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 161, PSMA-LCDR2 as set forth in SEQ ID NO: 162, and PSMA-LCDR3 as set forth in SEQ ID NO: 163; or
iv)所述PSMA-VH包含如SEQ ID NO：176所示的PSMA-HCDR1，如SEQ ID NO：177所示的PSMA-HCDR2，和如SEQ ID NO：178所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：179所示的PSMA-LCDR1，如SEQ ID NO：180所示的PSMA-LCDR2，和如SEQ ID NO：181所示的PSMA-LCDR3；或iv) the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 176, PSMA-HCDR2 as shown in SEQ ID NO: 177, and PSMA-HCDR3 as shown in SEQ ID NO: 178; and Said PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 179, PSMA-LCDR2 as set forth in SEQ ID NO: 180, and PSMA-LCDR3 as set forth in SEQ ID NO: 181; or
v)所述PSMA-VH包含如SEQ ID NO：213所示的PSMA-HCDR1，如SEQ ID NO：214所示的PSMA-HCDR2，和如SEQ ID NO：215所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：216所示的PSMA-LCDR1，如SEQ ID NO：217所示的PSMA-LCDR2，和如SEQ ID NO：218所示的PSMA-LCDR3。v) the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 213, PSMA-HCDR2 as shown in SEQ ID NO: 214, and PSMA-HCDR3 as shown in SEQ ID NO: 215; and The PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 216, PSMA-LCDR2 as shown in SEQ ID NO: 217, and PSMA-LCDR3 as shown in SEQ ID NO: 218.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述PSMA-VH包含如SEQ ID NO：170所示的PSMA-HCDR1，如SEQ ID NO：171所示PSMA-HCDR2，和如SEQ ID NO：172所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：173所示的PSMA-LCDR1，如SEQ ID NO：174所示的PSMA-LCDR2，和如SEQ ID NO：175所示的PSMA-LCDR3。The PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and the PSMA- VL contains PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述PSMA-VH包含如SEQ ID NO：164所示的PSMA-HCDR1，如SEQ ID NO：165所示的PSMA-HCDR2，和如SEQ ID NO：166所示的PSMA-HCDR3；并且所 述PSMA-VL包含如SEQ ID NO：167所示的PSMA-LCDR1，如SEQ ID NO：168所示的PSMA-LCDR2，和如SEQ ID NO：169所示的PSMA-LCDR3。The PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and The PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO:167, PSMA-LCDR2 as shown in SEQ ID NO:168, and PSMA-LCDR3 as shown in SEQ ID NO:169.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：98的氨基酸序列或其变体，所述变体为在SEQ ID NO：98的FR区包含选自26A、29L、69L、71V、78A和93S组成的组中的一个或多个氨基酸取代，和所述CD28-VL包含SEQ ID NO：102的氨基酸序列或其变体，所述变体为在SEQ ID NO：102的FR区包含选自41D、42G、43T、44I和71Y组成的组中的一个或多个氨基酸取代；或(i) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 98 or a variant thereof, wherein the variant is in the FR region of SEQ ID NO: 98 and includes a sequence selected from 26A, 29L, 69L, 71V, 78A and 93S One or more amino acid substitutions in the group consisting of, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 102 or a variant thereof, the variant being in the FR region of SEQ ID NO: 102 and comprising 41D selected from the group consisting of One or more amino acid substitutions from the group consisting of , 42G, 43T, 44I and 71Y; or
(ii)所述CD28-VH包含SEQ ID NO：69的氨基酸序列或其变体，所述变体为在SEQ ID NO：69的FR区包含选自28S、69L、71V、73K和94S组成的组中的一个或多个氨基酸取代，和所述CD28-VL包含SEQ ID NO：70的氨基酸序列或其变体，所述变体为在SEQ ID NO：70的FR区包含选自43S和70K组成的组中的一个或多个氨基酸取代；或(ii) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof, wherein the variant contains a sequence selected from the group consisting of 28S, 69L, 71V, 73K and 94S in the FR region of SEQ ID NO: 69 One or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or a variant thereof, the variant being in the FR region of SEQ ID NO: 70 and including 43S and 70K One or more amino acid substitutions in the group consisting of; or
(iii)所述CD28-VH包含SEQ ID NO：133的氨基酸序列或其变体，所述变体为在SEQ ID NO：133的FR区包含选自25P、71V和78A组成的组中的一个或多个氨基酸取代，和所述CD28-VL包含SEQ ID NO：137的氨基酸序列或其变体，所述变体为在SEQ ID NO：137的FR区包含选自41D、42G、43T、44V和73L组成的组中的一个或多个氨基酸取代；或(iii) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 133 or a variant thereof, wherein the variant is one selected from the group consisting of 25P, 71V and 78A in the FR region of SEQ ID NO: 133 Or multiple amino acid substitutions, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 137 or a variant thereof, the variant is in the FR region of SEQ ID NO: 137. One or more amino acid substitutions in the group consisting of 73L; or
(iv)所述CD28-VH包含SEQ ID NO：53的氨基酸序列或其变体，所述变体为在SEQ ID NO：53的FR区包含选自1E、69L、71V、73K、78A和94S组成的组中的一个或多个氨基酸取代，和所述CD28-VL包含SEQ ID NO：52的氨基酸序列或其变体，所述变体为在SEQ ID NO：52的FR区包含选自43S和73F组成的组中的一个或多个氨基酸取代。(iv) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 53 or a variant thereof, wherein the variant contains a FR region selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S of SEQ ID NO: 53 consisting of one or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52 or a variant thereof, the variant being in the FR region of SEQ ID NO: 52 and comprising 43S selected from the group consisting of One or more amino acid substitutions from the group consisting of and 73F.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：95、35、94、96、97或98的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101、36、99、100、102、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列；或(i) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 35, 94, 96, 97 or 98, and the CD28-VL comprises SEQ ID NO: 101, 36, 99, 100, 102, 103 , 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 amino acid sequence; or
(ii)所述CD28-VH包含SEQ ID NO：68、33、63、64、65、66、67或69的氨基酸序列，和所述CD28-VL包含SEQ ID NO：80、34、70、71、72、73、74、75、76、77、78或79的氨基酸序列；或(ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69, and the CD28-VL comprises SEQ ID NO: 80, 34, 70, 71 , 72, 73, 74, 75, 76, 77, 78 or 79 amino acid sequence; or
(iii)所述CD28-VH包含SEQ ID NO：126、37、123、124、125、127、128、129、130、131、132或133的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、38、135、136、137、138、139、140、141、142或143的氨基酸序列；或(iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 37, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133, and the CD28-VL comprises SEQ ID NO. : the amino acid sequence of 134, 38, 135, 136, 137, 138, 139, 140, 141, 142 or 143; or
(iv)所述CD28-VH包含SEQ ID NO：44、31、45、46、47、48、49或53的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52、32、50或51的氨基酸序列。(iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 31, 45, 46, 47, 48, 49 or 53, and the CD28-VL comprises SEQ ID NO: 52, 32, 50 or 51 amino acid sequence.
在一些实施方式中，如前任一项所述的抗原结合分子，其中： In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：95、94、96、97或98的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101、99、100、102、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列；或(i) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98, and the CD28-VL comprises SEQ ID NO: 101, 99, 100, 102, 103, 104, 105 , the amino acid sequence of 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115; or
(ii)所述CD28-VH包含SEQ ID NO：68、63、64、65、66、67或69的氨基酸序列，和所述CD28-VL包含SEQ ID NO：80、70、71、72、73、74、75、76、77、78或79的氨基酸序列；或(ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 63, 64, 65, 66, 67 or 69, and the CD28-VL comprises SEQ ID NO: 80, 70, 71, 72, 73 , 74, 75, 76, 77, 78 or 79 amino acid sequence; or
(iii)所述CD28-VH包含SEQ ID NO：126、123、124、125、127、128、129、130、131、132或133的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、135、136、137、138、139、140、141、142或143的氨基酸序列；或(iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133, and the CD28-VL comprises SEQ ID NO: 134 , 135, 136, 137, 138, 139, 140, 141, 142 or 143 amino acid sequence; or
(iv)所述CD28-VH包含SEQ ID NO：44、45、46、47、48、49或53的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52、50或51的氨基酸序列。(iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 45, 46, 47, 48, 49 or 53, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52, 50 or 51.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
i)所述CD28-VH包含SEQ ID NO：95、94、96、97或98的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101、99、100或102的氨基酸序列，或i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102, or
所述CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101、99、100、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列，或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes SEQ ID NO: 101, 99, 100, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
所述CD28-VH包含SEQ ID NO：94的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99、100、101、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列，或The CD28-VH includes the amino acid sequence of SEQ ID NO: 94, and the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
所述CD28-VH包含SEQ ID NO：96的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99或100的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 96, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99 or 100, or
所述CD28-VH包含SEQ ID NO：97的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99、100或101的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 97, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99, 100 or 101, or
ii)所述CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述CD28-VL包含SEQ ID NO：80、72、73、74、75、76、77、78或79的氨基酸序列，或ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80, 72, 73, 74, 75, 76, 77, 78 or 79, or
所述CD28-VH包含SEQ ID NO：63的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70或71的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 63, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or 71, or
所述CD28-VH包含SEQ ID NO：64的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70、71或72的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 64, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
所述CD28-VH包含SEQ ID NO：65的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70、71或72的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 65, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
所述CD28-VH包含SEQ ID NO：66的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70、71、72、74、75、76或79氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 66, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71, 72, 74, 75, 76 or 79, or
所述CD28-VH包含SEQ ID NO：67的氨基酸序列，和所述CD28-VL包含SEQ ID NO：72、74、75、76或79的氨基酸序列，或 The CD28-VH comprises the amino acid sequence of SEQ ID NO: 67, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 74, 75, 76 or 79, or
iii)所述CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、135、136、137、138或139的氨基酸序列，或iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135, 136, 137, 138 or 139, or
所述CD28-VH包含SEQ ID NO：123的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、135或136的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 123, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135 or 136, or
所述CD28-VH包含SEQ ID NO：124的氨基酸序列，和所述CD28-VL包含SEQ ID NO：135的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 124, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 135; or
所述CD28-VH包含SEQ ID NO：125的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134或135的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 125, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134 or 135, or
所述CD28-VH包含SEQ ID NO：127的氨基酸序列，和所述CD28-VL包含SEQ ID NO：137、138或139的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 127, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 137, 138 or 139; or
所述CD28-VH包含SEQ ID NO：128的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、137、138、139或142的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 128, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 137, 138, 139 or 142; or
所述CD28-VH包含SEQ ID NO：129的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、138、139、140、141、142或143的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 129, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 138, 139, 140, 141, 142 or 143, or
所述CD28-VH包含SEQ ID NO：130的氨基酸序列，和所述CD28-VL包含SEQ ID NO：138、140、141、142或143的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 130, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
所述CD28-VH包含SEQ ID NO：131的氨基酸序列，和所述CD28-VL包含SEQ ID NO：138、140、141、142或143的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 131, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
iv)所述CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52、50或51的氨基酸序列，或iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52, 50 or 51, or
所述CD28-VH包含SEQ ID NO：45的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 45, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
所述CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或52的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 46, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 52, or
所述CD28-VH包含SEQ ID NO：47的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 47, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
所述CD28-VH包含SEQ ID NO：48的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列。The CD28-VH comprises the amino acid sequence of SEQ ID NO: 48, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：35的氨基酸序列，和所述CD28-VL包含SEQ ID NO：36的氨基酸序列；或(i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 35, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 36; or
(ii)所述CD28-VH包含SEQ ID NO：33的氨基酸序列，和所述CD28-VL包含SEQ ID NO：34的氨基酸序列；或(ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 33, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 34; or
(iii)所述CD28-VH包含SEQ ID NO：37的氨基酸序列，和所述CD28-VL包含SEQ ID NO：38的氨基酸序列；或(iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 37, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 38; or
(iv)所述CD28-VH包含SEQ ID NO：31的氨基酸序列，和所述CD28-VL包 含SEQ ID NO：32的氨基酸序列。(iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 31, and the CD28-VL contains Contains the amino acid sequence of SEQ ID NO: 32.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50的氨基酸序列；或(i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50; or
所述CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52; or
所述CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 46, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52; or
(ii)所述CD28-VH包含SEQ ID NO：65的氨基酸序列，和所述CD28-VL包含SEQ ID NO：71的氨基酸序列；或(ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 65, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 71; or
所述CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述CD28-VL包含SEQ ID NO：78的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 78; or
所述CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述CD28-VL包含SEQ ID NO：80的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80; or
(iii)所述CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101的氨基酸序列；或(iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101; or
所述CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述CD28-VL包含SEQ ID NO：106的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 106; or
所述CD28-VH包含SEQ ID NO：94的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 94, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101; or
(iv)所述CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134的氨基酸序列；或(iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134; or
所述的CD28-VH包含SEQ ID NO：125的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：135的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 125, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 135; or
所述的CD28-VH包含SEQ ID NO：128的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：142的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 128, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 142; or
所述CD28-VH包含SEQ ID NO：129的氨基酸序列，和所述CD28-VL包含SEQ ID NO：138的氨基酸序列。The CD28-VH comprises the amino acid sequence of SEQ ID NO: 129, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH的氨基酸序列如SEQ ID NO：44所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：50所示；或(i) The amino acid sequence of CD28-VH is shown in SEQ ID NO: 44, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 50; or
所述CD28-VH的氨基酸序列如SEQ ID NO：44所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：52所示；或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 44, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 52; or
所述CD28-VH的氨基酸序列如SEQ ID NO：46所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：52所示；或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 46, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 52; or
(ii)所述CD28-VH的氨基酸序列如SEQ ID NO：65所示，和所述CD28-VL 的氨基酸序列如SEQ ID NO：71所示；或(ii) The amino acid sequence of CD28-VH is shown in SEQ ID NO: 65, and the CD28-VL The amino acid sequence is shown in SEQ ID NO: 71; or
所述CD28-VH的氨基酸序列如SEQ ID NO：68所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：78所示；或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 68, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 78; or
所述CD28-VH的氨基酸序列如SEQ ID NO：68所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：80所示；或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 68, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 80; or
(iii)所述CD28-VH的氨基酸序列如SEQ ID NO：95所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：101所示；或(iii) The amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 101; or
所述CD28-VH的氨基酸序列如SEQ ID NO：95所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：106所示；或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 106; or
所述CD28-VH的氨基酸序列如SEQ ID NO：94所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：101所示；或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 94, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 101; or
(iv)所述CD28-VH的氨基酸序列如SEQ ID NO：126所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：134所示；或(iv) The amino acid sequence of the CD28-VH is shown in SEQ ID NO: 126, and the amino acid sequence of the CD28-VL is shown in SEQ ID NO: 134; or
所述的CD28-VH的氨基酸序列如SEQ ID NO：125所示，和所述的CD28-VL的氨基酸序列如SEQ ID NO：135所示；或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 125, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 135; or
所述的CD28-VH的氨基酸序列如SEQ ID NO：128所示，和所述的CD28-VL的氨基酸序列如SEQ ID NO：142所示；或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 128, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 142; or
所述CD28-VH的氨基酸序列如SEQ ID NO：129所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：138所示。The amino acid sequence of CD28-VH is shown in SEQ ID NO: 129, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 138.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50、51或52的氨基酸序列，或(i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50, 51 or 52, or
所述CD28-VH包含SEQ ID NO：45的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 45, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
所述CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或52的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 46, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 52, or
所述CD28-VH包含SEQ ID NO：47的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 47, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
所述CD28-VH包含SEQ ID NO：48的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 48, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
(ii)所述CD28-VH包含SEQ ID NO：63、64、65或66的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70的氨基酸序列；或(ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 63, 64, 65 or 66, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70; or
所述CD28-VH包含SEQ ID NO：63、64、65或66的氨基酸序列，和所述CD28-VL包含SEQ ID NO：71的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 63, 64, 65 or 66, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 71; or
所述CD28-VH包含SEQ ID NO：64或65的氨基酸序列，和所述CD28-VL包含SEQ ID NO：72的氨基酸序列；或 The CD28-VH comprises the amino acid sequence of SEQ ID NO: 64 or 65, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72; or
所述CD28-VH包含SEQ ID NO：66的氨基酸序列，和所述CD28-VL包含SEQ ID NO：71、72、74、75、76或79的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 66, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 71, 72, 74, 75, 76 or 79; or
所述CD28-VH包含SEQ ID NO：67的氨基酸序列，和所述CD28-VL包含SEQ ID NO：72、74、75、76或79的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 67, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 74, 75, 76 or 79; or
所述CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述CD28-VL包含SEQ ID NO：72、73、74、75、76、78、79或80的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 73, 74, 75, 76, 78, 79 or 80; or
(iii)所述CD28-VH包含SEQ ID NO：94的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99、100、101、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列；或(iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 94, and the CD28-VL comprises SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110 , 111, 112, 113, 114 or 115 amino acid sequence; or
所述CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99、100、101、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, The amino acid sequence of 112, 113, 114 or 115; or
所述CD28-VH包含SEQ ID NO：96的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99或100的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 96, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99 or 100; or
所述CD28-VH包含SEQ ID NO：97的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99、100或101的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 97, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99, 100 or 101; or
(iv)所述CD28-VH包含SEQ ID NO：123的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、135或136的氨基酸序列；或(iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 123, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135 or 136; or
所述CD28-VH包含SEQ ID NO：124的氨基酸序列，和所述CD28-VL包含SEQ ID NO：135的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 124, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 135; or
所述CD28-VH包含SEQ ID NO：125的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134或135的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 125, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134 or 135, or
所述CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、135、136、137、138或139的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135, 136, 137, 138 or 139; or
所述CD28-VH包含SEQ ID NO：127的氨基酸序列，和所述CD28-VL包含SEQ ID NO：137、138或139的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 127, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 137, 138 or 139; or
所述CD28-VH包含SEQ ID NO：128的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、137、138、139或142的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 128, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 137, 138, 139 or 142; or
所述CD28-VH包含SEQ ID NO：129的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、138、139、140、141、142或143的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 129, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 138, 139, 140, 141, 142 or 143; or
所述CD28-VH包含SEQ ID NO：130的氨基酸序列，和所述CD28-VL包含SEQ ID NO：138、140、141、142或143的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 130, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143; or
所述CD28-VH包含SEQ ID NO：131的氨基酸序列，和所述CD28-VL包含SEQ ID NO：138、140、141、142或143的氨基酸序列。The CD28-VH comprises the amino acid sequence of SEQ ID NO: 131, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述CD28-VL包 含SEQ ID NO：101的氨基酸序列；或(i) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, and the CD28-VL contains Containing the amino acid sequence of SEQ ID NO: 101; or
(ii)所述CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述CD28-VL包含SEQ ID NO：80的氨基酸序列；或(ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80; or
(iii)所述CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134的氨基酸序列；或(iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134; or
(iv)所述CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52的氨基酸序列。(iv) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
(i)所述CD28-VH的氨基酸序列如SEQ ID NO：95所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：101所示；或(i) The amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 101; or
(ii)所述CD28-VH的氨基酸序列如SEQ ID NO：68所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：80所示；或(ii) The amino acid sequence of CD28-VH is shown in SEQ ID NO: 68, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 80; or
(iii)所述CD28-VH的氨基酸序列如SEQ ID NO：126所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：134所示；或(iii) The amino acid sequence of the CD28-VH is shown in SEQ ID NO: 126, and the amino acid sequence of the CD28-VL is shown in SEQ ID NO: 134; or
(iv)所述CD28-VH的氨基酸序列如SEQ ID NO：44所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：52所示。(iv) The amino acid sequence of CD28-VH is shown in SEQ ID NO: 44, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 52.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述CD28-VH的氨基酸序列如SEQ ID NO：95所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：101所示。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 95, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 101 shown.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述CD28-VH的氨基酸序列如SEQ ID NO：68所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：80所示。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 68, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 80 shown.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
i)所述PSMA-VH包含SEQ ID NO：197、199、332、194、195、196或198的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：201、202、333或200的氨基酸序列；或i) The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, 199, 332, 194, 195, 196 or 198, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201, 202, 333 or 200 ;or
ii)所述PSMA-VH包含SEQ ID NO：204、184或203的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：206、185或205的氨基酸序列；或ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, 184 or 203, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206, 185 or 205; or
iii)所述PSMA-VH包含SEQ ID NO：188、182、189或190的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：191、183、192或193的氨基酸序列；或iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, 182, 189 or 190, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191, 183, 192 or 193; or
iv)所述PSMA-VH包含SEQ ID NO：207或186的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：208、187、209或210的氨基酸序列；或iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207 or 186, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208, 187, 209 or 210; or
v)所述PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：212的氨基酸序列；v) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212;
在一些实施方式中，如前任一项所述的抗原结合分子，其中： In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
i)所述PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：206的氨基酸序列；或i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
ii)所述PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：201的氨基酸序列；或ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201; or
所述PSMA-VH包含SEQ ID NO：199的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：202的氨基酸序列；或The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202; or
iii)所述PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：191的氨基酸序列；或iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191; or
iv)所述PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：208的氨基酸序列。iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：206的氨基酸序列。The PSMA-VH includes the amino acid sequence of SEQ ID NO:204, and the PSMA-VL includes the amino acid sequence of SEQ ID NO:206.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：201的氨基酸序列。The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述PSMA-VH包含SEQ ID NO：199的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：202的氨基酸序列。The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
i)所述PSMA-VH的氨基酸序列如SEQ ID NO：204所示，和所述PSMA-VL的氨基酸序列如SEQ ID NO：206所示；或i) The amino acid sequence of the PSMA-VH is as shown in SEQ ID NO: 204, and the amino acid sequence of the PSMA-VL is as shown in SEQ ID NO: 206; or
ii)所述PSMA-VH的氨基酸序列如SEQ ID NO：197所示，和所述PSMA-VL的氨基酸序列如SEQ ID NO：201所示；或ii) The amino acid sequence of the PSMA-VH is as shown in SEQ ID NO: 197, and the amino acid sequence of the PSMA-VL is as shown in SEQ ID NO: 201; or
所述PSMA-VH的氨基酸序列如SEQ ID NO：199所示，和所述PSMA-VL的氨基酸序列如SEQ ID NO：202所示；或The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 199, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 202; or
iii)所述PSMA-VH的氨基酸序列如SEQ ID NO：188所示，和所述PSMA-VL的氨基酸序列如SEQ ID NO：191所示；或iii) The amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 188, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 191; or
iv)所述PSMA-VH的氨基酸序列如SEQ ID NO：207所示，和所述PSMA-VL的氨基酸序列如SEQ ID NO：208所示。iv) The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 207, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 208.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述PSMA-VH的氨基酸序列如SEQ ID NO：204所示，和所述PSMA-VL的氨基酸序列如SEQ ID NO：206所示。The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 204, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 206.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述PSMA-VH的氨基酸序列如SEQ ID NO：197所示，和所述PSMA-VL的氨基酸序列如SEQ ID NO：201所示。 The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 197, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 201.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述PSMA-VH的氨基酸序列如SEQ ID NO：199所示，和所述PSMA-VL的氨基酸序列如SEQ ID NO：202所示。The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 199, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 202.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述特异性结合CD28的抗原结合模块或所述特异性结合PSMA的抗原结合模块包含能够形成二聚体的Titin链和Obscurin链。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding module that specifically binds CD28 or the antigen-binding module that specifically binds PSMA comprises a Titin chain and Obscurin capable of forming a dimer chain.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述特异性结合CD28的抗原结合模块包含能够形成二聚体的Titin链和Obscurin链。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding module that specifically binds CD28 comprises a Titin chain and an Obscurin chain capable of forming a dimer.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述特异性结合PSMA的抗原结合模块包含能够形成二聚体的Titin链和Obscurin链。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding module that specifically binds PSMA includes a Titin chain and an Obscurin chain capable of forming a dimer.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述Titin链包含选自由SEQ ID NO：334至SEQ ID NO：352组成的组的氨基酸序列，所述Obscurin链包含选自由SEQ ID NO：353至SEQ ID NO：393组成的组的氨基酸序列。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 334 to SEQ ID NO: 352, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of The amino acid sequence of the group consisting of SEQ ID NO: 353 to SEQ ID NO: 393.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述Titin链包含SEQ ID NO：350的氨基酸序列。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述Obscurin链包含SEQ ID NO：388的氨基酸序列。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 388.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述Titin链包含SEQ ID NO：350的氨基酸序列，和所述Obscurin链包含SEQ ID NO：388的氨基酸序列。In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 388.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含Fc区。In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含IgG Fc区。In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein the antigen-binding molecule comprises an IgG Fc region.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含IgG1Fc区。In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein the antigen-binding molecule comprises an IgG1 Fc region.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含Fc区，所述Fc区包含一个或多个能够减少Fc区与Fcγ受体结合的氨基酸取代。In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region comprising one or more amino acid substitutions capable of reducing the binding of the Fc region to Fcγ receptors.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含Fc区，所述Fc区是人IgG1Fc区，并且在234和235位置的氨基酸为A，编号依据为EU索引。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region is a human IgG1 Fc region, and the amino acids at positions 234 and 235 are A, and the numbering basis is EU index.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含Fc区，所述Fc区包含能够相互缔合的第一亚基Fc1和第二亚基Fc2，所述Fc1和Fc2各自独立地具有一个或多个减少Fc区同源二聚化的氨基酸取代。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, so The Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分 子包含Fc区，所述Fc区包含能够相互缔合的第一亚基Fc1和第二亚基Fc2，所述Fc1具有根据杵臼技术的凸起结构，和所述Fc2具有根据杵臼技术的孔结构。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule The subunit includes an Fc region, the Fc region includes a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 has a convex structure according to the pestle and mortar technology, and the Fc2 has a pore structure according to the pestle and mortar technology. .
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含Fc区，所述Fc区包含能够相互缔合的第一亚基Fc1和第二亚基Fc2，所述Fc1具有根据杵臼技术的凸起结构，和所述Fc2具有根据杵臼技术的孔结构，所述Fc1在366位置的氨基酸为W；并且所述Fc2在366位置的氨基酸为S、在368位置的氨基酸为A、和在407位置的氨基酸为V，编号依据为EU索引。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, so The Fc1 has a convex structure according to the pestle and mortar technology, and the Fc2 has a pore structure according to the pestle and mortar technology, the amino acid of the Fc1 at position 366 is W; and the amino acid of Fc2 at position 366 is S, and the amino acid at position 368 is S. The amino acid is A, and the amino acid at position 407 is V, and the numbering basis is the EU index.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含Fc区，所述Fc区包含能够相互缔合的第一亚基Fc1和第二亚基Fc2，所述Fc1具有根据杵臼技术的凸起结构，和所述Fc2具有根据杵臼技术的孔结构，其中，所述Fc1在354位置的氨基酸为C和在366位置的氨基酸为W；并且所述Fc2在349位置的氨基酸为C、在366位置的氨基酸为S、在368位置的氨基酸为A、和在407位置的氨基酸为V，编号依据为EU索引。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, so The Fc1 has a convex structure according to the pestle and mortar technology, and the Fc2 has a pore structure according to the pestle and mortar technology, wherein the amino acid at the 354 position of the Fc1 is C and the amino acid at the 366 position is W; and the Fc2 is at 349 The amino acid at position 366 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V. The numbering basis is the EU index.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含Fc区，所述Fc区包含能够相互缔合的第一亚基Fc1和第二亚基Fc2，其中，所述Fc1包含SEQ ID NO：220的氨基酸序列；和所述Fc2包含SEQ ID NO：221或222的氨基酸序列。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein , the Fc1 includes the amino acid sequence of SEQ ID NO: 220; and the Fc2 includes the amino acid sequence of SEQ ID NO: 221 or 222.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含Fc区，所述Fc区包含能够相互缔合的第一亚基Fc1和第二亚基Fc2，其中，所述Fc1包含SEQ ID NO：220的氨基酸序列；和所述Fc2包含SEQ ID NO：221的氨基酸序列。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein , the Fc1 includes the amino acid sequence of SEQ ID NO: 220; and the Fc2 includes the amino acid sequence of SEQ ID NO: 221.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含一个特异性结合CD28的抗原结合模块和一个特异性结合PSMA的抗原结合模块。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds CD28 and an antigen-binding module that specifically binds PSMA.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含一个特异性结合CD28的抗原结合模块和一个特异性结合PSMA的抗原结合模块，其中：In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds CD28 and an antigen-binding module that specifically binds PSMA, wherein:
所述特异性结合CD28的抗原结合模块是Fab，和所述特异性结合PSMA的抗原结合模块是经替换的Fab，其包含能够形成二聚体的Titin链和Obscurin链。The antigen-binding module that specifically binds to CD28 is a Fab, and the antigen-binding module that specifically binds to PSMA is a replaced Fab that contains a Titin chain and an Obscurin chain capable of forming dimers.
在一些实施方式中，如前任一项所述的抗原结合分子，所述抗原结合分子包含一条具有式(a)所示结构的第一链、一条具有式(b)所示结构的第二链、一条具有式(c)所示结构的第三链和一条具有式(d)所示结构的第四链，In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding molecule includes a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b) , a third chain having the structure shown in formula (c) and a fourth chain having the structure shown in formula (d),
(a)[CD28-VH]-[CH1]-[Fc1]，(a)[CD28-VH]-[CH1]-[Fc1],
(b)[CD28-VL]-[CL]，(b)[CD28-VL]-[CL],
(c)[PSMA-VH]-[连接子1]-[Titin链]-[Fc2]，(c)[PSMA-VH]-[linker 1]-[Titin chain]-[Fc2],
(d)[PSMA-VL]-[连接子2]-[Obscurin链]， (d)[PSMA-VL]-[Linker 2]-[Obscurin chain],
其中：所述连接子1和所述连接子2是相同或不同的肽连接子；式(a)、(b)、(c)和(d)所示的结构是从N端至C端排列的。Wherein: the linker 1 and the linker 2 are the same or different peptide linkers; the structures shown in formulas (a), (b), (c) and (d) are arranged from the N end to the C end. of.
在一些实施方式中，如前任一项所述的抗原结合分子，所述抗原结合分子包含一条具有式(a)所示结构的第一链、一条具有式(b)所示结构的第二链、一条具有式(c)所示结构的第三链和一条具有式(d)所示结构的第四链，In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding molecule includes a first chain having a structure represented by formula (a) and a second chain having a structure represented by formula (b) , a third chain having the structure shown in formula (c) and a fourth chain having the structure shown in formula (d),
(a)[CD28-VH]-[CH1]-[Fc1]，(a)[CD28-VH]-[CH1]-[Fc1],
(b)[CD28-VL]-[CL]，(b)[CD28-VL]-[CL],
(c)[PSMA-VH]-[连接子1]-[Titin链]-[Fc2]，(c)[PSMA-VH]-[linker 1]-[Titin chain]-[Fc2],
(d)[PSMA-VL]-[连接子2]-[Obscurin链]，(d)[PSMA-VL]-[Linker 2]-[Obscurin chain],
其中：所述连接子1和所述连接子2不存在，即所述连接子1和所述连接子2均是键；式(a)、(b)、(c)和(d)所示的结构是从N端至C端排列的。Wherein: the connector 1 and the connector 2 do not exist, that is, the connector 1 and the connector 2 are both bonds; as shown in formulas (a), (b), (c) and (d) The structure is arranged from the N-terminus to the C-terminus.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
i)所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：80的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或i) The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204 sequence, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 206; or
所述的CD28-VH包含SEQ ID NO：33的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：34的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 33, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 34; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：65的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：71的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 65, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 71; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：78的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 78; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：80的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：80的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：80的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：191的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：80的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：208的氨基酸序列；或 The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
ii)所述的CD28-VH包含SEQ ID NO：31的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：32的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或ii) The CD28-VH includes the amino acid sequence of SEQ ID NO: 31, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 32; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：50的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 50; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
所述的CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 206; or
所述的CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
所述的CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 206; or
所述的CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：191的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
所述的CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：208的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
iii)所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或iii) The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204 sequence, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或 The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：191的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：208的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
iv)所述的CD28-VH包含SEQ ID NO：37的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：38的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或iv) the CD28-VH includes the amino acid sequence of SEQ ID NO: 37, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 38; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列。The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 212.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：80的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列。The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 206.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述的CD28-VH的氨基酸序列如SEQ ID NO：68所示，和所述的CD28-VL的氨基酸序列如SEQ ID NO：80所示；并且所述的PSMA-VH的氨基酸序列如SEQ ID NO：204所示，和所述的PSMA-VL的氨基酸序列如SEQ ID NO：206所示。The amino acid sequence of CD28-VH is shown in SEQ ID NO: 68, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 80; and the amino acid sequence of PSMA-VH is shown in SEQ ID NO: 204 is shown, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 206.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的Titin链和Obscurin链可选自本披露中表3-1和表3-2中任意可以形成二聚体的Titin链和Obscurin链。在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的Titin链包含如SEQ ID NO：350的氨基酸序列。在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的Obscurin链包含如SEQ ID NO：388的氨基酸序列。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain and Obscurin chain can be selected from any Titin that can form dimers in Table 3-1 and Table 3-2 of the present disclosure. chain and Obscurin chain. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 388.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的连接子1和连接子2均为本领域已知的肽连接子，只要抗原结合分子能够展现出期望的抗原结合活性。例如，肽连接子可以是具有1-50或3-20个氨基酸残基的柔性肽。在一些实施方案中，所述肽连接子的长度为3-15个氨基酸残基。在一些实施方案中，所述肽连接子各自独立地具有(GGGGS)n的结构，其中n是1、2或3。在一些实施方案中，所述的连接子1和连接子2的序列为GGGGS(SEQ ID NO：223)。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the linker 1 and the linker 2 are both peptide linkers known in the art, as long as the antigen-binding molecule can exhibit the desired antigen binding active. For example, the peptide linker can be a flexible peptide having 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, the peptide linkers each independently have the structure (GGGGS)n, where n is 1, 2, or 3. In some embodiments, the sequences of linker 1 and linker 2 are GGGGS (SEQ ID NO: 223).
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的CH1为IgG 的CH1序列。在一些实施方案中，所述的CH1为IgG1的CH1。在一些实施方案中，所述的CH1包含SEQ ID NO：219的氨基酸序列。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein said CH1 is IgG The CH1 sequence. In some embodiments, the CH1 is CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 219.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的CL为抗体的轻链恒定区。在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的CL为kappa或lamada的轻链恒定区。在一些实施方式中，其中所述的CL包含SEQ ID NO：145的氨基酸序列。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein said CL is the light chain constant region of an antibody. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the CL is the light chain constant region of kappa or lamada. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 145.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子具有：In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule has:
一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：239的氨基酸序列的第二链、一条包含SEQ ID NO：249的氨基酸序列的第三链和一条包含SEQ ID NO：250的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 239, a third strand comprising the amino acid sequence of SEQ ID NO: 249 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of the amino acid sequence of 250; or
一条包含SEQ ID NO：226的氨基酸序列的第一链、一条包含SEQ ID NO：227的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 226, a second strand comprising the amino acid sequence of SEQ ID NO: 227, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 226 : The fourth strand of the amino acid sequence 229; or
一条包含SEQ ID NO：230的氨基酸序列的第一链、一条包含SEQ ID NO：231的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 230, a second strand comprising the amino acid sequence of SEQ ID NO: 231, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 230 : The fourth strand of the amino acid sequence 229; or
一条包含SEQ ID NO：230的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 230, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 232 : The fourth strand of the amino acid sequence 229; or
一条包含SEQ ID NO：233的氨基酸序列的第一链、一条包含SEQ ID NO：234的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 233, a second strand comprising the amino acid sequence of SEQ ID NO: 234, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 233 : The fourth strand of the amino acid sequence 229; or
一条包含SEQ ID NO：235的氨基酸序列的第一链、一条包含SEQ ID NO：236的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 235, a second strand comprising the amino acid sequence of SEQ ID NO: 236, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 235 : The fourth strand of the amino acid sequence 229; or
一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：238的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 238, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of the amino acid sequence 229; or
一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：239的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 239, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of the amino acid sequence 229; or
一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO：241的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 240, a second strand comprising the amino acid sequence of SEQ ID NO: 241, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 240 : The fourth strand of the amino acid sequence 229; or
一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO： 242的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或One contains the first strand of the amino acid sequence of SEQ ID NO: 240, one contains the first strand of the amino acid sequence of SEQ ID NO: or
一条包含SEQ ID NO：243的氨基酸序列的第一链、一条包含SEQ ID NO：244的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 243, a second strand comprising the amino acid sequence of SEQ ID NO: 244, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 243 : The fourth strand of the amino acid sequence 229; or
一条包含SEQ ID NO：245的氨基酸序列的第一链、一条包含SEQ ID NO：246的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 245, a second strand comprising the amino acid sequence of SEQ ID NO: 246, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 245 : The fourth strand of the amino acid sequence 229; or
一条包含SEQ ID NO：230的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：247的氨基酸序列的第三链和一条包含SEQ ID NO：248的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 230, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 247 and a third strand comprising the amino acid sequence of SEQ ID NO: 232 : The fourth strand of an amino acid sequence of 248; or
一条包含SEQ ID NO：230的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：249的氨基酸序列的第三链和一条包含SEQ ID NO：250的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 230, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 249 and a third strand comprising the amino acid sequence of SEQ ID NO: 232 : The fourth strand of the amino acid sequence of 250; or
一条包含SEQ ID NO：251的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：247的氨基酸序列的第三链和一条包含SEQ ID NO：248的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 251, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 247 and a third strand comprising the amino acid sequence of SEQ ID NO: 247 : The fourth strand of an amino acid sequence of 248; or
一条包含SEQ ID NO：251的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：249的氨基酸序列的第三链和一条包含SEQ ID NO：250的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 251, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 249 and a third strand comprising the amino acid sequence of SEQ ID NO: 249 : The fourth strand of the amino acid sequence of 250; or
一条包含SEQ ID NO：251的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：252的氨基酸序列的第三链和一条包含SEQ ID NO：253的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 251, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 252 and a third strand comprising the amino acid sequence of SEQ ID NO: 251 : The fourth strand of the amino acid sequence 253; or
一条包含SEQ ID NO：251的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：254的氨基酸序列的第三链和一条包含SEQ ID NO：255的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 251, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 254 and a third strand comprising the amino acid sequence of SEQ ID NO: 251 : The fourth strand of an amino acid sequence of 255; or
一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：239的氨基酸序列的第二链、一条包含SEQ ID NO：247的氨基酸序列的第三链和一条包含SEQ ID NO：248的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 239, a third strand comprising the amino acid sequence of SEQ ID NO: 247 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of an amino acid sequence of 248; or
一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：239的氨基酸序列的第二链、一条包含SEQ ID NO：252的氨基酸序列的第三链和一条包含SEQ ID NO：253的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 239, a third strand comprising the amino acid sequence of SEQ ID NO: 252 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of the amino acid sequence 253; or
一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：239的氨基酸序列的第二链、一条包含SEQ ID NO：254的氨基酸序列的第三链和一条包含SEQ ID NO：255的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 239, a third strand comprising the amino acid sequence of SEQ ID NO: 254 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of an amino acid sequence of 255; or
一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO： 242的氨基酸序列的第二链、一条包含SEQ ID NO：247的氨基酸序列的第三链和一条包含SEQ ID NO：248的氨基酸序列的第四链；或One contains the first strand of the amino acid sequence of SEQ ID NO: 240, one contains the first strand of the amino acid sequence of SEQ ID NO: or
一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO：242的氨基酸序列的第二链、一条包含SEQ ID NO：249的氨基酸序列的第三链和一条包含SEQ ID NO：250的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 240, a second strand comprising the amino acid sequence of SEQ ID NO: 242, a third strand comprising the amino acid sequence of SEQ ID NO: 249 and a third strand comprising the amino acid sequence of SEQ ID NO: 242 : The fourth strand of the amino acid sequence of 250; or
一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO：242的氨基酸序列的第二链、一条包含SEQ ID NO：252的氨基酸序列的第三链和一条包含SEQ ID NO：253的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 240, a second strand comprising the amino acid sequence of SEQ ID NO: 242, a third strand comprising the amino acid sequence of SEQ ID NO: 252 and a third strand comprising the amino acid sequence of SEQ ID NO: 240 : The fourth strand of the amino acid sequence 253; or
一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO：242的氨基酸序列的第二链、一条包含SEQ ID NO：254的氨基酸序列的第三链和一条包含SEQ ID NO：255的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 240, a second strand comprising the amino acid sequence of SEQ ID NO: 242, a third strand comprising the amino acid sequence of SEQ ID NO: 254 and a third strand comprising the amino acid sequence of SEQ ID NO: 240 : The fourth strand of the amino acid sequence of 255.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子具有：In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule has:
一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：239的氨基酸序列的第二链、一条包含SEQ ID NO：249的氨基酸序列的第三链和一条包含SEQ ID NO：250的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 239, a third strand comprising the amino acid sequence of SEQ ID NO: 249 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of the 250 amino acid sequence.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子具有：In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule has:
一条如SEQ ID NO：237所示的第一链、一条如SEQ ID NO：239所示的第二链、一条如SEQ ID NO：249所示的第三链和一条如SEQ ID NO：250所示的第四链。A first strand as shown in SEQ ID NO:237, a second strand as shown in SEQ ID NO:239, a third strand as shown in SEQ ID NO:249 and a third strand as shown in SEQ ID NO:250 The fourth chain shown.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含一个特异性结合CD28的抗原结合模块和一个特异性结合PSMA的抗原结合模块，所述特异性结合PSMA的抗原结合模块是Fab；所述特异性结合CD28的抗原结合模块是经替换的Fab，其包含能够形成二聚体的Titin链和Obscurin链。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds CD28 and an antigen-binding module that specifically binds PSMA, and the antigen-binding module that specifically binds PSMA The antigen-binding module is a Fab; the antigen-binding module that specifically binds to CD28 is a replaced Fab, which contains a Titin chain and an Obscurin chain capable of forming dimers.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含一条具有式(e)所示结构的第一链、一条具有式(f)所示结构的第二链、一条具有式(g)所示结构的第三链和一条具有式(h)所示结构的第四链，In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule includes a first chain having a structure shown in formula (e), a second chain having a structure shown in formula (f) chain, a third chain having the structure shown in formula (g) and a fourth chain having the structure shown in formula (h),
(e)[CD28-VH]-[连接子3]-[Titin链]-[Fc1]，(e)[CD28-VH]-[Linker 3]-[Titin chain]-[Fc1],
(f)[CD28-VL]-[连接子4]-[Obscurin链]，(f)[CD28-VL]-[linker 4]-[Obscurin chain],
(g)[PSMA-VH]-[CH1]-[Fc2]，(g)[PSMA-VH]-[CH1]-[Fc2],
(h)[PSMA-VL]-[CL]，(h)[PSMA-VL]-[CL],
其中：所述连接子3和连接子4是相同或不同的肽连接子；式(e)、(f)、(g)和(h)所示的结构是从N端至C端排列的。Wherein: the linker 3 and the linker 4 are the same or different peptide linkers; the structures shown in formulas (e), (f), (g) and (h) are arranged from the N-terminus to the C-terminus.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子包含一条具有式(e)所示结构的第一链、一条具有式(f)所示结构的第二链、一条 具有式(g)所示结构的第三链和一条具有式(h)所示结构的第四链，In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule includes a first chain having a structure shown in formula (e), a second chain having a structure shown in formula (f) chain, one a third chain having a structure represented by formula (g) and a fourth chain having a structure represented by formula (h),
(e)[CD28-VH]-[连接子3]-[Titin链]-[Fc1]，(e)[CD28-VH]-[Linker 3]-[Titin chain]-[Fc1],
(f)[CD28-VL]-[连接子4]-[Obscurin链]，(f)[CD28-VL]-[linker 4]-[Obscurin chain],
(g)[PSMA-VH]-[CH1]-[Fc2]，(g)[PSMA-VH]-[CH1]-[Fc2],
(h)[PSMA-VL]-[CL]，(h)[PSMA-VL]-[CL],
其中：所述连接子3和连接子4不存在，即所述连接子3和连接子4均为键；式(e)、(f)、(g)和(h)所示的结构是从N端至C端排列的。Among them: the connector 3 and the connector 4 do not exist, that is, the connector 3 and the connector 4 are both bonds; the structures shown in formulas (e), (f), (g) and (h) are from Arranged from N-terminus to C-terminus.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
i)所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或i) The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204 sequence, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：94的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 94, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：191的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：208的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
ii)所述的CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或ii) The CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
iii)所述的CD28-VH包含SEQ ID NO：37的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：38的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或iii) The CD28-VH includes the amino acid sequence of SEQ ID NO: 37, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 38; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL 包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL Comprising the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH comprising the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：129的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：138的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 129, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 138; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 206; or
所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：191的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：208的氨基酸序列。The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 208.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列。The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 201.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列。The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, And the PSMA-VL contains the amino acid sequence of SEQ ID NO: 206.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述的CD28-VH的氨基酸序列如SEQ ID NO：95所示，和所述的CD28-VL的氨基酸序列如SEQ ID NO：101所示；并且所述的PSMA-VH的氨基酸序列如SEQ ID NO：197所示，和所述的PSMA-VL的氨基酸序列如SEQ ID NO：201所示。The amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 101; and the amino acid sequence of PSMA-VH is shown in SEQ ID NO: 197 is shown, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 201.
在一些实施方式中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述的CD28-VH的氨基酸序列如SEQ ID NO：95所示，和所述的CD28-VL的氨基酸序列如SEQ ID NO：101所示；并且所述的PSMA-VH的氨基酸序列如SEQ ID NO：204所示，和所述的PSMA-VL的氨基酸序列如SEQ ID NO：206所示。The amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 101; and the amino acid sequence of PSMA-VH is shown in SEQ ID NO: 204 is shown, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 206.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的Titin链和 Obscurin链可选自本披露中表3-1和表3-2中任意可以形成二聚体的Titin链和Obscurin链。在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的Titin链包含如SEQ ID NO：350的氨基酸序列。在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的Obscurin链包含如SEQ ID NO：388的氨基酸序列。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain and The Obscurin chain can be selected from any Titin chain and Obscurin chain that can form dimers in Table 3-1 and Table 3-2 in this disclosure. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 388.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的连接子3和连接子4均为本领域已知的肽连接子，只要抗原结合分子能够展现出期望的抗原结合活性。例如，肽连接子可以是包含1-50或3-20个氨基酸残基的柔性肽。在一些实施方案中，所述肽连接子的长度为3-15个氨基酸残基。在一些实施方案中，所述肽连接子各自独立地具有(GGGGS)n的结构，其中n是1、2或3。在一些实施方案中，所述的连接子3和连接子4的序列为GGGGS(SEQ ID NO：223)。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the linker 3 and the linker 4 are both peptide linkers known in the art, as long as the antigen-binding molecule can exhibit the desired antigen binding active. For example, the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, the peptide linkers each independently have the structure (GGGGS)n, where n is 1, 2, or 3. In some embodiments, the sequences of linker 3 and linker 4 are GGGGS (SEQ ID NO: 223).
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的CH1为IgG的CH1序列。在一些实施方案中，所述的CH1为IgG1的CH1。在一些实施方案中，所述的CH1包含SEQ ID NO：219的氨基酸序列。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 219.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的CL为抗体的轻链恒定区。在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的CL为kappa或lamada的轻链恒定区。在一些实施方式中，其中所述的CL包含SEQ ID NO：145的氨基酸序列。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein said CL is the light chain constant region of an antibody. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the CL is the light chain constant region of kappa or lamada. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 145.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子具有：In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule has:
一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：274的氨基酸序列的第三链和一条包含SEQ ID NO：275的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 274 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of an amino acid sequence of 275; or
一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：272的氨基酸序列的第三链和一条包含SEQ ID NO：273的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 272 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence 273; or
一条包含SEQ ID NO：257的氨基酸序列的第一链、一条包含SEQ ID NO：258的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 257, a second strand comprising the amino acid sequence of SEQ ID NO: 258, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 257 : The fourth strand of the amino acid sequence of 260; or
一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence of 260; or
一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：263的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 263, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence of 260; or
一条包含SEQ ID NO：264的氨基酸序列的第一链、一条包含SEQ ID NO： 265的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或One contains the first strand of the amino acid sequence of SEQ ID NO: 264, one contains the first strand of the amino acid sequence of SEQ ID NO: or
一条包含SEQ ID NO：266的氨基酸序列的第一链、一条包含SEQ ID NO：267的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 266, a second strand comprising the amino acid sequence of SEQ ID NO: 267, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 266 : The fourth strand of the amino acid sequence of 260; or
一条包含SEQ ID NO：268的氨基酸序列的第一链、一条包含SEQ ID NO：269的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 268, a second strand comprising the amino acid sequence of SEQ ID NO: 269, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 268 : The fourth strand of the amino acid sequence of 260; or
一条包含SEQ ID NO：270的氨基酸序列的第一链、一条包含SEQ ID NO：271的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 270, a second strand comprising the amino acid sequence of SEQ ID NO: 271, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 271 : The fourth strand of the amino acid sequence of 260; or
一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：276的氨基酸序列的第三链和一条包含SEQ ID NO：277的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 276 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence 277; or
一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：278的氨基酸序列的第三链和一条包含SEQ ID NO：279的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 278 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence 279; or
一条包含SEQ ID NO：268的氨基酸序列的第一链、一条包含SEQ ID NO：269的氨基酸序列的第二链、一条包含SEQ ID NO：272的氨基酸序列的第三链和一条包含SEQ ID NO：273的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 268, a second strand comprising the amino acid sequence of SEQ ID NO: 269, a third strand comprising the amino acid sequence of SEQ ID NO: 272 and a third strand comprising the amino acid sequence of SEQ ID NO: 268 : The fourth strand of the amino acid sequence 273; or
一条包含SEQ ID NO：268的氨基酸序列的第一链、一条包含SEQ ID NO：269的氨基酸序列的第二链、一条包含SEQ ID NO：274的氨基酸序列的第三链和一条包含SEQ ID NO：275的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 268, a second strand comprising the amino acid sequence of SEQ ID NO: 269, a third strand comprising the amino acid sequence of SEQ ID NO: 274 and a third strand comprising the amino acid sequence of SEQ ID NO: 268 : The fourth strand of the amino acid sequence 275; or
一条包含SEQ ID NO：268的氨基酸序列的第一链、一条包含SEQ ID NO：269的氨基酸序列的第二链、一条包含SEQ ID NO：276的氨基酸序列的第三链和一条包含SEQ ID NO：277的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 268, a second strand comprising the amino acid sequence of SEQ ID NO: 269, a third strand comprising the amino acid sequence of SEQ ID NO: 276 and a third strand comprising the amino acid sequence of SEQ ID NO: 268 : The fourth strand of the amino acid sequence 277; or
一条包含SEQ ID NO：268的氨基酸序列的第一链、一条包含SEQ ID NO：269的氨基酸序列的第二链、一条包含SEQ ID NO：278的氨基酸序列的第三链和一条包含SEQ ID NO：279的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 268, a second strand comprising the amino acid sequence of SEQ ID NO: 269, a third strand comprising the amino acid sequence of SEQ ID NO: 278 and a third strand comprising the amino acid sequence of SEQ ID NO: 268 : The fourth strand of the amino acid sequence of 279.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子具有：In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule has:
一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：272的氨基酸序列的第三链和一条包含SEQ ID NO：273的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 272 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence of 273.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子具有： In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule has:
一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：274的氨基酸序列的第三链和一条包含SEQ ID NO：275的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 274 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence of 275.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子具有：In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule has:
一条如SEQ ID NO：261所示的第一链、一条如SEQ ID NO：262所示的第二链、一条如SEQ ID NO：272所示的第三链和一条如SEQ ID NO：273所示的第四链。A first strand as shown in SEQ ID NO:261, a second strand as shown in SEQ ID NO:262, a third strand as shown in SEQ ID NO:272 and a third strand as shown in SEQ ID NO:273 The fourth chain shown.
在一些实施方式中，如前任一项所述的抗原结合分子，其中所述抗原结合分子具有：In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule has:
一条如SEQ ID NO：261所示的第一链、一条如SEQ ID NO：262所示的第二链、一条如SEQ ID NO：274所示的第三链和一条如SEQ ID NO：275所示的第四链。A first strand as shown in SEQ ID NO: 261, a second strand as shown in SEQ ID NO: 262, a third strand as shown in SEQ ID NO: 274 and a third strand as shown in SEQ ID NO: 275 The fourth chain shown.
在另一方面，本披露还提供一种抗CD28抗体，其能够特异性结合CD28，所述的抗CD28抗体包含重链可变区CD28-VH和轻链可变区CD28-VL，其中：In another aspect, the present disclosure also provides an anti-CD28 antibody capable of specifically binding to CD28, the anti-CD28 antibody comprising a heavy chain variable region CD28-VH and a light chain variable region CD28-VL, wherein:
(i)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：35中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：36、103、104、105、106、107、108、109、110、111、112、113、114或115中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或(i) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28 in SEQ ID NO: 36, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 respectively -amino acid sequences of LCDR1, CD28-LCDR2 and CD28-LCDR3, or
(ii)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：33中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：34、73、74、75、76、77、78、79或80中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或(ii) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL contain CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 34, 73, 74, 75, 76, 77, 78, 79 or 80 respectively the amino acid sequence, or
(iii)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：37、129或132中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：38、139、141、142或143中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或(iii) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37, 129 or 132, and CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in the CD28-VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, 139, 141, 142 or 143, or
(iv)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：31中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：32、46、47、48或49中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列。 (iv) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32, 46, 47, 48 or 49.
在一些实施方式中，如前所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody as described above, wherein:
(i)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：35中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：36中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或(i) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 35, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 36, or
(ii)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：33中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：80中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或(ii) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 80, or
所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：33中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：34中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33, and the CD28-VL in the CD28-VL. CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 34, or
(iii)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：37中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：38中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列，或(iii) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 37, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 38, or
(iv)所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：31中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：32中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列；或(iv) CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in VL respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32; or
所述CD28-VH中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3分别包含SEQ ID NO：31中的CD28-HCDR1、CD28-HCDR2和CD28-HCDR3的氨基酸序列，和所述CD28-VL中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3分别包含SEQ ID NO：52中的CD28-LCDR1、CD28-LCDR2和CD28-LCDR3的氨基酸序列。CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in the CD28-VH respectively comprise the amino acid sequences of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 31, and the CD28-VL in the CD28-VL. CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 respectively comprise the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 52.
在一些实施方案中，所述CD28-HCDR1、CD28-HCDR2、CD28-HCDR3、CD28-LCDR1、CD28-LCDR2和CD28-LCDR3根据Kabat、IMGT、Chothia、AbM或Contact编号规则定义。In some embodiments, the CD28-HCDR1, CD28-HCDR2, CD28-HCDR3, CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 are defined according to Kabat, IMGT, Chothia, AbM or Contact numbering rules.
在一些实施方式中，如前所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody as described above, wherein:
(i)所述CD28-VH具有：包含SEQ ID NO：19的氨基酸序列的CD28-HCDR1、 包含SEQ ID NO：20的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：21的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：22的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：23、81、82、83、84、85、86、87、88、89、90、91、92或93的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：24的氨基酸序列的CD28-LCDR3；或(i) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 24 CD28-LCDR3; or
(ii)所述CD28-VH具有：包含SEQ ID NO：13的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：14的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：15的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：16的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：17、54、55、56、57、58、59、60、61或62的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：18的氨基酸序列的CD28-LCDR3；或(ii) The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15. HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, comprising the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 The sequence of CD28-LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18; or
(iii)所述CD28-VH具有：包含SEQ ID NO：25或117的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26或116的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或(iii) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27 CD28-HCDR3 of the sequence, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28- comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
(iv)所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9、39、40、41或42的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：11或43的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3。(iv) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42 amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or 43. ID NO: 12 amino acid sequence of CD28-LCDR3.
在一些实施方案中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
所述CD28-VH具有：包含SEQ ID NO：25的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and The CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
所述CD28-VH具有：包含SEQ ID NO：25的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：116的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and The CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
所述CD28-VH具有：包含SEQ ID NO：117的氨基酸序列的CD28-HCDR1、 包含SEQ ID NO：26的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, A CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
所述CD28-VH具有：包含SEQ ID NO：117的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：116的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3。The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and The CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 30 amino acid sequence of CD28-LCDR3.
在一些实施方案中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
(i)所述CD28-VH具有：包含SEQ ID NO：19的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：20的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：21的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：22的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：23的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：24的氨基酸序列的CD28-LCDR3；或(i) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23 and CD28- comprising the amino acid sequence of SEQ ID NO: 24 LCDR3; or
(ii)所述CD28-VH具有：包含SEQ ID NO：13的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：14的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：15的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：16的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：62的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：18的氨基酸序列的CD28-LCDR3；或(ii) The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15. HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 62 and CD28- comprising the amino acid sequence of SEQ ID NO: 18 LCDR3; or
(iii)所述CD28-VH具有：包含SEQ ID NO：25的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或(iii) The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27. HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28- comprising the amino acid sequence of SEQ ID NO: 30 LCDR3; or
(iv)所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：43的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3。(iv) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 12 LCDR3.
在一些实施方案中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
(i)所述CD28-VH包含如SEQ ID NO：19所示的CD28-HCDR1、如SEQ ID NO：20所示的CD28-HCDR2和如SEQ ID NO：21所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：22所示的CD28-LCDR1、如SEQ ID NO：23、81、82、83、84、85、86、87、88、89、90、91、92或93所示的CD28-LCDR2和如 SEQ ID NO：24所示的的CD28-LCDR3；或(i) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and The CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 22, such as SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 CD28-LCDR2 is shown and as CD28-LCDR3 shown in SEQ ID NO: 24; or
(ii)所述CD28-VH包含如SEQ ID NO：13所示的CD28-HCDR1、如SEQ ID NO：14所示的CD28-HCDR2和如SEQ ID NO：15所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：16所示的CD28-LCDR1、如SEQ ID NO：17、54、55、56、57、58、59、60、61或62所示的CD28-LCDR2和如SEQ ID NO：18所示的CD28-LCDR3；或(ii) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and The CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and CD28-LCDR3 shown in SEQ ID NO: 18; or
(iii)所述CD28-VH包含如SEQ ID NO：25或117所示的CD28-HCDR1、如SEQ ID NO：26或116所示的CD28-HCDR2和如SEQ ID NO：27所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：28所示的CD28-LCDR1、如SEQ ID NO：29、118、119、120、121或122所示的CD28-LCDR2和如SEQ ID NO：30所示的CD28-LCDR3；或(iii) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25 or 117, CD28-HCDR2 as shown in SEQ ID NO: 26 or 116, and CD28-HCDR1 as shown in SEQ ID NO: 27 HCDR3, and the CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 as set forth in SEQ ID NO: CD28-LCDR3 shown in 30; or
(iv)所述CD28-VH包含如SEQ ID NO：7所示的CD28-HCDR1、如SEQ ID NO：8所示的CD28-HCDR2和如SEQ ID NO：9、39、40、41或42所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：10所示的CD28-LCDR1、如SEQ ID NO：11或43所示的CD28-LCDR2和如SEQ ID NO：12所示的CD28-LCDR3。(iv) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 7, CD28-HCDR2 as shown in SEQ ID NO: 8 and CD28-HCDR2 as shown in SEQ ID NO: 9, 39, 40, 41 or 42 CD28-HCDR3 as shown, and the CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 11 or 43, and CD28-LCDR2 as shown in SEQ ID NO: 12 CD28-LCDR3.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
所述CD28-VH具有：包含SEQ ID NO：25的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and The CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
所述CD28-VH具有：包含SEQ ID NO：25的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：116的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and The CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
所述CD28-VH具有：包含SEQ ID NO：117的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 27, and The CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: CD28-LCDR3 with an amino acid sequence of 30; or
所述CD28-VH具有：包含SEQ ID NO：117的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：116的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨 基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3。The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 116 and amino acid sequence comprising SEQ ID NO: 27 CD28-HCDR3 with amino acid sequence, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 CD28-LCDR2 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO:30.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9、39、40、41或42的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：11的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3；或The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42. The amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12 The amino acid sequence of CD28-LCDR3; or
所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9、39、40、41或42的氨基酸序列的CD28-HCDR3，和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：43的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3。The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42. The amino acid sequence of CD28-HCDR3, and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 12 Amino acid sequence of CD28-LCDR3.
在一些实施方案中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
(i)所述CD28-VH包含如SEQ ID NO：19所示的CD28-HCDR1、如SEQ ID NO：20所示的CD28-HCDR2和如SEQ ID NO：21所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：22所示的CD28-LCDR1、如SEQ ID NO：23所示的CD28-LCDR2和如SEQ ID NO：24所示的CD28-LCDR3；或(i) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and The CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 22, CD28-LCDR2 as set forth in SEQ ID NO: 23, and CD28-LCDR3 as set forth in SEQ ID NO: 24; or
(ii)所述CD28-VH包含如SEQ ID NO：13所示的CD28-HCDR1、如SEQ ID NO：14所示的CD28-HCDR2和如SEQ ID NO：15所示的CD28-HCDR3，和所述CD28-VL如SEQ ID NO：16所示的CD28-LCDR1、如SEQ ID NO：62所示的CD28-LCDR2和如SEQ ID NO：18所示的CD28-LCDR3；或(ii) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and The CD28-VL is CD28-LCDR1 set forth in SEQ ID NO: 16, CD28-LCDR2 set forth in SEQ ID NO: 62, and CD28-LCDR3 set forth in SEQ ID NO: 18; or
(iii)所述CD28-VH包含如SEQ ID NO：25所示的CD28-HCDR1、如SEQ ID NO：26所示的CD28-HCDR2和如SEQ ID NO：27所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：28所示的CD28-LCDR1、如SEQ ID NO：29所示的CD28-LCDR2和如SEQ ID NO：30所示的CD28-LCDR3；或(iii) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 25, CD28-HCDR2 as shown in SEQ ID NO: 26 and CD28-HCDR3 as shown in SEQ ID NO: 27, and The CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 28, CD28-LCDR2 as set forth in SEQ ID NO: 29, and CD28-LCDR3 as set forth in SEQ ID NO: 30; or
(iv)所述CD28-VH包含如SEQ ID NO：7所示的CD28-HCDR1、如SEQ ID NO：8所示的CD28-HCDR2和如SEQ ID NO：9所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：10所示的CD28-LCDR1、如SEQ ID NO：43所示的CD28-LCDR2和如SEQ ID NO：12所示的CD28-LCDR3。(iv) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO:7, CD28-HCDR2 as shown in SEQ ID NO:8 and CD28-HCDR3 as shown in SEQ ID NO:9, and The CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 10, CD28-LCDR2 as shown in SEQ ID NO: 43, and CD28-LCDR3 as shown in SEQ ID NO: 12.
在一些实施方案中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
所述CD28-VH包含如SEQ ID NO：19所示的CD28-HCDR1、如SEQ ID NO：20所示的CD28-HCDR2和如SEQ ID NO：21所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：22所示的CD28-LCDR1、如SEQ ID NO：23所示的CD28-LCDR2和如SEQ ID NO：24所示的CD28-LCDR3。 The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and the CD28- VL includes CD28-LCDR1 as shown in SEQ ID NO:22, CD28-LCDR2 as shown in SEQ ID NO:23, and CD28-LCDR3 as shown in SEQ ID NO:24.
在一些实施方案中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
所述CD28-VH包含如SEQ ID NO：13所示的CD28-HCDR1、如SEQ ID NO：14所示的CD28-HCDR2和如SEQ ID NO：15所示的CD28-HCDR3，和所述CD28-VL如SEQ ID NO：16所示的CD28-LCDR1、如SEQ ID NO：62所示的CD28-LCDR2和如SEQ ID NO：18所示的CD28-LCDR3。The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and the CD28- VL is CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 62, and CD28-LCDR3 as shown in SEQ ID NO: 18.
在一些实施方式中，如前任一项所述的抗CD28抗体，所述CD28-HCDR1、CD28-HCDR2、CD28-HCDR3、CD28-LCDR1、CD28-LCDR2和CD28-LCDR3是根据Kabat编号规则定义的。In some embodiments, the anti-CD28 antibody of any one of the preceding items, the CD28-HCDR1, CD28-HCDR2, CD28-HCDR3, CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 are defined according to the Kabat numbering rule.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述的CD28-VH和CD28-VL是人源化的，均包含人抗体的FR区。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the CD28-VH and CD28-VL are humanized and both comprise the FR region of a human antibody.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
(i)所述CD28-VH具有：包含SEQ ID NO：19的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：20的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：21的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、26A、29L、69L、71V、78A和93S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：22的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：23、81、82、83、84、85、86、87、88、89、90、91、92或93的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：24的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自41D、42G、43T、44I和71Y组成的组中的一个或多个氨基酸取代；或(i) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 with the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and the FR region of the CD28-VL comprises one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44I and 71Y; or
(ii)所述CD28-VH具有：包含SEQ ID NO：13的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：14的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：15的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、28S、69L、71V、73K和94S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：16的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：17、54、55、56、57、58、59、60、61或62的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：18的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自43S和70K组成的组中的一个或多个氨基酸取代；或(ii) The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15. HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 with the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and the amino acid sequence of SEQ ID NO: 18 CD28-LCDR3, and the FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 43S and 70K; or
(iii)所述CD28-VH具有：包含SEQ ID NO：25或117的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26或116的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、25P、71V和78A组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自41D、42G、43T、44V和73L组成的组中的一个或多个氨基酸取代；或(iii) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116 and the amino acid sequence comprising SEQ ID NO: 27 The sequence of CD28-HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 71V and 78A; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 having the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 having the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and CD28-LCDR3 having the amino acid sequence of SEQ ID NO: 30, and said The FR region of CD28-VL contains one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44V and 73L; or
(iv)所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、 包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9、39、40、41或42的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、69L、71V、73K、78A和94S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：11或43的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3；且所述CD28-VL的FR区包含选自43S和73F组成的组中的一个或多个氨基酸取代。(iv) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or 42, and the FR region of the CD28-VH comprises a FR region selected from 1E, One or more amino acid substitutions in the group consisting of 69L, 71V, 73K, 78A and 94S; and the CD28-VL has: a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 11 or CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 12 and CD28-LCDR3 with the amino acid sequence of SEQ ID NO: 12; and the FR region of CD28-VL includes one or more amino acid substitutions selected from the group consisting of 43S and 73F.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
(i)所述CD28-VH具有：包含SEQ ID NO：19的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：20的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：21的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、26A、29L、69L、71V、78A和93S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：22的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：23的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：24的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自41D、42G、43T、44I和71Y组成的组中的一个或多个氨基酸取代；或(i) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 26A, 29L, 69L, 71V, 78A and 93S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, and the FR region of CD28-VL includes the selected One or more amino acid substitutions from the group consisting of 41D, 42G, 43T, 44I and 71Y; or
(ii)所述CD28-VH具有：包含SEQ ID NO：13的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：14的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：15的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、28S、69L、71V、73K和94S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：16的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：62的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：18的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自43S和70K组成的组中的一个或多个氨基酸取代；或(ii) The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 14, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 15. HCDR3, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID NO: CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 62, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 62, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 18, and the FR region of the CD28-VL contains a protein selected from the group consisting of 43S and one or more amino acid substitutions in the group consisting of 70K; or
(iii)所述CD28-VH具有：包含SEQ ID NO：25的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：26的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、25P、71V和78A组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：29的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3，且所述CD28-VL的FR区包含选自41D、42G、43T、44V和73L组成的组中的一个或多个氨基酸取代；或(iii) The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 26, and CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 27. HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 71V and 78A; and the CD28-VL has: the amino acid sequence comprising SEQ ID NO: 28 CD28-LCDR1, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30, and the FR region of the CD28-VL comprises 41D, 42G, 43T One or more amino acid substitutions in the group consisting of , 44V and 73L; or
(iv)所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1、包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：9的氨基酸序列的CD28-HCDR3，且所述CD28-VH的FR区包含选自1E、69L、71V、73K、78A和94S组成的组中的一个或多个氨基酸取代；和所述CD28-VL具有：包含SEQ  ID NO：10的氨基酸序列的CD28-LCDR1、包含SEQ ID NO：43的氨基酸序列的CD28-LCDR2和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3；且所述CD28-VL的FR区包含选自43S和73F组成的组中的一个或多个氨基酸取代。(iv) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3, and the FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S; and the CD28-VL has: comprising SEQ CD28-LCDR1 with the amino acid sequence of ID NO: 10, CD28-LCDR2 with the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR3 with the amino acid sequence of SEQ ID NO: 12; and the FR region of CD28-VL includes One or more amino acid substitutions selected from the group consisting of 43S and 73F.
在一些实施方式中，如前任一项所述的抗CD28抗体，其在25℃条件下以小于2×10-8M、8×10-9M或5×10-9M的KD结合人CD28，所述KD是通过表面等离子体共振法测量的。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items binds to human CD28 with a KD of less than 2×10 -8 M, 8×10 -9 M or 5×10 -9 M at 25°C. , the KD is measured by surface plasmon resonance method.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：98的氨基酸序列或其变体，所述变体为在SEQ ID NO：98的FR区包含且所述CD28-VH的FR区包含选自26A、29L、69L、71V、78A和93S组成的组中的一个或多个氨基酸取代，和所述CD28-VL包含SEQ ID NO：102的氨基酸序列或其变体，所述变体为在SEQ ID NO：102的FR区包含选自41D、42G、43T、44I和71Y组成的组中的一个或多个氨基酸取代；或(i) The CD28-VH includes the amino acid sequence of SEQ ID NO: 98 or a variant thereof, the variant is included in the FR region of SEQ ID NO: 98 and the FR region of the CD28-VH includes a sequence selected from 26A , 29L, 69L, 71V, 78A and 93S, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 102 or a variant thereof, the variant being in SEQ ID NO. The FR region of NO: 102 contains one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44I and 71Y; or
(ii)所述CD28-VH包含SEQ ID NO：69的氨基酸序列或其变体，所述变体为在SEQ ID NO：69的FR区包含选自28S、69L、71V、73K和94S组成的组中的一个或多个氨基酸取代，和所述CD28-VL包含SEQ ID NO：70的氨基酸序列或其变体，所述变体为在SEQ ID NO：70的FR区包含选自43S和70K组成的组中的一个或多个氨基酸取代；或(ii) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof, wherein the variant contains a sequence selected from the group consisting of 28S, 69L, 71V, 73K and 94S in the FR region of SEQ ID NO: 69 One or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or a variant thereof, the variant being in the FR region of SEQ ID NO: 70 and including 43S and 70K One or more amino acid substitutions in the group consisting of; or
(iii)所述CD28-VH包含SEQ ID NO：133的氨基酸序列或其变体，所述变体为在SEQ ID NO：133的FR区包含选自25P、71V和78A组成的组中的一个或多个氨基酸取代，和所述CD28-VL包含SEQ ID NO：137的氨基酸序列或其变体，所述变体为在SEQ ID NO：137的FR区包含选自41D、42G、43T、44V和73L组成的组中的一个或多个氨基酸取代；或(iii) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 133 or a variant thereof, wherein the variant is one selected from the group consisting of 25P, 71V and 78A in the FR region of SEQ ID NO: 133 Or multiple amino acid substitutions, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 137 or a variant thereof, the variant is in the FR region of SEQ ID NO: 137. One or more amino acid substitutions in the group consisting of 73L; or
(iv)所述CD28-VH包含SEQ ID NO：53的氨基酸序列或其变体，所述变体为在SEQ ID NO：53的FR区包含选自1E、69L、71V、73K、78A和94S组成的组中的一个或多个氨基酸取代，和所述CD28-VL包含SEQ ID NO：50的氨基酸序列或其变体，所述变体为在SEQ ID NO：50的FR区包含选自43S和73F组成的组中的一个或多个氨基酸取代；或(iv) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 53 or a variant thereof, wherein the variant contains a FR region selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S of SEQ ID NO: 53 consisting of one or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or a variant thereof, the variant being in the FR region of SEQ ID NO: 50 and comprising 43S selected from the group consisting of One or more amino acid substitutions in the group consisting of 73F; or
所述CD28-VH包含SEQ ID NO：53的氨基酸序列或其变体，所述变体为在SEQ ID NO：53的FR区包含选自1E、69L、71V、73K、78A和94S组成的组中的一个或多个氨基酸取代，和所述CD28-VL包含SEQ ID NO：52的氨基酸序列或其变体，所述变体为在SEQ ID NO：52的FR区包含选自43S和73F组成的组中的一个或多个氨基酸取代。The CD28-VH comprises the amino acid sequence of SEQ ID NO: 53 or a variant thereof, wherein the variant is a group selected from the group consisting of 1E, 69L, 71V, 73K, 78A and 94S in the FR region of SEQ ID NO: 53. One or more amino acid substitutions in, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52 or a variant thereof, wherein the variant is composed of a FR region selected from 43S and 73F of SEQ ID NO: 52 One or more amino acids in the group are substituted.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：95、35、94、96、97或98的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101、36、99、100、102、103、104、105、 106、107、108、109、110、111、112、113、114或115的氨基酸序列；或(i) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 35, 94, 96, 97 or 98, and the CD28-VL comprises SEQ ID NO: 101, 36, 99, 100, 102, 103 ,104,105, The amino acid sequence of 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115; or
(ii)所述CD28-VH包含SEQ ID NO：68、33、63、64、65、66、67或69的氨基酸序列，和所述CD28-VL包含SEQ ID NO：80、34、70、71、72、73、74、75、76、77、78或79的氨基酸序列；或(ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69, and the CD28-VL comprises SEQ ID NO: 80, 34, 70, 71 , 72, 73, 74, 75, 76, 77, 78 or 79 amino acid sequence; or
(iii)所述CD28-VH包含SEQ ID NO：126、37、123、124、125、127、128、129、130、131、132或133的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、38、135、136、137、138、139、140、141、142或143的氨基酸序列；或(iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 37, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133, and the CD28-VL comprises SEQ ID NO. : the amino acid sequence of 134, 38, 135, 136, 137, 138, 139, 140, 141, 142 or 143; or
(iv)所述CD28-VH包含SEQ ID NO：44、31、45、46、47、48、49或53的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52、32、50或51的氨基酸序列。(iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 31, 45, 46, 47, 48, 49 or 53, and the CD28-VL comprises SEQ ID NO: 52, 32, 50 or 51 amino acid sequence.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：95、94、96、97或98的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101、99、100、102、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列；或(i) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98, and the CD28-VL comprises SEQ ID NO: 101, 99, 100, 102, 103, 104, 105 , the amino acid sequence of 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115; or
(ii)所述CD28-VH包含SEQ ID NO：68、63、64、65、66、67或69的氨基酸序列，和所述CD28-VL包含SEQ ID NO：80、70、71、72、73、74、75、76、77、78或79的氨基酸序列；或(ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 63, 64, 65, 66, 67 or 69, and the CD28-VL comprises SEQ ID NO: 80, 70, 71, 72, 73 , 74, 75, 76, 77, 78 or 79 amino acid sequence; or
(iii)所述CD28-VH包含SEQ ID NO：126、123、124、125、127、128、129、130、131、132或133的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、135、136、137、138、139、140、141、142或143的氨基酸序列；或(iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133, and the CD28-VL comprises SEQ ID NO: 134 , 135, 136, 137, 138, 139, 140, 141, 142 or 143 amino acid sequence; or
(iv)所述CD28-VH包含SEQ ID NO：44、45、46、47、48或49的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52、50、51或53的氨基酸序列。(iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 45, 46, 47, 48 or 49, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52, 50, 51 or 53.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：35的氨基酸序列，和所述CD28-VL包含SEQ ID NO：36的氨基酸序列；或(i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 35, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 36; or
(ii)所述CD28-VH包含SEQ ID NO：33的氨基酸序列，和所述CD28-VL包含SEQ ID NO：34的氨基酸序列；或(ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 33, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 34; or
(iii)所述CD28-VH包含SEQ ID NO：37的氨基酸序列，和所述CD28-VL包含SEQ ID NO：38的氨基酸序列；或(iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 37, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 38; or
(iv)所述CD28-VH包含SEQ ID NO：31的氨基酸序列，和所述CD28-VL包含SEQ ID NO：32的氨基酸序列。(iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 31, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 32.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：95、94、96、97或98的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101、99、100或102的氨基酸序列，或(i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102, or
所述CD28-VH包含SEQ ID NO：94的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99、100、101、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列，或 The CD28-VH includes the amino acid sequence of SEQ ID NO: 94, and the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
所述CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99、100、101、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列，或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
所述CD28-VH包含SEQ ID NO：96的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99或100的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 96, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99 or 100, or
所述CD28-VH包含SEQ ID NO：97的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99、100或101的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 97, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99, 100 or 101, or
(ii)所述CD28-VH包含SEQ ID NO：63的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70或71的氨基酸序列，或(ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 63, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or 71, or
所述CD28-VH包含SEQ ID NO：64的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70、71或72的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 64, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
所述CD28-VH包含SEQ ID NO：65的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70、71或72的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 65, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
所述CD28-VH包含SEQ ID NO：66的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70、71、72、74、75、76或79氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 66, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71, 72, 74, 75, 76 or 79, or
所述CD28-VH包含SEQ ID NO：67的氨基酸序列，和所述CD28-VL包含SEQ ID NO：72、74、75、76或79的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 67, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 74, 75, 76 or 79, or
所述CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述CD28-VL包含SEQ ID NO：72、73、74、75、76、77、78、79或80的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79 or 80, or
(iii)所述CD28-VH包含SEQ ID NO：123的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、135或136的氨基酸序列，或(iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 123, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135 or 136, or
所述CD28-VH包含SEQ ID NO：124的氨基酸序列，和所述CD28-VL包含SEQ ID NO：135的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 124, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 135; or
所述CD28-VH包含SEQ ID NO：125的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134或135的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 125, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134 or 135, or
所述CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、135、136、137、138或139的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135, 136, 137, 138 or 139, or
所述CD28-VH包含SEQ ID NO：127的氨基酸序列，和所述CD28-VL包含SEQ ID NO：137、138或139的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 127, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 137, 138 or 139; or
所述CD28-VH包含SEQ ID NO：128的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、137、138、139或142的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 128, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 137, 138, 139 or 142; or
所述CD28-VH包含SEQ ID NO：129的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、138、139、140、141、142或143的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 129, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 138, 139, 140, 141, 142 or 143, or
所述CD28-VH包含SEQ ID NO：130的氨基酸序列，和所述CD28-VL包含SEQ ID NO：138、140、141、142或143的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 130, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
所述CD28-VH包含SEQ ID NO：131的氨基酸序列，和所述CD28-VL包含 SEQ ID NO：138、140、141、142或143的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 131, and the CD28-VL comprises The amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
(iv)所述CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50、51或52的氨基酸序列，或(iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50, 51 or 52, or
所述CD28-VH包含SEQ ID NO：45的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 45, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
所述CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或52的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 46, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 52, or
所述CD28-VH包含SEQ ID NO：47的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 47, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
所述CD28-VH包含SEQ ID NO：48的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列。The CD28-VH comprises the amino acid sequence of SEQ ID NO: 48, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中：In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein:
(i)所述CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101的氨基酸序列；或(i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101; or
(ii)所述CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述CD28-VL包含SEQ ID NO：80的氨基酸序列；或(ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80; or
(iii)所述CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134的氨基酸序列；或(iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134; or
(iv)所述CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52的氨基酸序列。(iv) The CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述CD28-VH的氨基酸序列如SEQ ID NO：95所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：101所示。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 95, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 101 shown.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述CD28-VH的氨基酸序列如SEQ ID NO：68所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：80所示。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 68, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 80 shown.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述CD28-VH的氨基酸序列如SEQ ID NO：126所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：134所示。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 126, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 134 shown.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述CD28-VH的氨基酸序列如SEQ ID NO：44所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：52所示。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the amino acid sequence of CD28-VH is as shown in SEQ ID NO: 44, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 52 shown.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述的抗CD28抗体包含重链恒定区和轻链恒定区。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain constant region and a light chain constant region.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述重链恒定区为人IgG1恒定区。 In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein the heavy chain constant region is a human IgGl constant region.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述重链恒定区包含如SEQ ID NO：144的氨基酸序列。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 144.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述轻链恒定区包含如SEQ ID NO：145的氨基酸序列。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO: 145.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述的抗CD28抗体包含重链和轻链，其中：In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein:
所述重链包含与SEQ ID NO：146具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的氨基酸序列，和所述轻链包含与SEQ ID NO：147具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的氨基酸序列；或The heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence, and the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, An amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical; or
所述重链包含与SEQ ID NO：148具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的氨基酸序列，和所述轻链包含与SEQ ID NO：149具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的氨基酸序列；或The heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence, and the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, An amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical; or
所述重链包含与SEQ ID NO：150具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的氨基酸序列，和所述轻链包含与SEQ ID NO：151具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的氨基酸序列；或The heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence, and the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, An amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical; or
所述重链包含与SEQ ID NO：152具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的氨基酸序列，和所述轻链包含与SEQ ID NO：153具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的氨基酸序列。The heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% sequence identity to an amino acid sequence, and the light chain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, Amino acid sequences with 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述的抗CD28抗体包含重链和轻链，其中：In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein:
所述重链包含SEQ ID NO：146的氨基酸序列，和所述轻链包含SEQ ID NO：147的氨基酸序列；The heavy chain includes the amino acid sequence of SEQ ID NO: 146, and the light chain includes the amino acid sequence of SEQ ID NO: 147;
所述重链包含SEQ ID NO：148的氨基酸序列，和所述轻链包含SEQ ID NO：149的氨基酸序列；或The heavy chain comprises the amino acid sequence of SEQ ID NO: 148, and the light chain comprises the amino acid sequence of SEQ ID NO: 149; or
所述重链包含SEQ ID NO：150的氨基酸序列，和所述轻链包含SEQ ID NO：151的氨基酸序列；或The heavy chain comprises the amino acid sequence of SEQ ID NO: 150, and the light chain comprises the amino acid sequence of SEQ ID NO: 151; or
所述重链包含SEQ ID NO：152的氨基酸序列，和所述轻链包含SEQ ID NO：153的氨基酸序列。 The heavy chain includes the amino acid sequence of SEQ ID NO:152, and the light chain includes the amino acid sequence of SEQ ID NO:153.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述的抗CD28抗体包含重链和轻链，其中所述重链包含SEQ ID NO：150的氨基酸序列，和所述轻链包含SEQ ID NO：151的氨基酸序列。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 150, and the light chain The chain contains the amino acid sequence of SEQ ID NO: 151.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述的抗CD28抗体包含重链和轻链，其中所述重链包含SEQ ID NO：148的氨基酸序列，和所述轻链包含SEQ ID NO：149的氨基酸序列。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 148, and the light chain The chain contains the amino acid sequence of SEQ ID NO: 149.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述的抗CD28抗体是抗体片段。In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein the anti-CD28 antibody is an antibody fragment.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述的抗CD28抗体是抗体片段，其中所述的抗体片段为Fab、Fab'、F(ab')2、Fd、Fv、scFv、dsFv或dAb。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody is an antibody fragment, wherein the antibody fragment is Fab, Fab', F(ab')2, Fd, Fv , scFv, dsFv or dAb.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述的抗CD28抗体是双特异性抗体。In some embodiments, the anti-CD28 antibody of any one of the preceding items, wherein the anti-CD28 antibody is a bispecific antibody.
在一些实施方式中，如前任一项所述的抗CD28抗体，其中所述的抗CD28抗体是双特异性抗体，所述双特异性抗体特异性结合CD28和PSMA。In some embodiments, the anti-CD28 antibody as described in any one of the preceding items, wherein the anti-CD28 antibody is a bispecific antibody, and the bispecific antibody specifically binds to CD28 and PSMA.
在另一个方面，本披露提供了一种药物组合物，其含有：(例如治疗有效量的)前述任一项所述的抗原结合分子或前述任一项所述的抗CD28抗体，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。在一些实施方案中，所述的药物组合物中还包含至少一种(例如一种)第二治疗剂。在一个实施例中，所述第二治疗剂是有利地与PSMA/CD28双特异性抗原结合分子组合的任何药剂(包括抗肿瘤药剂、放射疗法、抗体药物缀合物、双特异性抗体、与抗肿瘤药剂缀合的双特异性抗体、免疫检查点抑制剂或其组合)。在一些实施方案中，所述第二治疗剂是能够特异性结合CD3的抗体；优选地，所述第二治疗剂是能够特异性结合CD3的双特异性抗体；更优选地，所述第二治疗剂是能够特异性结合CD3和肿瘤细胞表面抗原(TAA)的双特异性抗体；最优选地，所述第二治疗剂是能够特异性结合CD3和PSMA的双特异性的抗体。在一些实施方案中，所述第二治疗剂是免疫检查点抑制剂，其中所述的免疫检查点抑制剂靶向PD-1或CTLA4；优选地，其中所述的免疫检查点抑制剂是抗PD-1抗体。In another aspect, the present disclosure provides a pharmaceutical composition comprising: (e.g., a therapeutically effective amount) the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing, and a or more pharmaceutically acceptable carriers, diluents, buffers or excipients. In some embodiments, the pharmaceutical composition further includes at least one (eg, one) second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with a PSMA/CD28 bispecific antigen-binding molecule (including antineoplastic agents, radiotherapy, antibody drug conjugates, bispecific antibodies, and antineoplastic agent-conjugated bispecific antibodies, immune checkpoint inhibitors, or combinations thereof). In some embodiments, the second therapeutic agent is an antibody capable of specifically binding CD3; preferably, the second therapeutic agent is a bispecific antibody capable of specifically binding CD3; more preferably, the second therapeutic agent is a bispecific antibody capable of specifically binding CD3. The therapeutic agent is a bispecific antibody that specifically binds to CD3 and tumor cell surface antigen (TAA); most preferably, the second therapeutic agent is a bispecific antibody that specifically binds to CD3 and PSMA. In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor targets PD-1 or CTLA4; preferably, the immune checkpoint inhibitor is an anti- PD-1 antibodies.
在另一个方面，本披露还提供分离的核酸，其编码前述任一项所述的抗原结合分子或前述任一项所述的抗CD28抗体。In another aspect, the present disclosure also provides an isolated nucleic acid encoding the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing.
在另一个方面，本披露还提供载体，其包含前述的分离的核酸。In another aspect, the present disclosure also provides a vector comprising the aforementioned isolated nucleic acid.
在另一个方面，本披露还提供一种宿主细胞，其包含前述的载体。In another aspect, the present disclosure also provides a host cell comprising the aforementioned vector.
在另一个方面，本披露还提供一种治疗疾病的方法，所述方法包括向受试者施用(例如治疗有效量的)前述任一项所述的抗原结合分子或前述任一项所述的抗CD28抗体或如前任一项所述的组合物。In another aspect, the present disclosure also provides a method of treating a disease, the method comprising administering (e.g., a therapeutically effective amount) to a subject the antigen-binding molecule of any of the foregoing or the antigen-binding molecule of any of the foregoing. Anti-CD28 antibody or a composition as described in any one of the preceding items.
在另一个方面，本披露还提供前述任一项所述的抗原结合分子或前述任一项 所述的抗体或其组合物在制备治疗疾病的药物中的用途。In another aspect, the present disclosure also provides the antigen-binding molecule of any of the foregoing or any of the foregoing. The use of the antibody or its composition in the preparation of medicines for treating diseases.
在另一个方面，本披露还提供用作药物的前述任一项所述的抗原结合分子或前述任一项所述的抗CD28抗体或如前任一项所述的组合物。在一些实施方式中，所述药物用于治疗疾病。In another aspect, the present disclosure also provides the antigen-binding molecule of any of the foregoing, the anti-CD28 antibody of any of the foregoing, or the composition of any of the foregoing for use as a medicament. In some embodiments, the medicament is used to treat disease.
在一些实施方式中，如前任一项所述的疾病是肿瘤。在一些实施方式中，如前任一项所述的肿瘤为前列腺癌、肾癌、尿路上皮癌、乳腺癌、膀胱癌、原发性肝癌、胰腺癌、黑色素瘤、胶质母细胞瘤(例如多形性胶质母细胞瘤)、骨肉瘤、卵巢癌、食管癌、宫颈鳞状细胞癌、肺癌(非小细胞肺癌、小细胞肺癌、肺鳞状细胞癌、肺腺癌)、结直肠癌(包括结肠癌和直肠癌)、子宫内膜癌、皮肤癌、头颈部鳞状细胞癌、脑癌、胃食管癌(胃食管腺癌)、肝脏转移性癌、多发性骨髓瘤、淋巴瘤、急性髓样白血病(AML)、急性淋巴母细胞性白血病(ALL)、慢性淋巴细胞白血病(CLL)或B细胞淋巴瘤。In some embodiments, the disease of any of the preceding items is a tumor. In some embodiments, the tumor of any one of the preceding items is prostate cancer, renal cancer, urothelial cancer, breast cancer, bladder cancer, primary liver cancer, pancreatic cancer, melanoma, glioblastoma (e.g. Glioblastoma multiforme), osteosarcoma, ovarian cancer, esophageal cancer, cervical squamous cell carcinoma, lung cancer (non-small cell lung cancer, small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma), colorectal cancer (including colon and rectal cancer), endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, gastroesophageal cancer (gastroesophageal adenocarcinoma), metastatic liver cancer, multiple myeloma, lymphoma , acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) or B-cell lymphoma.
在一些实施方式中，如前所述的肿瘤的细胞或靠近该肿瘤的血管内皮细胞表达PSMA。In some embodiments, PSMA is expressed by cells of a tumor as described above or by vascular endothelial cells proximate the tumor.
在一些实施方式中，如前所述的肿瘤是实体瘤。In some embodiments, the tumor as described above is a solid tumor.
在一些实施方式中，如前所述的肿瘤是实体瘤，所述实体瘤为前列腺癌、肾癌、尿路上皮癌、乳腺癌、膀胱癌、原发性肝癌、胰腺癌、黑色素瘤、胶质母细胞瘤(例如多形性胶质母细胞瘤)、骨肉瘤、卵巢癌、食管癌、宫颈鳞状细胞癌、肺癌(非小细胞肺癌、小细胞肺癌、肺鳞状细胞癌、肺腺癌)、结直肠癌(包括结肠癌和直肠癌)、子宫内膜癌、皮肤癌、头颈部鳞状细胞癌、脑癌、胃食管癌(胃食管腺癌)、肝脏转移性癌。In some embodiments, the tumor as described above is a solid tumor, and the solid tumor is prostate cancer, renal cancer, urothelial cancer, breast cancer, bladder cancer, primary liver cancer, pancreatic cancer, melanoma, glaucoma, Blastoma (such as glioblastoma multiforme), osteosarcoma, ovarian cancer, esophageal cancer, cervical squamous cell carcinoma, lung cancer (non-small cell lung cancer, small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma cancer), colorectal cancer (including colon cancer and rectal cancer), endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, gastroesophageal cancer (gastroesophageal adenocarcinoma), liver metastatic cancer.
在一些实施方式中，如前所述的肿瘤是前列腺癌。In some embodiments, the tumor as described above is prostate cancer.
在一些实施方式中，如前所述的肿瘤是非实体瘤。In some embodiments, the tumor as described above is a non-solid tumor.
在一些实施方式中，如前所述的肿瘤是非实体瘤，所述非实体瘤为多发性骨髓瘤、淋巴瘤、急性髓样白血病(AML)、急性淋巴母细胞性白血病(ALL)、慢性淋巴细胞白血病(CLL)或B细胞淋巴瘤。In some embodiments, the tumor as described above is a non-solid tumor, the non-solid tumor being multiple myeloma, lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia Cellular leukemia (CLL) or B-cell lymphoma.
在一些实施方式中，如前所述的前列腺癌是难治性前列腺癌、前列腺上皮内瘤、雄激素非依赖性前列腺癌、恶性前列腺癌、复发性前列腺癌或去势抵抗性前列腺癌。In some embodiments, the prostate cancer as described above is refractory prostate cancer, prostatic intraepithelial neoplasia, androgen-independent prostate cancer, malignant prostate cancer, recurrent prostate cancer, or castration-resistant prostate cancer.
在一些实施方案中，前述的疾病为与PSMA相关的疾病；在一些实施方案中，前述的疾病为表达PSMA的疾病。In some embodiments, the aforementioned disease is a disease associated with PSMA; in some embodiments, the aforementioned disease is a disease expressing PSMA.
在一些实施方案中，如前所述治疗疾病的方法，其进一步包括使用第二治疗剂。在一些实施方案中，所述所述第二治疗剂包括抗肿瘤药剂、放射疗法、抗体药物缀合物、双特异性抗体、与抗肿瘤药剂缀合的双特异性抗体、免疫检查点抑制剂或其组合。在一些实施方案中，所述第二治疗剂是能够特异性结合CD3的抗体；优选地，所述第二治疗剂是能够特异性结合CD3的双特异性抗体；更优选地， 所述第二治疗剂是能够特异性结合CD3和肿瘤细胞表面抗原(TAA)的双特异性抗体；最优选地，所述第二治疗剂是能够特异性结合CD3和PSMA的双特异性的抗体。在一些实施方案中，所述第二治疗剂是免疫检查点抑制剂，其中所述的免疫检查点抑制剂靶向PD-1或CTLA4；优选地，其中所述的免疫检查点抑制剂是抗PD-1抗体。In some embodiments, the method of treating a disease as described above further includes using a second therapeutic agent. In some embodiments, the second therapeutic agent includes an anti-tumor agent, radiation therapy, antibody drug conjugates, bispecific antibodies, bispecific antibodies conjugated to an anti-tumor agent, immune checkpoint inhibitors or combination thereof. In some embodiments, the second therapeutic agent is an antibody capable of specifically binding CD3; preferably, the second therapeutic agent is a bispecific antibody capable of specifically binding CD3; more preferably, The second therapeutic agent is a bispecific antibody that can specifically bind to CD3 and tumor cell surface antigen (TAA); most preferably, the second therapeutic agent is a bispecific antibody that can specifically bind to CD3 and PSMA. . In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor targets PD-1 or CTLA4; preferably, the immune checkpoint inhibitor is an anti- PD-1 antibodies.
在一些实施方案中，如前任一项所述的第二治疗剂与本披露任一项所述的抗原结合分子同时施用、序贯施用或分开施用。In some embodiments, the second therapeutic agent of any one of the preceding and the antigen-binding molecule of any of this disclosure are administered simultaneously, sequentially, or separately.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含至少一个特异性结合PSMA的抗原结合模块和至少一个特异性结合CD3的抗原结合模块。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding moiety that specifically binds PSMA and at least one antigen-binding moiety that specifically binds CD3. Antigen binding module.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含至少一个特异性结合PSMA的抗原结合模块和至少一个特异性结合CD3的抗原结合模块，其中所述的特异性结合PSMA的抗原结合模块与特异性结合CD28和PSMA的抗原结合分子中结合PSMA的抗原结合模块结合的表位不同。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding moiety that specifically binds PSMA and at least one antigen-binding moiety that specifically binds CD3. Antigen-binding module, wherein the antigen-binding module that specifically binds to PSMA binds to a different epitope than the antigen-binding module that specifically binds to CD28 and PSMA in the antigen-binding molecule that specifically binds to CD28 and PSMA.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含至少一个特异性结合PSMA的抗原结合模块和至少一个特异性结合CD3的抗原结合模块，所述特异性结合PSMA的抗原结合模块包含重链可变区PSMA-VH和轻链可变区PSMA-VL，所述特异性结合CD3的抗原结合模块包含重链可变区CD3-VH和轻链可变区CD3-VL。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding moiety that specifically binds PSMA and at least one antigen-binding moiety that specifically binds CD3. Antigen-binding module, the antigen-binding module that specifically binds to PSMA includes the heavy chain variable region PSMA-VH and the light chain variable region PSMA-VL, and the antigen-binding module that specifically binds to CD3 includes the heavy chain variable region CD3 -VH and light chain variable region CD3-VL.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
i)所述PSMA-VH具有：包含SEQ ID NO：164的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：165的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：166的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：167的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：168的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：169的氨基酸序列的PSMA-LCDR3；或i) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 166 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 168, and PSMA comprising the amino acid sequence of SEQ ID NO: 169 -LCDR3; or
ii)所述PSMA-VH具有：包含SEQ ID NO：170的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：171的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：172的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：173的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：174的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：175的氨基酸序列的PSMA-LCDR3；或ii) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 172 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 174, and PSMA comprising the amino acid sequence of SEQ ID NO: 175 -LCDR3; or
iii)所述PSMA-VH具有：包含SEQ ID NO：158的氨基酸序列的 PSMA-HCDR1，包含SEQ ID NO：159的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：160的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：161的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：162的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：163的氨基酸序列的PSMA-LCDR3；或iii) The PSMA-VH has: comprising the amino acid sequence of SEQ ID NO: 158 PSMA-HCDR1, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 159, and PSMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 160; and said PSMA-VL has: comprising the amino acid sequence of SEQ ID NO: 161 PSMA-LCDR1, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 162, and PSMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 163; or
iv)所述PSMA-VH具有：包含SEQ ID NO：176的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：177的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：178的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：179的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：180的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：181的氨基酸序列的PSMA-LCDR3；或iv) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 176, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 177, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 178 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 179, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 180, and PSMA comprising the amino acid sequence of SEQ ID NO: 181 -LCDR3; or
v)所述PSMA-VH具有：包含SEQ ID NO：301的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：302的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：303的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：304的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：305的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：306的氨基酸序列的PSMA-LCDR3。v) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 301, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 302, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 303 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 304, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 305, and PSMA comprising the amino acid sequence of SEQ ID NO: 306 -LCDR3.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
所述PSMA-VH具有：包含SEQ ID NO：170的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：171的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：172的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：173的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：174的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：175的氨基酸序列的PSMA-LCDR3。The PSMA-VH has: PSMA-HCDR1 including the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 including the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR3 including the amino acid sequence of SEQ ID NO: 172; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 174, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 175 .
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
所述PSMA-VH具有：包含SEQ ID NO：164的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：165的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：166的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：167的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：168的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：169的氨基酸序列的PSMA-LCDR3The PSMA-VH has: PSMA-HCDR1 including the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 including the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR3 including the amino acid sequence of SEQ ID NO: 166; And the PSMA-VL has: PSMA-LCDR1 including the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 including the amino acid sequence of SEQ ID NO: 168, and PSMA-LCDR3 including the amino acid sequence of SEQ ID NO: 169
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
i)所述PSMA-VH包含如SEQ ID NO：164所示的PSMA-HCDR1，如SEQ ID NO：165所示的PSMA-HCDR2，和如SEQ ID NO：166所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：167所示的PSMA-LCDR1，如SEQ ID NO： 168所示的PSMA-LCDR2，和如SEQ ID NO：169所示的PSMA-LCDR3；或i) the PSMA-VH comprises PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and The PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 167, as shown in SEQ ID NO: PSMA-LCDR2 as shown in 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169; or
ii)所述PSMA-VH包含如SEQ ID NO：170所示的PSMA-HCDR1，如SEQ ID NO：171所示的PSMA-HCDR2，和如SEQ ID NO：172所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：173所示的PSMA-LCDR1，如SEQ ID NO：174所示的PSMA-LCDR2，和如SEQ ID NO：175所示的PSMA-LCDR3；或ii) the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175; or
iii)所述PSMA-VH包含如SEQ ID NO：158所示的PSMA-HCDR1，如SEQ ID NO：159所示的PSMA-HCDR2，和如SEQ ID NO：160所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：161所示的PSMA-LCDR1，如SEQ ID NO：162所示的PSMA-LCDR2，和如SEQ ID NO：163所示的PSMA-LCDR3；或iii) the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 158, PSMA-HCDR2 as shown in SEQ ID NO: 159, and PSMA-HCDR3 as shown in SEQ ID NO: 160; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 161, PSMA-LCDR2 as shown in SEQ ID NO: 162, and PSMA-LCDR3 as shown in SEQ ID NO: 163; or
iv)所述PSMA-VH包含如SEQ ID NO：176所示的PSMA-HCDR1，如SEQ ID NO：177所示的PSMA-HCDR2，和如SEQ ID NO：178所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：179所示的PSMA-LCDR1，如SEQ ID NO：180所示的PSMA-LCDR2，和如SEQ ID NO：181所示的PSMA-LCDR3；或iv) the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 176, PSMA-HCDR2 as shown in SEQ ID NO: 177, and PSMA-HCDR3 as shown in SEQ ID NO: 178; and Said PSMA-VL includes PSMA-LCDR1 as set forth in SEQ ID NO: 179, PSMA-LCDR2 as set forth in SEQ ID NO: 180, and PSMA-LCDR3 as set forth in SEQ ID NO: 181; or
v)所述PSMA-VH包含如SEQ ID NO：301所示的PSMA-HCDR1，如SEQ ID NO：302所示的PSMA-HCDR2，和如SEQ ID NO：303所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：304所示的PSMA-LCDR1，如SEQ ID NO：305所示的PSMA-LCDR2，和如SEQ ID NO：306所示的PSMA-LCDR3。v) the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 301, PSMA-HCDR2 as shown in SEQ ID NO: 302, and PSMA-HCDR3 as shown in SEQ ID NO: 303; and The PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 304, PSMA-LCDR2 as shown in SEQ ID NO: 305, and PSMA-LCDR3 as shown in SEQ ID NO: 306.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
所述PSMA-VH包含如SEQ ID NO：170所示的PSMA-HCDR1，如SEQ ID NO：171所示的PSMA-HCDR2，和如SEQ ID NO：172所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：173所示的PSMA-LCDR1，如SEQ ID NO：174所示的PSMA-LCDR2，和如SEQ ID NO：175所示的PSMA-LCDR3。The PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and the PSMA -VL contains PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
所述PSMA-VH包含如SEQ ID NO：164所示的PSMA-HCDR1，如SEQ ID NO：165所示的PSMA-HCDR2，和如SEQ ID NO：166所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：167所示的PSMA-LCDR1，如SEQ ID NO：168所示的PSMA-LCDR2，和如SEQ ID NO：169所示的PSMA-LCDR3。The PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and the PSMA -VL contains PSMA-LCDR1 as shown in SEQ ID NO: 167, PSMA-LCDR2 as shown in SEQ ID NO: 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
i)所述PSMA-VH包含SEQ ID NO：197、199、332、194、195、196或198的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：201、202、333或200的氨基酸序列；或i) The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, 199, 332, 194, 195, 196 or 198, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201, 202, 333 or 200 ;or
ii)所述PSMA-VH包含SEQ ID NO：204、184或203的氨基酸序列，和所述 PSMA-VL包含SEQ ID NO：206、185或205的氨基酸序列；或ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, 184 or 203, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206, 185 or 205; or
iii)所述PSMA-VH包含SEQ ID NO：188、182、189或190的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：191、183、192或193的氨基酸序列；或iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, 182, 189 or 190, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191, 183, 192 or 193; or
iv)所述PSMA-VH包含SEQ ID NO：207或186的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：208、187、209或210的氨基酸序列；或iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207 or 186, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208, 187, 209 or 210; or
v)所述PSMA-VH包含SEQ ID NO：313的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：314的氨基酸序列。v) The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 313, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 314.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
i)所述PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：201的氨基酸序列；或i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201; or
ii)所述PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：206的氨基酸序列；或ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
iii)所述PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：191的氨基酸序列；或iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191; or
iv)所述PSMA-VH包含SEQ ID NO：199的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：202的氨基酸序列；或iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202; or
v)所述PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：208的氨基酸序列。v) The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：206的氨基酸序列。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, and said PSMA -VL contains the amino acid sequence of SEQ ID NO: 206.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：201的氨基酸序列。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, and said PSMA -VL contains the amino acid sequence of SEQ ID NO:201.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述PSMA-VH包含SEQ ID NO：199的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：202的氨基酸序列。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199, and said PSMA -VL contains the amino acid sequence of SEQ ID NO:202.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述特异性结合CD3的抗原结合模块包含重链可变区CD3-VH和轻链可变区CD3-VL，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
i)所述CD3-VH具有：包含SEQ ID NO：283的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：284的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：285的氨基酸序列的CD3-HCDR3；并且所述CD3-VL具有：包含SEQ ID NO：286的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：287的氨基酸序列的CD3-LCDR2， 和包含SEQ ID NO：288的氨基酸序列的CD3-LCDR3；或i) The CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 283, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 284, and CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 285 HCDR3; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 286, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 288; or
ii)所述CD3-VH具有：包含SEQ ID NO：307的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：308的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：309的氨基酸序列的CD3-HCDR3；并且所述CD3-VL具有：包含SEQ ID NO：310的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：311的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：312的氨基酸序列的CD3-LCDR3。ii) The CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 307, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 308, and CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 309 HCDR3; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 310, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 311, and CD3 comprising the amino acid sequence of SEQ ID NO: 312 -LCDR3.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述特异性结合CD3的抗原结合模块包含重链可变区CD3-VH和轻链可变区CD3-VL，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
所述CD3-VH具有：包含SEQ ID NO：283的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：284的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：285的氨基酸序列的CD3-HCDR3；并且所述CD3-VL具有：包含SEQ ID NO：286的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：287的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：288的氨基酸序列的CD3-LCDR3。The CD3-VH has: CD3-HCDR1 including the amino acid sequence of SEQ ID NO: 283, CD3-HCDR2 including the amino acid sequence of SEQ ID NO: 284, and CD3-HCDR3 including the amino acid sequence of SEQ ID NO: 285; And the CD3-VL has: CD3-LCDR1 including the amino acid sequence of SEQ ID NO: 286, CD3-LCDR2 including the amino acid sequence of SEQ ID NO: 287, and CD3-LCDR3 including the amino acid sequence of SEQ ID NO: 288 .
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述特异性结合CD3的抗原结合模块包含重链可变区CD3-VH和轻链可变区CD3-VL，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
i)所述CD3-VH包含如SEQ ID NO：283所示的CD3-HCDR1，如SEQ ID NO：284所示的CD3-HCDR2，和如SEQ ID NO：285所示的CD3-HCDR3；并且所述CD3-VL包含如SEQ ID NO：286所示的CD3-LCDR1，如SEQ ID NO：287所示的CD3-LCDR2，和如SEQ ID NO：288所示的CD3-LCDR3；或i) the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and The CD3-VL includes CD3-LCDR1 as set forth in SEQ ID NO: 286, CD3-LCDR2 as set forth in SEQ ID NO: 287, and CD3-LCDR3 as set forth in SEQ ID NO: 288; or
ii)所述CD3-VH包含如SEQ ID NO：307所示的CD3-HCDR1，如SEQ ID NO：308所示的CD3-HCDR2，和如SEQ ID NO：309所示的CD3-HCDR3；并且所述CD3-VL包含如SEQ ID NO：310所示的CD3-LCDR1，如SEQ ID NO：311所示的CD3-LCDR2，和如SEQ ID NO：312所示的CD3-LCDR3。ii) the CD3-VH includes CD3-HCDR1 as set forth in SEQ ID NO: 307, CD3-HCDR2 as set forth in SEQ ID NO: 308, and CD3-HCDR3 as set forth in SEQ ID NO: 309; and The CD3-VL includes CD3-LCDR1 as shown in SEQ ID NO:310, CD3-LCDR2 as shown in SEQ ID NO:311, and CD3-LCDR3 as shown in SEQ ID NO:312.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述特异性结合CD3的抗原结合模块包含重链可变区CD3-VH和轻链可变区CD3-VL，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and the light chain variable region CD3-VL, where:
所述CD3-VH包含如SEQ ID NO：283所示的CD3-HCDR1，如SEQ ID NO：284所示的CD3-HCDR2，和如SEQ ID NO：285所示的CD3-HCDR3；并且所述CD3-VL包含如SEQ ID NO：286所示的CD3-LCDR1，如SEQ ID NO：287所示的CD3-LCDR2，和如SEQ ID NO：288所示的CD3-LCDR3。The CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and the CD3 -VL contains CD3-LCDR1 as shown in SEQ ID NO:286, CD3-LCDR2 as shown in SEQ ID NO:287, and CD3-LCDR3 as shown in SEQ ID NO:288.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，所述CD3-VH包含SEQ ID NO：289的氨基酸序列，和所述CD3-VL包含SEQ ID NO：290的氨基酸序列；或In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, said CD3-VH comprising the amino acid sequence of SEQ ID NO: 289, and said CD3- VL contains the amino acid sequence of SEQ ID NO: 290; or
所述CD3-VH包含SEQ ID NO：315的氨基酸序列，和所述CD3-VL包含SEQ  ID NO：316的氨基酸序列。The CD3-VH comprises the amino acid sequence of SEQ ID NO: 315, and the CD3-VL comprises SEQ Amino acid sequence of ID NO:316.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
所述PSMA-VH包含如SEQ ID NO：170所示的PSMA-HCDR1，如SEQ ID NO：171所示的PSMA-HCDR2，和如SEQ ID NO：172所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：173所示的PSMA-LCDR1，如SEQ ID NO：174所示的PSMA-LCDR2，和如SEQ ID NO：175所示的PSMA-LCDR3；且The PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and the PSMA - VL contains PSMA-LCDR1 as set forth in SEQ ID NO: 173, PSMA-LCDR2 as set forth in SEQ ID NO: 174, and PSMA-LCDR3 as set forth in SEQ ID NO: 175; and
所述CD3-VH包含如SEQ ID NO：283所示的CD3-HCDR1，如SEQ ID NO：284所示的CD3-HCDR2，和如SEQ ID NO：285所示的CD3-HCDR3；并且所述CD3-VL包含如SEQ ID NO：286所示的CD3-LCDR1，如SEQ ID NO：287所示的CD3-LCDR2，和如SEQ ID NO：288所示的CD3-LCDR3。The CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and the CD3 -VL contains CD3-LCDR1 as shown in SEQ ID NO:286, CD3-LCDR2 as shown in SEQ ID NO:287, and CD3-LCDR3 as shown in SEQ ID NO:288.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
所述PSMA-VH包含如SEQ ID NO：164所示的PSMA-HCDR1，如SEQ ID NO：165所示的PSMA-HCDR2，和如SEQ ID NO：166所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：167所示的PSMA-LCDR1，如SEQ ID NO：168所示的PSMA-LCDR2，和如SEQ ID NO：169所示的PSMA-LCDR3；且The PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and the PSMA -VL contains PSMA-LCDR1 as set forth in SEQ ID NO: 167, PSMA-LCDR2 as set forth in SEQ ID NO: 168, and PSMA-LCDR3 as set forth in SEQ ID NO: 169; and
所述CD3-VH包含如SEQ ID NO：283所示的CD3-HCDR1，如SEQ ID NO：284所示的CD3-HCDR2，和如SEQ ID NO：285所示的CD3-HCDR3；并且所述CD3-VL包含如SEQ ID NO：286所示的CD3-LCDR1，如SEQ ID NO：287所示的CD3-LCDR2，和如SEQ ID NO：288所示的CD3-LCDR3。The CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 283, CD3-HCDR2 as shown in SEQ ID NO: 284, and CD3-HCDR3 as shown in SEQ ID NO: 285; and the CD3 -VL contains CD3-LCDR1 as shown in SEQ ID NO:286, CD3-LCDR2 as shown in SEQ ID NO:287, and CD3-LCDR3 as shown in SEQ ID NO:288.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
所述PSMA-VH包含如SEQ ID NO：301所示的PSMA-HCDR1，如SEQ ID NO：302所示的PSMA-HCDR2，和如SEQ ID NO：303所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：304所示的PSMA-LCDR1，如SEQ ID NO：305所示的PSMA-LCDR2，和如SEQ ID NO：306所示的PSMA-LCDR3；且The PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 301, PSMA-HCDR2 as shown in SEQ ID NO: 302, and PSMA-HCDR3 as shown in SEQ ID NO: 303; and the PSMA -VL contains PSMA-LCDR1 as shown in SEQ ID NO:304, PSMA-LCDR2 as shown in SEQ ID NO:305, and PSMA-LCDR3 as shown in SEQ ID NO:306; and
所述CD3-VH具有：包含SEQ ID NO：307的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：308的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：309的氨基酸序列的CD3-HCDR3；并且所述CD3-VL具有：包含SEQ ID NO：310的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：311的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：312的氨基酸序列的CD3-LCDR3。The CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 307, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 308, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO: 309; And the CD3-VL has: CD3-LCDR1 including the amino acid sequence of SEQ ID NO: 310, CD3-LCDR2 including the amino acid sequence of SEQ ID NO: 311, and CD3-LCDR3 including the amino acid sequence of SEQ ID NO: 312 .
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
所述PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：206的氨基酸序列；且 The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; and
所述CD3-VH包含SEQ ID NO：289的氨基酸序列，和所述CD3-VL包含SEQ ID NO：290的氨基酸序列。The CD3-VH comprises the amino acid sequence of SEQ ID NO: 289, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 290.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
所述PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：201的氨基酸序列；且The PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 201; and
所述CD3-VH包含SEQ ID NO：289的氨基酸序列，和所述CD3-VL包含SEQ ID NO：290的氨基酸序列。The CD3-VH comprises the amino acid sequence of SEQ ID NO: 289, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 290.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
所述PSMA-VH包含SEQ ID NO：199的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：202的氨基酸序列；且The PSMA-VH includes the amino acid sequence of SEQ ID NO: 199, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 202; and
所述CD3-VH包含SEQ ID NO：289的氨基酸序列，和所述CD3-VL包含SEQ ID NO：290的氨基酸序列。The CD3-VH comprises the amino acid sequence of SEQ ID NO: 289, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 290.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein:
所述PSMA-VH包含SEQ ID NO：313的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：314的氨基酸序列；且The PSMA-VH includes the amino acid sequence of SEQ ID NO: 313, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 314; and
所述CD3-VH包含SEQ ID NO：315的氨基酸序列，和所述CD3-VL包含SEQ ID NO：316的氨基酸序列。The CD3-VH comprises the amino acid sequence of SEQ ID NO: 315, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 316.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含Fc区。在一些实施方案中，所述Fc区为IgG Fc区。在一些实施方案中，所述Fc区为IgG1Fc区。在一些实施方案中，所述Fc区所述Fc区包含一个或多个能够减少Fc区与Fcγ受体结合的氨基酸取代。在一些实施方案中，所述Fc区是人IgG1Fc区，并且在234和235位置的氨基酸为A，编号依据为EU索引。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an Fc region. In some embodiments, the Fc region is an IgG Fc region. In some embodiments, the Fc region is an IgGl Fc region. In some embodiments, the Fc region contains one or more amino acid substitutions that reduce binding of the Fc region to Fcγ receptors. In some embodiments, the Fc region is a human IgGl Fc region, and the amino acids at positions 234 and 235 are A, numbered according to the EU index.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含Fc区，所述Fc区包含能够相互缔合的第一亚基Fc1和第二亚基Fc2，所述Fc1和Fc2各自独立地具有一个或多个减少Fc区同源二聚化的氨基酸取代。在一些实施方案中，所述Fc1具有根据杵臼技术的凸起结构，和所述Fc2具有根据杵臼技术的孔结构。在一些实施方案中，所述Fc1在366位置的氨基酸为W；并且所述Fc2在366位置的氨基酸为S、在368位置的氨基酸为A、和在407位置的氨基酸为V，编号依据为EU索引。在一些实施方案中，所述Fc1在354位置的氨基酸为C和在366位置的氨基酸为W；并且所述Fc2在349位置的氨基酸为C、在366位置的氨基酸为S、在368位置的氨基酸为A、和在407位置的氨基酸为V，编号依据为EU索引。在一些实施方案中，所述Fc1包含 SEQ ID NO：220的氨基酸序列；所述Fc2包含SEQ ID NO：221的氨基酸序列。在一些实施方案中，所述Fc1包含SEQ ID NO：220的氨基酸序列；所述Fc2包含SEQ ID NO：222的氨基酸序列。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an Fc region comprising a first subunit Fcl capable of associating with each other and a second subunit Fc2, each of said Fc1 and Fc2 independently having one or more amino acid substitutions that reduce homodimerization of the Fc region. In some embodiments, the Fc1 has a convex structure according to the pestle and mortar technology, and the Fc2 has a pore structure according to the pestle and mortar technology. In some embodiments, the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbering according to EU index. In some embodiments, the Fc1 amino acid at position 354 is C and the amino acid at position 366 is W; and the Fc2 amino acid at position 349 is C, the amino acid at position 366 is S, and the amino acid at position 368 is W is A, and the amino acid at position 407 is V, and the numbering basis is the EU index. In some embodiments, the Fcl comprises The amino acid sequence of SEQ ID NO: 220; the Fc2 includes the amino acid sequence of SEQ ID NO: 221. In some embodiments, the Fc1 comprises the amino acid sequence of SEQ ID NO:220; the Fc2 comprises the amino acid sequence of SEQ ID NO:222.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含一个特异性结合PSMA的抗原结合模块和一个特异性结合CD3的抗原结合模块，所述的特异性结合PSMA的抗原结合模块和特异性结合CD3的抗原结合模块均是scFv。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an antigen-binding moiety that specifically binds PSMA and an antigen-binding moiety that specifically binds CD3. module, the antigen-binding module that specifically binds to PSMA and the antigen-binding module that specifically binds to CD3 are both scFv.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含一条具有式(a)所示结构的第一链和一条具有式(b)所示结构的第二链，In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
(a)[PSMA-VH]-[连接子6]-[PSMA-VL]-[连接子7]-[CD3-VH]-[连接子8]-[CD3-VL]-[连接子9]-[Fc2]，(a) [PSMA-VH]-[Linker 6]-[PSMA-VL]-[Linker 7]-[CD3-VH]-[Linker 8]-[CD3-VL]-[Linker 9] -[Fc2],
(b)[Fc1]，(b)[Fc1],
式(a)和(b)所示的结构是从N端至C端排列的，所述连接子6、连接子7、连接子8和/或连接子9是相同或不同的肽连接子。The structures shown in formulas (a) and (b) are arranged from the N end to the C end, and the linker 6, linker 7, linker 8 and/or linker 9 are the same or different peptide linkers.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含一条具有式(a)所示结构的第一链和一条具有式(b)所示结构的第二链，In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
(a)[PSMA-VH]-[连接子6]-[PSMA-VL]-[连接子7]-[CD3-VH]-[连接子8]-[CD3-VL]-[连接子9]-[Fc2]，(a) [PSMA-VH]-[Linker 6]-[PSMA-VL]-[Linker 7]-[CD3-VH]-[Linker 8]-[CD3-VL]-[Linker 9] -[Fc2],
(b)[Fc1]，(b)[Fc1],
式(a)和(b)所示的结构是从N端至C端排列的，所述连接子6、连接子7、连接子8和/或连接子9是相同或不同的肽连接子；其中：The structures shown in formulas (a) and (b) are arranged from the N end to the C end, and the linker 6, linker 7, linker 8 and/or linker 9 are the same or different peptide linkers; in:
所述PSMA-VH包含SEQ ID NO：197的氨基酸序列；和所述所述PSMA-VL包含SEQ ID NO：201的氨基酸序列；且所述CD3-VH包含SEQ ID NO：289的氨基酸序列；和所述所述CD3-V L包含SEQ ID NO：290的氨基酸序列。The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197; and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289; and The CD3-V L includes the amino acid sequence of SEQ ID NO: 290.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含一条具有式(a)所示结构的第一链和一条具有式(b)所示结构的第二链，In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
(a)[PSMA-VH]-[连接子6]-[PSMA-VL]-[连接子7]-[CD3-VH]-[连接子8]-[CD3-VL]-[连接子9]-[Fc2]，(a) [PSMA-VH]-[Linker 6]-[PSMA-VL]-[Linker 7]-[CD3-VH]-[Linker 8]-[CD3-VL]-[Linker 9] -[Fc2],
(b)[Fc1]，(b)[Fc1],
式(a)和(b)所示的结构是从N端至C端排列的，所述连接子6、连接子7、连接子8和/或连接子9是相同或不同的肽连接子；其中：The structures shown in formulas (a) and (b) are arranged from the N end to the C end, and the linker 6, linker 7, linker 8 and/or linker 9 are the same or different peptide linkers; in:
所述PSMA-VH包含SEQ ID NO：199的氨基酸序列；和所述所述PSMA-VL包含SEQ ID NO：201的氨基酸序列；且所述CD3-VH包含SEQ ID NO：289的 氨基酸序列；和所述所述CD3-V L包含SEQ ID NO：290的氨基酸序列。The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199; and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 289 Amino acid sequence; and said CD3-V L comprises the amino acid sequence of SEQ ID NO: 290.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含一条具有式(a)所示结构的第一链和一条具有式(b)所示结构的第二链，In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (a) and a first chain having a structure represented by formula ( b) The second strand of the structure shown,
(a)[PSMA-VH]-[连接子6]-[PSMA-VL]-[连接子7]-[CD3-VH]-[连接子8]-[CD3-VL]-[连接子9]-[Fc2]，(a) [PSMA-VH]-[Linker 6]-[PSMA-VL]-[Linker 7]-[CD3-VH]-[Linker 8]-[CD3-VL]-[Linker 9] -[Fc2],
(b)[Fc1]，(b)[Fc1],
式(a)和(b)所示的结构是从N端至C端排列的，所述连接子6和连接子8是序列相同的肽连接子；连接子7和连接子9是序列不同的肽连接子。The structures shown in formulas (a) and (b) are arranged from the N end to the C end. The linker 6 and the linker 8 are peptide linkers with the same sequence; the linker 7 and the linker 9 have different sequences. Peptide linker.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，所述连接子6、连接子7、连接子8和连接子9可以是任意适宜的肽连接子，只要抗原结合分子能够展现出期望的抗原结合活性。例如，肽连接子可以是包含1-50或3-20个氨基酸残基的柔性肽。在一些实施方式中，所述的肽连接子各自独立地具有L1-(GGGGS)n-L2的结构，其中，L1是键、S、GS、GGS(SEQ ID NO：399)或GGGS(SEQ ID NO：400)，n是0、1、2、3、4、5、6、7、8、9或10，L2是键、G、GG、GGG(SEQ ID NO：295)或GGGG(SEQ ID NO：256)，并且所述肽连接子不是键。在一些实施方案中，所述肽连接子的长度为3-15个氨基酸残基。In some embodiments, the second therapeutic agent as described in any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, and the linker 6, linker 7, linker 8, and linker 9 can be any Suitable peptide linkers are as long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linkers each independently have the structure of L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, S, GS, GGS (SEQ ID NO: 399) or GGGS (SEQ ID NO: 400), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is key, G, GG, GGG (SEQ ID NO: 295) or GGGG (SEQ ID NO: 256), and the peptide linker is not a bond. In some embodiments, the peptide linker is 3-15 amino acid residues in length.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述的连接子6和连接子8包含SEQ ID NO：293的氨基酸序列(GGGGSGGGGSGGGGS)。在一些实施方案中，其中所述的连接子7包含SEQ ID NO：294的氨基酸序列(SGGGGS)。在一些实施方案中，其中所述的连接子9包含SEQ ID NO：295的氨基酸序列(GGG)。In some embodiments, the second therapeutic agent as described in any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said linker 6 and linker 8 comprise the amino acid sequence of SEQ ID NO: 293 (GGGGGSGGGGSGGGGS). In some embodiments, the linker 7 includes the amino acid sequence of SEQ ID NO: 294 (SGGGGS). In some embodiments, the linker 9 includes the amino acid sequence of SEQ ID NO: 295 (GGG).
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述的Fc1包含SEQ ID NO：220的氨基酸序列。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein said Fc1 comprises the amino acid sequence of SEQ ID NO: 220.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述的Fc2包含SEQ ID NO：221的氨基酸序列。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the Fc2 comprises the amino acid sequence of SEQ ID NO: 221.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述特异性结合PSMA和CD3的双特异性抗体具有：一条包含SEQ ID NO：296的氨基酸序列的第一链和一条包含SEQ ID NO：220的氨基酸序列的第二链。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the bispecific antibody that specifically binds PSMA and CD3 has: a bar comprising SEQ ID The first strand contains the amino acid sequence of SEQ ID NO: 296 and the second strand contains the amino acid sequence of SEQ ID NO: 220.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述特异性结合PSMA和CD3的双特异性抗体具有：In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the bispecific antibody that specifically binds PSMA and CD3 has:
一条包含SEQ ID NO：297的氨基酸序列的第一链和一条包含SEQ ID NO：220的氨基酸序列的第二链。A first strand comprising the amino acid sequence of SEQ ID NO: 297 and a second strand comprising the amino acid sequence of SEQ ID NO: 220.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和 CD3的双特异性抗体，其包含一个特异性结合PSMA的抗原结合模块和一个特异性结合CD3的抗原结合模块，所述特异性结合PSMA的抗原结合模块或特异性结合CD3的抗原结合模块包含能够形成二聚体的Titin链和Obscurin链。In some embodiments, the second therapeutic agent of any one of the preceding items specifically binds PSMA and Bispecific antibodies to CD3, which comprise an antigen-binding module that specifically binds PSMA and an antigen-binding module that specifically binds CD3, and the antigen-binding module that specifically binds PSMA or the antigen-binding module that specifically binds CD3 includes an antigen-binding module that specifically binds to PSMA. Titin chain and Obscurin chain forming a dimer.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含一个特异性结合PSMA的抗原结合模块和一个特异性结合CD3的抗原结合模块，所述特异性结合PSMA的抗原结合模块是Fab；所述特异性结合CD3的抗原结合模块是经替换的Fab，其包含能够形成二聚体的Titin链和Obscurin链。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising an antigen-binding moiety that specifically binds PSMA and an antigen-binding moiety that specifically binds CD3. Module, the antigen-binding module that specifically binds to PSMA is a Fab; the antigen-binding module that specifically binds to CD3 is a replaced Fab, which includes a Titin chain and an Obscurin chain capable of forming dimers.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述特异性结合CD3的抗原结合模块是Fab；所述特异性结合PSMA的抗原结合模块是经替换的Fab，其包含能够形成二聚体的Titin链和Obscurin链。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the antigen-binding moiety that specifically binds CD3 is a Fab; said specifically binds PSMA The antigen-binding module is a replaced Fab containing a Titin chain and an Obscurin chain capable of forming dimers.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含一条具有式(c)所示结构的第一链、一条具有式(d)所示结构的第二链、一条具有式(e)所示结构的第三链和一条具有式(f)所示结构的第四链，In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (c), a first chain having a structure represented by formula (c), d) a second chain having the structure shown in formula (e), a third chain having the structure shown in formula (e) and a fourth chain having the structure shown in formula (f),
(c)[PSMA-VH]-[CH1]-[Fc1]，(c)[PSMA-VH]-[CH1]-[Fc1],
(d)[PSMA-VL]-[CL]，(d)[PSMA-VL]-[CL],
(e)[CD3-VH]-[连接子10]-[Obscurin链]-[Fc2]，(e)[CD3-VH]-[linker 10]-[Obscurin chain]-[Fc2],
(f)[CD3-VL]-[连接子11]-[Titin链]，(f)[CD3-VL]-[linker 11]-[Titin chain],
式(c)、(d)、(e)和(f)所示的结构是从N端至C端排列的，所述连接子10和连接子11是相同或不同的肽连接子。The structures shown in formulas (c), (d), (e) and (f) are arranged from the N-terminus to the C-terminus, and the linker 10 and the linker 11 are the same or different peptide linkers.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含一条具有式(c)所示结构的第一链、一条具有式(d)所示结构的第二链、一条具有式(e)所示结构的第三链和一条具有式(f)所示结构的第四链，In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising a first chain having a structure represented by formula (c), a first chain having a structure represented by formula (c), d) a second chain having the structure shown in formula (e), a third chain having the structure shown in formula (e) and a fourth chain having the structure shown in formula (f),
(c)[PSMA-VH]-[CH1]-[Fc1]，(c)[PSMA-VH]-[CH1]-[Fc1],
(d)[PSMA-VL]-[CL]，(d)[PSMA-VL]-[CL],
(e)[CD3-VH]-[连接子10]-[Obscurin链]-[Fc2]，(e)[CD3-VH]-[linker 10]-[Obscurin chain]-[Fc2],
(f)[CD3-VL]-[连接子11]-[Titin链]，(f)[CD3-VL]-[linker 11]-[Titin chain],
式(c)、(d)、(e)和(f)所示的结构是从N端至C端排列的，所述连接子10和连接子11是相同或不同的肽连接子；其中：The structures shown in formulas (c), (d), (e) and (f) are arranged from the N end to the C end, and the linker 10 and the linker 11 are the same or different peptide linkers; wherein:
所述PSMA-VH包含SEQ ID NO：204的氨基酸序列；和所述所述PSMA-VL包含SEQ ID NO：206的氨基酸序列；且所述CD3-VH包含SEQ ID NO：289的氨基酸序列；和所述所述CD3-VL包含SEQ ID NO：290的氨基酸序列。The PSMA-VH includes the amino acid sequence of SEQ ID NO: 204; and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 206; and the CD3-VH includes the amino acid sequence of SEQ ID NO: 289; and The CD3-VL includes the amino acid sequence of SEQ ID NO: 290.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和 CD3的双特异性抗体，其中所述的Titin链和Obscurin链为本披露中表3-1和表3-2中任意可以形成二聚体的Titin链和Obscurin链。在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的Titin链包含如SEQ ID NO：350的氨基酸序列。在一些实施方式中，如前任一项所述的抗原结合分子，其中所述的Obscurin链包含如SEQ ID NO：388的氨基酸序列。In some embodiments, the second therapeutic agent of any one of the preceding items specifically binds PSMA and A bispecific antibody to CD3, wherein the Titin chain and Obscurin chain are any Titin chain and Obscurin chain in Table 3-1 and Table 3-2 of the present disclosure that can form dimers. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 350. In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the Obscurin chain comprises an amino acid sequence such as SEQ ID NO: 388.
在一些实施方案中，如前所述任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述连接子10和连接子11可以是任意适宜的肽连接子，只要抗原结合分子能够展现出期望的抗原结合活性。例如，肽连接子可以是包含1-50或3-20个氨基酸残基的柔性肽。在一些实施方式中，所述的肽连接子各自独立地具有L1-(GGGGS)n-L2的结构，其中，L1是键、S、GS、GGS(SEQ ID NO：399)或GGGS(SEQ ID NO：400)，n是0、1、2、3、4、5、6、7、8、9或10，L2是键、G、GG、GGG(SEQ ID NO：295)或GGGG(SEQ ID NO：256)，并且所述肽连接子不是键。在一些实施方案中，所述肽连接子的长度为3-15个氨基酸残基。在一些实施方案中，所述连接子10和所述连接子11包含如SEQ ID NO：223所示的氨基酸序列。In some embodiments, the second therapeutic agent as described in any one of the preceding is a bispecific antibody that specifically binds PSMA and CD3, wherein the linker 10 and linker 11 can be any suitable peptide linkage as long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linkers each independently have the structure of L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, S, GS, GGS (SEQ ID NO: 399) or GGGS (SEQ ID NO: 400), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is key, G, GG, GGG (SEQ ID NO: 295) or GGGG (SEQ ID NO: 256), and the peptide linker is not a bond. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, the linker 10 and the linker 11 comprise the amino acid sequence set forth in SEQ ID NO:223.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述的CH1为IgG的CH1序列。在一些实施方案中，所述的CH1为IgG1的CH1。在一些实施方案中，所述的CH1包含SEQ ID NO：219的氨基酸序列。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the CH1 is the CH1 sequence of an IgG. In some embodiments, the CH1 is CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 219.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其中所述的CL为kappa或lamada的轻链恒定区。在一些实施方式中，其中所述的CL包含SEQ ID NO：145的氨基酸序列。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, wherein the CL is the light chain constant region of kappa or lamada. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 145.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其具有：一条包含SEQ ID NO：298的氨基酸序列的第一链、一条包含SEQ ID NO：275的氨基酸序列的第二链、一条包含SEQ ID NO：291的氨基酸序列的第三链和一条包含SEQ ID NO：292的氨基酸序列的第四链。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, having: a first chain comprising the amino acid sequence of SEQ ID NO: 298, a first chain comprising A second strand containing the amino acid sequence of SEQ ID NO: 275, a third strand containing the amino acid sequence of SEQ ID NO: 291 and a fourth strand containing the amino acid sequence of SEQ ID NO: 292.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其具有IgG-scFv结构。In some embodiments, the second therapeutic agent of any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, having an IgG-scFv structure.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含两条式(a)所示结构的第一链和两条式(b)所示结构的第二链：In some embodiments, the second therapeutic agent as described in any one of the preceding items is a bispecific antibody that specifically binds PSMA and CD3, comprising two first chains of the structure represented by formula (a) and two formulas ( b) Second strand of the structure shown:
(a)[PSMA-VH]-[IgG恒定区]-[连接子12]-[CD3-VL]-[连接子13]-[CD3-VH]；(a) [PSMA-VH]-[IgG constant region]-[linker 12]-[CD3-VL]-[linker 13]-[CD3-VH];
(b)[PSMA-VL]-CL；(b)[PSMA-VL]-CL;
式(a)和(b)所示的结构是从N端至C端排列的，所述连接子12和连接子13是相同或不同的肽连接子。The structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus, and the linker 12 and the linker 13 are the same or different peptide linkers.
在一些实施方案中，如前任一项所述的第二治疗剂是特异性结合PSMA和 CD3的双特异性抗体，其具有IgG-scFv结构，其包含两条式(a)所示结构的第一链和两条式(b)所示结构的第二链：In some embodiments, the second therapeutic agent of any one of the preceding items specifically binds PSMA and A bispecific antibody to CD3, which has an IgG-scFv structure, which includes two first chains of the structure represented by formula (a) and two second chains of the structure represented by formula (b):
(a)[PSMA-VH]-[IgG恒定区]-[连接子12]-[CD3-VL]-[连接子13]-[CD3-VH]；(a) [PSMA-VH]-[IgG constant region]-[linker 12]-[CD3-VL]-[linker 13]-[CD3-VH];
(b)[PSMA-VL]-CL；(b)[PSMA-VL]-CL;
式(a)和(b)所示的结构是从N端至C端排列的，所述连接子12和连接子13是相同或不同的肽连接子；其中：The structures shown in formulas (a) and (b) are arranged from the N-terminus to the C-terminus, and the linker 12 and the linker 13 are the same or different peptide linkers; wherein:
所述PSMA-VH包含SEQ ID NO：313的氨基酸序列；和所述所述PSMA-VL包含SEQ ID NO：314的氨基酸序列；且所述CD3-VH包含SEQ ID NO：315的氨基酸序列；和所述所述CD3-VL包含SEQ ID NO：316的氨基酸序列。The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 313; and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 314; and the CD3-VH comprises the amino acid sequence of SEQ ID NO: 315; and The CD3-VL includes the amino acid sequence of SEQ ID NO: 316.
在一些实施方案中，所述特异性结合PSMA和CD3的双特异性抗体包含两条SEQ ID NO：299的氨基酸序列和两条SEQ ID NO：300的氨基酸序列。In some embodiments, the bispecific antibody that specifically binds PSMA and CD3 comprises two amino acid sequences of SEQ ID NO: 299 and two amino acid sequences of SEQ ID NO: 300.
在一些实施方案中，如前任一项所述的第二治疗剂是免疫检查点抑制剂，其中所述的免疫检查点抑制剂靶向PD-1或CTLA4。In some embodiments, the second therapeutic agent of any one of the preceding items is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor targets PD-1 or CTLA4.
在一些实施方案中，如前任一项所述的第二治疗剂是免疫检查点抑制剂，其中所述的免疫检查点抑制剂是抗PD-1抗体。In some embodiments, the second therapeutic agent of any one of the preceding items is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
在一些实施方案中，如前任一项所述的第二治疗剂是抗PD-1抗体，其包括任何已知的具有治疗活性的抗PD-1抗体，包括但不限于卡瑞利珠单抗(Camrelizumab)、派姆单抗(Keytruda)、纳武单抗(Opdivo)和西米普利单抗(cemiplimab)。In some embodiments, the second therapeutic agent of any one of the preceding items is an anti-PD-1 antibody, including any known therapeutically active anti-PD-1 antibody, including but not limited to camrelizumab (Camrelizumab), pembrolizumab (Keytruda), nivolumab (Opdivo) and cemiplimab (cemiplimab).
在一些实施方案中，如前任一项所述的第二治疗剂是抗PD-1抗体，其包含重链可变区PD-1-VH和轻链可变区PD-1-VL，其中：In some embodiments, the second therapeutic agent of any one of the preceding items is an anti-PD-1 antibody comprising a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, wherein:
所述PD-1-VH具有：包含SEQ ID NO：319的氨基酸序列的PD-1-HCDR1、包含SEQ ID NO：320的氨基酸序列的PD-1-HCDR2和包含SEQ ID NO：321的氨基酸序列的PD-1-HCDR3，和所述PD-1-VL具有：包含SEQ ID NO：322的氨基酸序列的PD-1-LCDR1、包含SEQ ID NO：323的氨基酸序列的PD-1-LCDR2和包含SEQ ID NO：324的氨基酸序列的PD-1-LCDR3。The PD-1-VH has: PD-1-HCDR1 including the amino acid sequence of SEQ ID NO: 319, PD-1-HCDR2 including the amino acid sequence of SEQ ID NO: 320 and the amino acid sequence including SEQ ID NO: 321 PD-1-HCDR3, and the PD-1-VL has: PD-1-LCDR1 comprising the amino acid sequence of SEQ ID NO: 322, PD-1-LCDR2 comprising the amino acid sequence of SEQ ID NO: 323 and PD-1-LCDR3 of the amino acid sequence of SEQ ID NO: 324.
在一些实施方案中，如任一项所述的第二治疗剂是抗PD-1抗体，其包含重链可变区PD-1-VH和轻链可变区PD-1-VL，其中：In some embodiments, the second therapeutic agent of any one is an anti-PD-1 antibody comprising a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, wherein:
所述PD-1-VH包含SEQ ID NO：325的氨基酸序列，和所述PD-1-VL包含SEQ ID NO：326的氨基酸序列。The PD-1-VH includes the amino acid sequence of SEQ ID NO: 325, and the PD-1-VL includes the amino acid sequence of SEQ ID NO: 326.
在一些实施方案中，如前任一项所述的第二治疗剂是抗PD-1抗体，其包含SEQ ID NO：328的氨基酸序列的重链，和SEQ ID NO：329的氨基酸序列的轻链。In some embodiments, the second therapeutic agent of any one of the preceding items is an anti-PD-1 antibody comprising a heavy chain of the amino acid sequence of SEQ ID NO: 328, and a light chain of the amino acid sequence of SEQ ID NO: 329 .
本披露提供的抗原结合分子，具有治疗活性、安全性、药物代谢动力学特性和成药性(如稳定性)好的特点。The antigen-binding molecules provided by the present disclosure have the characteristics of good therapeutic activity, safety, pharmacokinetic properties and druggability (such as stability).
附图说明 Description of the drawings
图1A：PSMA/CD28双特异性抗体的式1的结构示意图；Figure 1A: Schematic structural diagram of Formula 1 of PSMA/CD28 bispecific antibody;
图1B：PSMA/CD28双特异性抗体的式2的结构示意图；Figure 1B: Schematic structural diagram of Formula 2 of PSMA/CD28 bispecific antibody;
图1C：PSMA/CD3双特异性抗体的式1的结构示意图；Figure 1C: Schematic structural diagram of Formula 1 of PSMA/CD3 bispecific antibody;
图1D：PSMA/CD3双特异性抗体的式5的结构示意图；其中Ob代表Obscurin。Figure 1D: Schematic structural diagram of Formula 5 of PSMA/CD3 bispecific antibody; where Ob represents Obscurin.
图2：固定PSMA/CD3双特异性抗体CC-1的浓度，不同浓度的PSMA/CD28双特异性抗体对CHO-huPSMA细胞的杀伤结果。Figure 2: Fixed concentration of PSMA/CD3 bispecific antibody CC-1, killing results of different concentrations of PSMA/CD28 bispecific antibody on CHO-huPSMA cells.
图3：在PSMA/CD3双抗、PSMA/CD28双抗和CHO-huPSMA细胞的共同作用下，PBMC中细胞因子IFN-γ分泌的变化。Figure 3: Changes in the secretion of cytokine IFN-γ in PBMCs under the combined action of PSMA/CD3 double antibodies, PSMA/CD28 double antibodies and CHO-huPSMA cells.
图4：在PSMA/CD3双抗、PSMA/CD28双抗和CHO-huPSMA细胞的共同作用下，PBMC中细胞因子IL-6分泌的变化。Figure 4: Changes in the secretion of cytokine IL-6 in PBMCs under the combined action of PSMA/CD3 double antibodies, PSMA/CD28 double antibodies and CHO-huPSMA cells.
具体实施方式Detailed ways
术语the term
本文所用的术语只是为了描述实施方案的目的，并非旨在进行限制。除非另外定义，本文所用的全部技术术语和科学术语具有与本披露所属领域的普通技术人员通常所理解的相同意义。The terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
除非上下文另外清楚要求，否则在整个说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为具有包含意义，而不是排他性或穷举性意义；也即，“包括但不仅限于”的意义。除非另有说明，“包含”包括了“由……组成”。例如，对于包含SEQ ID NO：1的氨基酸序列的PSMA-HCDR1，其明确的涵盖氨基酸序列如SEQ ID NO：1所示的PSMA-HCDR1。Unless the context clearly requires otherwise, throughout the specification and claims, the words "include," "have," "includes," and the like are to be understood in an inclusive sense and not in an exclusive or exhaustive sense; that is, " including but not limited to”. Unless otherwise stated, "comprising" includes "consisting of." For example, for PSMA-HCDR1 containing the amino acid sequence of SEQ ID NO: 1, it clearly covers PSMA-HCDR1 with the amino acid sequence shown in SEQ ID NO: 1.
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).
术语“和/或”，例如“X和/或Y”应当理解为意指“X和Y”或“X或Y”并且应当被用来提供对两种含义或任一含义的明确支持。The term "and/or", such as "X and/or Y" should be understood to mean "X and Y" or "X or Y" and should be used to provide clear support for both meanings or either meaning.
术语“CD28”(分化簇28，Tp44)是指来自任何脊椎动物来源的任何CD28蛋白，该脊椎动物来源包括哺乳动物诸如灵长类动物(例如，人)、非人灵长类动物(例如，食蟹猕猴)和啮齿动物(例如，小鼠和大鼠)。CD28在T细胞上表达，并提供T细胞活化和存活所需的共刺激信号。除T细胞受体(TCR)外，通过CD28刺激T细胞也可以为产生各种白介素提供有效的信号。CD28是CD80(B7.1)蛋白和CD86(B7.2)蛋白的受体，并且是在幼稚T细胞上组成性表达的唯一B7受体。人CD28的氨基酸序列显示在UniProt(www.uniprot.org)登录号P10747中。The term "CD28" (cluster of differentiation 28, Tp44) refers to any CD28 protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., macaques) and rodents (e.g., mice and rats). CD28 is expressed on T cells and provides costimulatory signals required for T cell activation and survival. In addition to T cell receptors (TCR), stimulation of T cells through CD28 can also provide effective signals for the production of various interleukins. CD28 is the receptor for the CD80 (B7.1) and CD86 (B7.2) proteins and is the only B7 receptor constitutively expressed on naive T cells. The amino acid sequence of human CD28 is shown in UniProt (www.uniprot.org) accession number P10747.
术语“氨基酸”是指天然存在的和合成的氨基酸，以及以与天然存在的氨基酸类似的方式起作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的那些氨基酸，以及后来修饰的那些氨基酸，例如羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指与天然存在的氨基酸具有相同基本化学结构 (即与氢、羧基、氨基和R基团结合的α碳)的化合物，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。此类类似物具有修饰的R基团(例如，正亮氨酸)或修饰的肽骨架，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指具有与氨基酸的一般化学结构不同的结构，但是以与天然存在的氨基酸类似的方式起作用的化学化合物。The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs have the same basic chemical structure as naturally occurring amino acids (i.e. alpha carbon bonded to hydrogen, carboxyl, amino and R groups) compounds such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones but retain the same basic chemical structure as the naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of amino acids, but act in a manner similar to naturally occurring amino acids.
术语“氨基酸突变”包括氨基酸取代，缺失，插入和修饰。可以进行取代、缺失、插入和修饰的任意组合来实现最终构建体，只要最终构建体拥有期望的特性，例如降低或对Fc受体的结合。氨基酸序列缺失和插入包括在多肽链的氨基端和/或羧基端的缺失和插入。具体的氨基酸突变可以是氨基酸取代。在一个实施方式中，氨基酸突变是非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。氨基酸取代包括由非天然存在的氨基酸或由20种天然氨基酸的衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟赖氨酸)替换。可以使用本领域中公知的遗传或化学方法生成氨基酸突变。遗传方法可以包括定点诱变、PCR，基因合成等。预计基因工程以外的改变氨基酸侧链基团的方法，如化学修饰也是可用的。本文中可使用各种名称来指示同一氨基酸突变。本文中，可采用位置+氨基酸残基的方式表示特定位点的氨基酸残基，例如366W，则表示在366位点上的氨基酸残基为W。T366W则表示第366位点上的氨基酸残基由原来的T突变为了W。The term "amino acid mutation" includes amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to achieve the final construct, as long as the final construct possesses the desired properties, such as reduction or binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and/or carboxyl terminus of the polypeptide chain. Specific amino acid mutations may be amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, ie, one amino acid is replaced by another amino acid with different structural and/or chemical properties. Amino acid substitutions include substitutions by non-naturally occurring amino acids or by derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) . Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is expected that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be available. Various names may be used herein to refer to the same amino acid mutation. In this article, the amino acid residue at a specific position can be represented by position + amino acid residue. For example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 has been mutated from the original T to W.
术语“抗原结合分子”以最广义使用，涵盖各种特异性结合抗原的分子，包括但不限于抗体、其他具有抗原结合活性的多肽以及两者融合而成的抗体融合蛋白，只要它们展现出期望的抗原结合活性。本文的抗原结合分子包含可变区(VH)和可变区(VL)，其共同构成抗原结合域。示例性的，本文中的抗原结合分子是双特异性抗原结合分子(例如：双特异性抗体)。The term "antigen-binding molecule" is used in the broadest sense and covers a variety of molecules that specifically bind to antigens, including but not limited to antibodies, other polypeptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of the two, as long as they exhibit the desired Antigen-binding activity. The antigen-binding molecules herein comprise a variable region (VH) and a variable region (VL), which together constitute an antigen-binding domain. Exemplarily, the antigen-binding molecule herein is a bispecific antigen-binding molecule (eg, bispecific antibody).
术语“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。例如，天然IgG抗体是约150,000道尔顿的异四聚糖蛋白，由二硫键结合的两条相同轻链和两条相同重链构成。从N至C端，每条重链具有一个可变区(VH)，又称作可变重域、重链可变区，接着是三个恒定域(CH1、CH2和CH3)。类似地，从N至C端，每条轻链具有一个可变区(VL)，又称作可变轻域，或轻链可变域，接着是一个恒定轻域(轻链恒定区、CL)。The term "antibody" is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibodies fragments (or antigen-binding fragments, or antigen-binding portions) as long as they exhibit the desired antigen-binding activity. For example, natural IgG antibodies are heterotetrameric proteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as variable heavy domain, heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C terminus, each light chain has a variable region (VL), also called a variable light domain, or light chain variable domain, followed by a constant light domain (light chain constant region, CL ).
术语“双特异性抗体”指能够对两个不同抗原或同一抗原的至少两个不同抗原表位特异性结合的抗体(包括抗体或其抗原结合片段，如单链抗体)。现有技术已公开了各种结构的双特异性抗体，根据IgG分子的完整性可分为IgG样双特异性抗体和抗体片段型双特异性抗体，根据抗原结合区域的数量可分为二价、三价、四价或更多价的双特异性抗体，根据结构是否对称可分为对称结构双特异性抗体 和不对称结构双特异性抗体。其中，基于抗体片段的双特异性抗体，例如缺乏Fc片段的Fab片段，其通过将2个或多个Fab片段结合在一个分子中形成双特异性抗体，其具有较低的免疫原性，且分子量小，具有较高的肿瘤组织渗透性，该类型的典型的抗体结构如F(ab)2、scFv-Fab、(scFv)2-Fab；IgG样双特异性抗体(例如具有Fc片段)，这类抗体相对分子量较大，Fc片段有助于抗体的纯化，并提高其溶解性、稳定性，Fc部分还可能会与受体FcRn结合，增加抗体血清半衰期，典型的双特异性抗体结构模型如KiH、CrossMAb、Triomab quadroma、FcΔAdp、ART-Ig、BiMAb、Biclonics、BEAT、DuoBody、Azymetric、XmAb、2:1TCBs、1Fab-IgG TDB、FynomAb、two-in-one/DAF、scFv-Fab-IgG、DART-Fc、LP-DART、CODV-Fab-TL、HLE-BiTE、F(ab)2-CrossMAb、IgG-(scFv)2、Bs4Ab、DVD-Ig、Tetravalent-DART-Fc、(scFv)4-Fc、CODV-Ig、mAb2、F(ab)4-CrossMAb等(参见Aran F.Labrijn等，Nature Reviews Drug Discovery volume 18，pages585–608(2019)；Chen S1等，J Immunol Res.2019Feb 11；2019:4516041)。The term "bispecific antibody" refers to an antibody (including antibodies or antigen-binding fragments thereof, such as single-chain antibodies) that can specifically bind to two different antigens or at least two different epitopes of the same antigen. Bispecific antibodies of various structures have been disclosed in the prior art. According to the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment type bispecific antibodies. According to the number of antigen-binding regions, they can be divided into bivalent antibodies. , trivalent, quadrivalent or more valent bispecific antibodies, which can be divided into symmetric structure bispecific antibodies according to whether the structure is symmetrical or not. and asymmetrically structured bispecific antibodies. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining 2 or more Fab fragments in one molecule, have lower immunogenicity, and Small molecular weight, high tumor tissue penetration, typical antibody structures of this type such as F(ab)2, scFv-Fab, (scFv)2-Fab; IgG-like bispecific antibodies (for example, with Fc fragment), This type of antibody has a relatively large molecular weight. The Fc fragment helps purify the antibody and improves its solubility and stability. The Fc part may also bind to the receptor FcRn, increasing the serum half-life of the antibody. Typical bispecific antibody structural model Such as KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 1Fab-IgG TDB, FynomAb, two-in-one/DAF, scFv-Fab-IgG , DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4 -Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11; 2019:4516041).
术语“可变区”或“可变域”指抗原结合分子中结合抗原的域。本文中，特异性结合PSMA的抗原结合模块中的重链可变区标示为PSMA-VH，轻链可变区标示为PSMA-VL；特异性结合CD28的抗原结合模块中的重链可变区标示为CD28-VH，轻链可变区标示为CD28-VL。VH和VL各包含四个保守的框架区(FR)和三个互补决定区(CDR)。其中，术语“互补决定区”或“CDR”指可变结构域内主要促成与抗原结合的区域；“框架”或“FR”是指除CDR残基之外的可变结构域残基。VH包含3个CDR区：HCDR1、HCDR2和HCDR3；VL包含3个CDR区：LCDR1、LCDR2和LCDR3。本文中，PSMA-VH中的3个CDR区分别标示为PSMA-HCDR1、PSMA-HCDR2和PSMA-HCDR3；PSMA-VL中的3个CDR区分别标示为PSMA-LCDR1、PSMA-LCDR2和PSMA-LCDR3；CD28-VH中的3个CDR区分别标示为CD28-HCDR1、CD28-HCDR2和CD28-HCDR3；CD28-VL中的3个CDR区分别标示为CD28-LCDR1、CD28-LCDR2和CD28-LCDR3。每个VH和VL从N端到C端依次为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。单个VH或VL可能足以赋予抗原结合特异性。The term "variable region" or "variable domain" refers to the domain of an antigen-binding molecule that binds the antigen. In this article, the heavy chain variable region in the antigen-binding module that specifically binds to PSMA is labeled PSMA-VH, and the light chain variable region is labeled PSMA-VL; the heavy-chain variable region in the antigen-binding module that specifically binds to CD28 is labeled PSMA-VH. It is labeled CD28-VH and the light chain variable region is labeled CD28-VL. VH and VL each contain four conserved framework regions (FR) and three complementarity determining regions (CDR). Among them, the term "complementarity determining region" or "CDR" refers to the region within the variable domain that mainly contributes to binding to the antigen; "framework" or "FR" refers to the variable domain residues other than CDR residues. VH contains 3 CDR areas: HCDR1, HCDR2 and HCDR3; VL contains 3 CDR areas: LCDR1, LCDR2 and LCDR3. In this article, the three CDR regions in PSMA-VH are labeled PSMA-HCDR1, PSMA-HCDR2 and PSMA-HCDR3 respectively; the three CDR regions in PSMA-VL are labeled PSMA-LCDR1, PSMA-LCDR2 and PSMA-LCDR3 respectively. ; The three CDR regions in CD28-VH are labeled CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 respectively; the three CDR regions in CD28-VL are labeled respectively CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3. Each VH and VL from N end to C end are: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen binding specificity.
可以通过各种公知方案来确定CDR的氨基酸序列边界，例如：“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则、“ABM”编号规则、“contact”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)；Front Immunol.2018Oct 16；9:2278)等；各种编号系统之间的对应关系是本领域技术人员熟知的。本披露的编号规则如下表1中所示。The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, such as: "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health , Bethesda, MD), "Chothia" numbering rule, "ABM" numbering rule, "contact" numbering rule (see Martin, ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001) and ImMunoGenTics (IMGT) numbering Rules (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art of. The numbering scheme for this disclosure is set forth in Table 1 below.
表1.CDR编号系统之间的关系
Table 1. Relationship between CDR numbering systems
除非另有说明，本披露实施例中的可变区和CDR序列均适用“Kabat”编号规则。尽管在具体的实施方案中，采用了一种编号系统(如Kabat)来限定氨基酸残基，但是其他编号系统所的对应技术方案将视为等同技术方案。Unless otherwise stated, the variable region and CDR sequences in the embodiments of the present disclosure are all subject to the "Kabat" numbering rule. Although in specific embodiments, one numbering system (such as Kabat) is used to define amino acid residues, the corresponding technical solutions in other numbering systems will be regarded as equivalent technical solutions.
术语“抗体片段”指不同于完整抗体的分子，其包含完整抗体的部分，所述部分保留了完整抗体的抗原结合能力。抗体片段的实例包括但不限于Fv、Fab、Fab’、Fab’-SH、F(ab′)2、单域抗体、单链Fab(scFab)、双抗体、线性抗体、单链抗体分子(例如scFv)；以及由抗体片段形成的多特异性抗体。The term "antibody fragment" refers to a molecule other than an intact antibody that contains portions of an intact antibody that retain the antigen-binding ability of the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
术语“Fc区”或“片段可结晶区”用于定义抗体重链的C末端区域，包括天然Fc区和改造的Fc区。在一些实施方式中，Fc区包含了相同或不同的两个亚基。在一些实施方式中，人IgG重链的Fc区定义为从Cys226位置处的氨基酸残基或从Pro230延伸至其羧基末端。用于本文所述抗体的合适天然序列Fc区包括人IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4。除非另有说明，Fc区的编号规则为EU索引。The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including native Fc regions and engineered Fc regions. In some embodiments, the Fc region contains two subunits that are the same or different. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. Suitable native sequence Fc regions for use in the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise stated, the numbering convention for the Fc region is the EU index.
术语“Titin链”是指Titin蛋白中一段长度为78-118个氨基酸的包含Titin Ig-样152结构域的肽段或其功能变体，所述Titin链能够与Obscurin链结合形成二聚化复合物。术语“Obscurin链”是指Obscurin蛋白上一段长度为87-117个氨基酸的包含Obscurin Ig-样1结构域的肽段或其功能变体，或Obscurin-样1蛋白上一段长度为78-118个氨基酸的包含Obscurin-样Ig-样1结构域的肽段或其功能变体，所述Obscurin链能够与Titin链结合形成二聚化复合物。本披露的Titin链与Obscurin链可用于替换Fab中的CH1和CL，形成经替换的Fab(Fab-S)，该替换不影响抗原结合分子与抗原的结合。The term "Titin chain" refers to a peptide segment in the Titin protein containing a Titin Ig-like 152 domain or a functional variant thereof with a length of 78-118 amino acids. The Titin chain can combine with the Obscurin chain to form a dimerization complex. things. The term "Obscurin chain" refers to a peptide segment of the Obscurin protein containing the Obscurin Ig-like 1 domain or a functional variant thereof with a length of 87-117 amino acids, or a segment of the Obscurin-like 1 protein with a length of 78-118 amino acids. A peptide segment of an amino acid containing an Obscurin-like Ig-like 1 domain or a functional variant thereof, and the Obscurin chain can combine with the Titin chain to form a dimerization complex. The Titin chain and Obscurin chain disclosed in the present disclosure can be used to replace CH1 and CL in Fab to form a substituted Fab (Fab-S). This substitution does not affect the binding of the antigen-binding molecule to the antigen.
术语“嵌合”抗体指这样的一种抗体，其中重和/或轻链的一部分源自特定的来源或物种，而重和/或轻链的剩余部分源自不同来源或物种。The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a specific source or species and the remaining portion of the heavy and/or light chain is derived from a different source or species.
术语“人源化”抗体是保留非人抗体的反应性同时在人中具有较低免疫原性的抗体。例如，可以通过保留非人CDR区并用其人对应物(即，恒定区以及可变区的框架区部分)替换抗体的其余部分来实现。The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. This can be accomplished, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions as well as the framework portions of the variable regions).
术语“亲和力”是指分子(例如，抗体)的单个结合部位与其结合配体(例如，抗原)之间非共价相互作用的总体的强度。除非另外指明，如本文所用，“结合亲和力”是指内部结合亲和力，其反映出结合对(例如，抗体与抗原)的成员之间1:1 相互作用。分子X对其配体Y的亲和力通常可以由平衡解离常数(KD)表示。亲和力可以通过本领域已知的常规方法(包括本文所述的那些)测量。术语“kassoc”或“ka”指特定抗体-抗原相互作用的缔合速率，而如本文所使用的术语“kdis”或“kd”意在是指特定抗体-抗原相互作用的解离速率。如本文所使用的，术语“KD”指平衡解离常数，其获得自kd与ka的比率(即kd/ka)并且表示为摩尔浓度(M)。可以使用本领域已知的方法测定抗体的KD值，例如表面等离子体共振法、ELISA或溶液平衡滴定法(SET)。The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the internal binding affinity that reflects a 1:1 between the members of a binding pair (e.g., antibody to antigen) interaction. The affinity of a molecule X for its ligand Y can generally be expressed by the equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. The term "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, while the term "kdis" or "kd" as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as molar concentration (M). The KD value of an antibody can be determined using methods known in the art, such as surface plasmon resonance, ELISA, or solution equilibrium titration (SET).
术语“单克隆抗体”指基本上均质的抗体的群或其成员，即在该群中包含的抗体分子的氨基酸序列是相同的，除了可能少量存在的天然突变以外。相比之下，多克隆抗体制剂通常包含在其可变结构域具有不同氨基酸序列的多种不同抗体，其通常特异性针对不同表位。“单克隆”表示从基本上均质的抗体群体获得的抗体的特征，并且不应解释为要求通过任何特定方法来生产抗体。在一些实施方式中，本披露提供的抗体是单克隆抗体。The term "monoclonal antibody" refers to a population of antibodies or members thereof that are substantially homogeneous, ie, the antibody molecules contained in the population are identical in amino acid sequence, except for natural mutations that may be present in minor amounts. In contrast, polyclonal antibody preparations typically contain multiple different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes. "Monoclonal" refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.
术语“抗原”是指能够由抗原结合分子(例如抗体)选择性识别或结合的分子或分子部分。抗原可具有一个或多个能够与不同的抗原结合分子(例如抗体)相互作用的表位。The term "antigen" refers to a molecule or portion of a molecule capable of being selectively recognized or bound by an antigen-binding molecule (eg, an antibody). An antigen may have one or more epitopes capable of interacting with different antigen-binding molecules (eg, antibodies).
术语“表位”指能够与抗体或其抗原结合片段特异性结合的抗原上的区域(area或region)。表位可以由连续氨基酸(线性表位)形成或包含非连续氨基酸(构象表位)，例如因抗原的折叠(即通过蛋白质性质的抗原的三级折叠)而使得非连续的氨基酸在空间上得以接近。构象表位和线性表位的差别在于：在变性溶剂的存在下，抗体对构象表位的结合丧失。表位包含处于独特空间构象的至少3，至少4，至少5，至少6，至少7，或8-10个氨基酸。筛选结合特定表位的抗体(即那些结合相同表位的)可以使用本领域例行方法来进行，例如但不限于丙氨酸扫描，肽印迹(见Meth.Mol.Biol.248(2004)443-463)，肽切割分析，表位切除，表位提取，抗原的化学修饰(见Prot.Sci.9(2000)487-496)，和交叉阻断(见“Antibodies”，Harlow and Lane(Cold Spring Harbor Press,Cold Spring Harb.,NY))。The term "epitope" refers to an area or region on an antigen that is capable of specifically binding to an antibody or antigen-binding fragment thereof. Epitopes may be formed from contiguous amino acids (linear epitopes) or contain non-contiguous amino acids (conformational epitopes), for example due to the folding of the antigen (i.e. the tertiary folding of the antigen by its proteinaceous nature) such that the non-contiguous amino acids are spatially separated. near. The difference between conformational epitopes and linear epitopes is that in the presence of denaturing solvents, the antibody's binding to the conformational epitope is lost. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443 -463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigen (see Prot.Sci.9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press,Cold Spring Harb.,NY)).
术语“能够特异性结合”、“特异性结合”或“结合”是指相比其他抗原或表位，抗体能够以更高的亲和力结合至某个抗原或表位。通常，抗体以约1×10-7M或更小(例如约1×10-8M或更小)的平衡解离常数(KD)结合抗原或表位。在一些实施方式中，抗体与抗原结合的KD为该抗体结合至非特异性抗原(例如BSA、酪蛋白)的KD的10％或更低(例如1％)。可使用已知的方法来测量KD，例如通过FACS或表面等离子体共振测定法所测量的。然而，特异性结合至抗原或其表位的抗体可能对其它相关的抗原具有交叉反应性，例如，对来自其它物种(同源)(诸如人或猴，例如食蟹猕猴(Macaca fascicularis)(cynomolgus，cyno)、黑猩猩(Pan troglodytes)(chimpanzee，chimp))或狨猴(Callithrix jacchus)(commonmarmoset，marmoset)的相应抗原具有交叉反应性。 The terms "capable of specifically binding", "specifically binding" or "binding" refer to the ability of an antibody to bind to an antigen or epitope with higher affinity than to other antigens or epitopes. Typically, antibodies bind an antigen or epitope with an equilibrium dissociation constant (KD) of about 1×10 −7 M or less (eg, about 1×10 −8 M or less). In some embodiments, the KD of the antibody binding to the antigen is 10% or less (eg, 1%) of the KD of the antibody binding to a non-specific antigen (eg, BSA, casein). KD can be measured using known methods, such as by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or its epitope may be cross-reactive to other related antigens, e.g., to antibodies from other species (homologous) such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus). , cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset) corresponding antigens are cross-reactive.
术语“不结合”是指抗体不能够以上述特异性结合的方式结合至某个抗原或其表位。例如，当抗体以约1×10-6M或更大的平衡解离常数(KD)结合抗原或其表位。The term "does not bind" means that the antibody is unable to bind to an antigen or its epitope in the specific binding manner described above. For example, when the antibody binds the antigen or its epitope with an equilibrium dissociation constant (KD) of about 1×10 -6 M or greater.
术语“抗原结合模块”指特异性结合目标抗原或其表位的多肽分子。具体的抗原结合模块包括抗体的抗原结合域，例如包含重链可变区和轻链可变区。术语“特异性结合CD28的抗原结合模块”是指能够以足够的亲和力结合CD28或其表位的模块，使得含有该模块的分子可用作靶向CD28的诊断剂和/或治疗剂。例如，特异性结合CD28的抗原结合模块具有以下的平衡解离常数(KD)：<约2×10-8M，其是通过表面等离子体共振测定法测量的。抗原结合模块包括如本文定义的抗体片段，例如Fab，经替换的Fab或scFv。The term "antigen-binding module" refers to a polypeptide molecule that specifically binds to a target antigen or an epitope thereof. Specific antigen-binding modules include the antigen-binding domain of an antibody, for example, including a heavy chain variable region and a light chain variable region. The term "antigen-binding module that specifically binds CD28" refers to a module that is capable of binding CD28 or an epitope thereof with sufficient affinity such that molecules containing the module can be used as diagnostic and/or therapeutic agents targeting CD28. For example, an antigen-binding module that specifically binds CD28 has the following equilibrium dissociation constant (KD): <approximately 2×10 −8 M, as measured by surface plasmon resonance assay. Antigen binding moieties include antibody fragments as defined herein, such as Fab, substituted Fab or scFv.
术语“连接子”指连接两个多肽片段的连接单元。在本文中，同一结构式中出现的连接子可以是相同或不同的。连接子可以是肽连接子，其包含一个或多个氨基酸，典型的约1-30个、2-24个或3-15个氨基酸。应用于本文的连接子可以是相同或不同的。当“-”出现在结构式中，其表示两侧的单元直接通过共价键连接。The term "linker" refers to a linking unit that joins two polypeptide fragments. In this article, linkers appearing in the same structural formula may be the same or different. The linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. The linkers used herein may be the same or different. When "-" appears in a structural formula, it means that the units on both sides are directly connected by covalent bonds.
“Tm”是溶解变性温度(内源荧光)。当蛋白质变性(加热或变性剂作用)时，三级结构打开，芳香族氨基酸微环境发生变化，导致发射荧光光谱改变。本披露中，Tm1是指荧光变化到最大值的一半时的温度。"Tm" is the solution denaturation temperature (intrinsic fluorescence). When proteins are denatured (heated or denatured), the tertiary structure is opened and the aromatic amino acid microenvironment changes, resulting in a change in the emission fluorescence spectrum. In this disclosure, Tm1 refers to the temperature at which the fluorescence changes to half of its maximum value.
“Tonset”是变性起始温度。意指蛋白质开始变性时的温度，即荧光值开始变化时的温度。"Tonset" is the denaturation starting temperature. It means the temperature at which the protein begins to denature, that is, the temperature at which the fluorescence value begins to change.
“Tagg”是聚集起始温度。通过静态光散射，在266nm和473nm两个波长下检测聚集体，监测到样品开始聚集时的温度。Tagg 266指的是266nm下监测到聚集起始温度。"Tagg" is the aggregation onset temperature. By static light scattering, aggregates are detected at two wavelengths, 266 nm and 473 nm, and the temperature at which the sample begins to aggregate is monitored. Tagg 266 refers to the aggregation onset temperature monitored at 266nm.
术语“核酸”在本文中可与术语“多核苷酸”互换使用，并且是指呈单链或双链形式的脱氧核糖核苷酸或核糖核苷酸及其聚合物。所述术语涵盖含有已知核苷酸类似物或修饰的骨架残基或连接的核酸，所述核酸是合成的、天然存在的和非天然存在的，具有与参考核酸相似的结合特性，并且以类似于参考核苷酸的方式代谢。此类类似物的实例包括但不限于硫代磷酸酯、氨基磷酸酯、甲基膦酸酯、手性-甲基膦酸酯、2-O-甲基核糖核苷酸、肽-核酸(PNA)。“分离的”核酸指已经与其天然环境的组分分开的核酸分子。编码所述抗原结合分子的分离的核酸指编码抗体重链和轻链(或其片段)的一个或更多个核酸分子，包括在单一载体或分开的载体中的这样的一个或更多个核酸分子，和存在于宿主细胞中一个或更多个位置的这样的一个或更多个核酸分子。除非另有说明，否则特定的核酸序列还隐含地涵盖其保守修饰的变体(例如，简并密码子取代)和互补序列以及明确指明的序列。具体地，如下详述，简并密码子取代可以通过产生如下序列而获得，在这些序列中，一个或多个所选的(或全部)密码子的第三位被简并碱基和/或脱氧肌苷残基取代。 The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring and non-naturally occurring, have similar binding properties to the reference nucleic acid, and are Metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNA ). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acid encoding said antigen-binding molecule refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such one or more nucleic acids in a single vector or separate vectors molecule, and such one or more nucleic acid molecules present at one or more locations in a host cell. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences thereof as well as sequences explicitly indicated. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a degenerate base and/or Deoxyinosine residue substitution.
术语“多肽”和“蛋白质”在本文中可互换使用，指氨基酸残基的聚合物。该术语适用于氨基酸聚合物，其中一个或多个氨基酸残基是天然存在的氨基酸相应的人工化学模拟物，以及适用于天然存在的氨基酸聚合物和非天然存在的氨基酸聚合物。除非另外说明，否则特定的多肽序列还隐含地涵盖其保守修饰的变体。The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are the corresponding artificial chemical mimetics of naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless stated otherwise, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
术语序列“同一性”指，当对两条序列进行最佳比对时，两条序列的氨基酸/核酸在等价位置相同的程度(百分比)。在比对过程中，必要时可允许引入间隙以获取最大序列同一性百分比，但任何保守性取代不视为构成序列同一性的一部分。为测定序列同一性百分比，比对可以通过本领域技术已知的技术来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。The term sequence "identity" refers to the extent (percentage) that the amino acids/nucleic acids of two sequences are identical at equivalent positions when the two sequences are optimally aligned. During the alignment process, gaps may be allowed to be introduced when necessary to achieve maximum percent sequence identity, but any conservative substitutions are not considered to form part of the sequence identity. To determine percent sequence identity, alignment can be accomplished by techniques known to those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximal alignment over the full length of the sequences being compared.
术语“融合”或“连接”是指部件(例如抗原结合模块和Fc结构域)直接地或经由连接子共价连接。The term "fusion" or "linking" means that the components (eg, the antigen-binding module and the Fc domain) are covalently linked, either directly or via a linker.
术语“载体”意指能够转运与其连接的另一多核苷酸的多核苷酸分子。一种类型的载体是“质粒”，其是指环状双链DNA环，其中可以连接附加的DNA区段。另一种类型的载体是病毒载体，例如腺相关病毒载体(AAV或AAV2)，其中另外的DNA区段可以连接到病毒基因组中。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)。其他载体(例如，非附加型哺乳动物载体)可以在引入宿主细胞中后整合到宿主细胞的基因组中，从而与宿主基因组一起复制。术语“表达载体”或“表达构建体”是指适用于对宿主细胞进行转化且含有指导和/或控制(连同宿主细胞一起)与其可操作地连接的一个或多个异源编码区的表达的核酸序列的载体。表达构建体可以包括但不限于影响或控制转录、翻译且在存在内含子时影响与其可操作地连接的编码区的RNA剪接的序列。The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA circle into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be introduced into the host cell and integrated into the host cell's genome, thereby replicating with the host genome. The term "expression vector" or "expression construct" refers to a vector suitable for transformation of a host cell and containing the direction and/or control of the expression of one or more heterologous coding regions operably linked thereto (together with the host cell). Nucleic acid sequence vectors. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, RNA splicing of the coding region operably linked thereto.
术语“宿主细胞”，“宿主细胞系”和“宿主细胞培养物”可互换使用，并且指已经导入外源核酸的细胞，包括此类细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞及自其衍生的后代，而不考虑传代的次数。后代在核酸内容物上可以与亲本细胞不完全相同，而是可以含有突变。本文中，该术语包括突变体后代，其与在原代转化细胞中筛选或选择的细胞具有相同的功能或生物学活性。宿主细胞包括原核和真核宿主细胞，其中真核宿主细胞包括但不限于哺乳动物细胞、昆虫细胞系植物细胞和真菌细胞。哺乳动物宿主细胞包括人、小鼠、大鼠、犬、猴、猪、山羊、牛、马和仓鼠细胞，包括但不限于中国仓鼠卵巢(CHO)细胞、NSO、SP2细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，Hep G2)、A549细胞、3T3细胞和HEK-293细胞。真菌细胞包括酵母和丝状真菌细胞，包括例如巴氏毕赤酵母(Pichiapastoris)、芬兰毕赤酵母(Pichia finlandica)、海藻毕赤酵母(Pichia trehalophila)、科克拉 马毕赤酵母(Pichia koclamae)、膜状毕赤酵母(Pichia membranaefaciens)、小毕赤酵母(Pichia minuta)(Ogataea minuta、Pichia lindneri)、仙人掌毕赤酵母(Pichiaopuntiae)、耐热毕赤酵母(Pichia thermotolerans)、柳毕赤酵母(Pichia salictaria)、Pichia guercuum、皮杰普毕赤酵母(Pichia pijperi)、具柄毕赤酵母(Pichia stiptis)、甲醇毕赤酵母(Pichia methanolica)、毕赤酵母属、酿酒酵母(Saccharomycescerevisiae)、酿酒酵母属、多形汉逊酵母(Hansenula polymorpha)、克鲁维酵母属、乳酸克鲁维酵母(Kluyveromyces lactis)、白色念珠菌(Candida albicans)、构巢曲霉(Aspergillus nidulans)、黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、里氏木霉(Trichoderma reesei)、勒克氏菌(Chrysosporium lucknowense)、镰刀菌属(Fusarium sp.)、禾谷镰刀菌(Fusarium gramineum)、菜镰刀菌(Fusarium venenatum)、小立碗藓(Physcomitrella patens)和粗糙脉孢菌(Neurospora crassa)。毕赤酵母属、任何酿酒酵母属、多形汉逊酵母(Hansenula polymorpha)、任何克鲁维酵母属、白色念珠菌(Candida albicans)、任何曲霉属、里氏木霉(Trichoderma reesei)、勒克霉菌(Chrysosporium lucknowense)、任何镰刀菌属、解脂耶氏酵母(Yarrowia lipolytica)和粗糙脉孢菌(Neurospora crassa)。本专利的宿主细胞不包括在专利法中不授权的客体。The terms "host cell,""host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parent cells, but may contain mutations. As used herein, the term includes mutant progeny that possess the same functional or biological activity as cells screened or selected in primary transformed cells. Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell line plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, canine, monkey, porcine, goat, bovine, equine, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Cokra Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans), Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp., Saccharomycescerevisiae, Saccharomyces genus, Hansenula polymorpha, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans ), Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum ), Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia pastoris, any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Luck Chrysosporium lucknowense, any species of Fusarium, Yarrowia lipolytica and Neurospora crassa. The host cells of this patent do not include subjects that are not authorized under the patent law.
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。"Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance does or does not occur.
术语“药物组合物”表示含有一种或多种本文所述的抗原结合分子或抗体与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。The term "pharmaceutical composition" means a mixture containing one or more antigen-binding molecules or antibodies described herein together with other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients.
术语“药学上可接受的载体”指药物制剂中与活性成分不同的，且对受试者无毒的成分。药学上可接受载剂包括但不限于缓冲剂、赋形剂、稳定剂或防腐剂。The term "pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical formulation that is distinct from the active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
术语“受试者”或“个体”包括人类和非人类动物。非人动物包括所有脊椎动物(例如哺乳动物和非哺乳动物)例如非人灵长类(例如，食蟹猴)、绵羊、狗、牛、鸡、两栖动物和爬行动物。除非明确指出，否则所述术语“患者”或“受试者”在本文中可互换地使用。如本文所使用的，术语“食蟹猴(cyno)”或“食蟹猴(cynomolgus)”是指食蟹猴(Macaca fascicularis)。在某些实施方案中，个体或受试者是人。The term "subject" or "individual" includes humans and non-human animals. Non-human animals include all vertebrates (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless expressly stated otherwise, the terms "patient" or "subject" are used interchangeably herein. As used herein, the term "cyno" or "cynomolgus" refers to the crab-eating monkey (Macaca fascicularis). In certain embodiments, the individual or subject is a human.
“施用”或“给予”，当其应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。"Administration" or "administration", when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, means the administration of an exogenous drug, therapeutic, diagnostic or composition to an animal, human , contact with subjects, cells, tissues, organs or biological fluids.
术语“样本”是指从受试者分离的类似流体、细胞、或组织的采集物，以及存在于受试者体内的流体、细胞或组织。示例性样本为生物流体，诸如血液、血清和浆膜液、血浆、淋巴液、尿液、唾液、囊液、泪液、排泄物、痰、分泌组织和器官的粘膜分泌物、阴道分泌物、腹水、胸膜、心包、腹膜、腹腔和其它体腔的流体、由支气管灌洗液收集的流体、滑液、与受试者或生物来源接触的液体溶液， 例如细胞和器官培养基(包括细胞或器官条件培养基)、灌洗液等，组织活检样本、细针穿刺、手术切除的组织、器官培养物或细胞培养物。The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph fluid, urine, saliva, cyst fluid, tears, excreta, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites , fluids from the pleura, pericardium, peritoneum, peritoneal cavity and other body cavities, fluids collected from bronchial lavage, synovial fluids, fluid solutions in contact with subjects or biological sources, For example, cell and organ culture media (including cell or organ conditioned media), lavage fluid, etc., tissue biopsy samples, fine needle aspiration, surgically resected tissue, organ culture or cell culture.
“治疗(treatment或treat)”和“处理”(及其语法变型)指试图施加至所治疗个体的临床干预，并且可以为了预防目的、或者在临床病理学的过程期间进行实施。治疗的期望效果包括但不限于预防疾病的发生或复发，减轻症状，减轻/减少疾病的任何直接或间接病理后果，预防转移，降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后。在一些实施方案中，使用本披露的分子来延迟疾病的形成或减缓疾病的进展。"Treatment" and "treatment" (and their grammatical variations) refer to a clinical intervention attempted to be applied to the individual being treated, and may be administered for preventive purposes, or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and regressing or improving prognosis . In some embodiments, molecules of the present disclosure are used to delay the development of a disease or slow the progression of a disease.
术语“复发(recurrence)”、“复发(relapse)”“复发(relapsed)”是指癌症或疾病在疾病消失的临床评估之后的恢复。远处癌转移或局部复发的诊断可视为复发。The terms "recurrence", "relapse" and "relapsed" refer to the return of cancer or disease following clinical assessment of disease resolution. The diagnosis of distant cancer metastasis or local recurrence may be considered recurrence.
术语“难治性”或“抵抗性”是指对治疗无反应的癌症或疾病。The term "refractory" or "resistant" refers to a cancer or disease that does not respond to treatment.
“有效量”一般是足以降低症状的严重程度和/或频率、消除这些症状及/或潜在病因、预防症状和/或其潜在病因出现和/或改良或改善由疾病状态引起或与其相关的损伤的量。在一些实施例中，有效量是治疗有效量或预防有效量。“治疗有效量”是足以治疗疾病状态或症状、尤其与该疾病状态相关的状态或症状，或者以其他方式预防、阻碍、延迟或逆转该疾病状态或以任何方式与该疾病相关的任何其他不理想症状的进展的量。“预防有效量”是当给予受试者时将具有预定预防效应，例如预防或延迟该疾病状态的发作(或复发)，或者降低该疾病状态或相关症状的发作(或复发)可能性的量。完全治疗或预防效未必在给予一个剂量之后便发生，可能在给予一系列剂量之后发生。因而，治疗或预防有效量可以一次或多次给予的方式给予。“治疗有效量”和“预防有效量”可取决于多种因素变化：诸如个体的疾病状态、年龄、性别和体重，以及治疗剂或治疗剂组合在个体中引发期望的应答的能力。有效治疗剂或治疗剂组合的示例性指标包括例如患者改善的健康状况。An "effective amount" is generally one sufficient to reduce the severity and/or frequency of symptoms, eliminate those symptoms and/or underlying causes, prevent the occurrence of symptoms and/or their underlying causes, and/or ameliorate or ameliorate impairments caused by or associated with the disease state. amount. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is one sufficient to treat a disease state or symptom, particularly a condition or symptom associated with that disease state, or to otherwise prevent, hinder, delay or reverse the disease state or any other adverse effect in any way related to the disease. The ideal amount of symptomatic progression. A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. . Complete therapeutic or prophylactic effect does not necessarily occur after administration of one dose but may occur after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations. "Therapeutically effective amount" and "prophylactically effective amount" may vary depending on a variety of factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.
术语“免疫检查点”意指在CD4T细胞和CD8T细胞的细胞表面上的一组分子。这些分子可以有效地充当下调或抑制抗肿瘤免疫应答的“刹车”。免疫检查点分子包括但不限于程序性死亡1(PD-1)、细胞毒T淋巴细胞抗原4(CTLA-4)、B7H1、B7H4、OX-40、CD137、CD40和LAG-3，它们直接抑制免疫细胞。The term "immune checkpoint" means a group of molecules on the cell surface of CD4 T cells and CD8 T cells. These molecules can effectively act as "brakes" to downregulate or inhibit anti-tumor immune responses. Immune checkpoint molecules include, but are not limited to, programmed death 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), B7H1, B7H4, OX-40, CD137, CD40, and LAG-3, which directly inhibit Immune Cells.
用于本披露的免疫检查点抑制剂为抑制免疫检查点分子的功能的物质。对免疫检查点抑制剂没有特别限制，只要抑制剂为可抑制免疫检查点分子的功能(信号)的物质即可。可以作为可用于本披露方法中的免疫检查点抑制剂包括但不限于PD-1、PD-L2、CTLA-4、TIM-3、LAG-3、VISTA、BTLA、TIGIT、LAIR1、CD160、2B4、CEACAM(例如，CEACAM-1、CEACAM-3和/或CEACAM-5)和/或TGFRβ的抑制剂。对抑制性分子的抑制可以通过在DNA、RNA或蛋白质水平抑制而实施。在实施方案中，抑制性核酸(例如，dsRNA、siRNA或shRNA)可以用来抑制抑制性分子的表达。在其他实施方案中，抑制性信号的抑制剂是与抑制性分子结合的多肽，例如，可溶性配体或抗体。本披露中使用的免疫检查点抑制剂的实例可以 包括但不特别限于抗PD-1抗体、抗PD-L1抗体和抗CTLA-4抗体，并且可以优选抗PD-1抗体和抗PD-L1抗体。Immune checkpoint inhibitors for use in the present disclosure are substances that inhibit the function of immune checkpoint molecules. The immune checkpoint inhibitor is not particularly limited as long as the inhibitor is a substance that can inhibit the function (signal) of the immune checkpoint molecule. Immune checkpoint inhibitors that can be used in the methods of the present disclosure include, but are not limited to, PD-1, PD-L2, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, Inhibitors of CEACAM (eg, CEACAM-1, CEACAM-3 and/or CEACAM-5) and/or TGFRβ. Inhibition of inhibitory molecules can be effected by inhibition at the DNA, RNA or protein level. In embodiments, inhibitory nucleic acids (eg, dsRNA, siRNA, or shRNA) can be used to inhibit the expression of inhibitory molecules. In other embodiments, the inhibitor of an inhibitory signal is a polypeptide that binds to an inhibitory molecule, e.g., a soluble ligand or an antibody. Examples of immune checkpoint inhibitors used in this disclosure can This includes, but is not particularly limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, and anti-PD-1 antibodies and anti-PD-L1 antibodies may be preferred.
本披露的抗原结合分子Antigen binding molecules of the present disclosure
本披露提供了抗原结合分子，其具有诸多有利的特性，例如良好的体外杀伤活性、治疗活性、安全性、药物代谢动力学特性和成药性(如产率、纯度和稳定性等)。The present disclosure provides antigen-binding molecules that have many advantageous properties, such as good in vitro killing activity, therapeutic activity, safety, pharmacokinetic properties and druggability (such as yield, purity and stability, etc.).
示例性的抗原结合分子Exemplary antigen binding molecules
本披露的抗原结合分子，包括特异性结合CD28和PSMA的双特异性抗原结合分子(例如双特异性抗体)和抗CD28抗体。特别的，本披露的抗原结合分子具有如下任一的性质：Antigen-binding molecules of the present disclosure include bispecific antigen-binding molecules (eg, bispecific antibodies) and anti-CD28 antibodies that specifically bind to CD28 and PSMA. In particular, the antigen-binding molecules of the present disclosure have any of the following properties:
a.对CD28的高亲和力。在一些实施方式中，所述抗CD28抗体以小于1nM的EC50结合人CD28，所述EC50是通过ELISA测量的。a. High affinity for CD28. In some embodiments, the anti-CD28 antibody binds human CD28 with an EC50 of less than 1 nM, as measured by ELISA.
b.对细胞表面的CD28和PSMA的高亲和力。b. High affinity for CD28 and PSMA on the cell surface.
c.对T细胞的体外激活活性。在一些实施方案中，所述的抗原结合分子在第一激活信号存在时，以小于0.1μg/mL的EC50激活表达IL-2的Jurkat细胞。c. In vitro activation activity on T cells. In some embodiments, the antigen-binding molecule activates IL-2-expressing Jurkat cells with an EC50 of less than 0.1 μg/mL in the presence of a first activation signal.
d.与PSMA/CD3双特异性抗体联用，诱导低水平的细胞因子(IL6和IFNγ)释放。d. Combined with PSMA/CD3 bispecific antibody to induce low-level cytokine (IL6 and IFNγ) release.
e.更强的体内治疗活性。在一些实施方案中，与PSMA/CD3双特异性抗体或抗PD-1抗体联用，具有更好的体内治疗活性。e. Stronger therapeutic activity in vivo. In some embodiments, combination with PSMA/CD3 bispecific antibody or anti-PD-1 antibody has better in vivo therapeutic activity.
f.与PSMA/CD3双特异性抗体联用，可显著增加PSMA/CD3双特异性抗体对肿瘤细胞的杀伤作用。f. Combined with PSMA/CD3 bispecific antibody, it can significantly increase the killing effect of PSMA/CD3 bispecific antibody on tumor cells.
本披露提供了一种抗原结合分子，其包含至少一个特异性结合CD28的抗原结合模块和至少一个特异性结合PSMA的抗原结合模块，所述特异性结合CD28的抗原结合模块包含CD28-VH和CD28-VL，所述特异性结合PSMA的抗原结合模块包含PSMA-VH和PSMA-VL。本披露还提供了一种抗CD28抗体，其能够特异性结合CD28，所述的抗体包含CD28-VH和CD28-VL。具体地，本文的实施例披露了抗体系列81、94、97和129。以下以抗体97和94为例描述本文的抗体。The present disclosure provides an antigen-binding molecule comprising at least one antigen-binding module that specifically binds to CD28 and at least one antigen-binding module that specifically binds to PSMA, the antigen-binding module that specifically binds to CD28 comprises CD28-VH and CD28 -VL, the antigen-binding module that specifically binds PSMA includes PSMA-VH and PSMA-VL. The present disclosure also provides an anti-CD28 antibody capable of specifically binding to CD28, the antibody comprising CD28-VH and CD28-VL. Specifically, the Examples herein disclose antibody series 81, 94, 97, and 129. The antibodies of this article are described below using antibodies 97 and 94 as examples.
特异性结合CD28和PSMA的抗原结合分子或抗CD28抗体，其中：An antigen-binding molecule or anti-CD28 antibody that specifically binds CD28 and PSMA, wherein:
(i)所述CD28-VH包含如SEQ ID NO：19所示的CD28-HCDR1、如SEQ ID NO：20所示的CD28-HCDR2和如SEQ ID NO：21所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：22所示的CD28-LCDR1、如SEQ ID NO：23、81、82、83、84、85、86、87、88、89、90、91、92或93所示的CD28-LCDR2和如SEQ ID NO：24所示的的CD28-LCDR3；或(i) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and The CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 22, as shown in SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 CD28-LCDR2 as shown and CD28-LCDR3 as shown in SEQ ID NO: 24; or
(ii)所述CD28-VH包含如SEQ ID NO：13所示的CD28-HCDR1、如SEQ ID NO：14所示的CD28-HCDR2和如SEQ ID NO：15所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：16所示的CD28-LCDR1、如SEQ ID NO：17、54、 55、56、57、58、59、60、61或62所示的CD28-LCDR2和如SEQ ID NO：18所示的CD28-LCDR3。(ii) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and The CD28-VL includes CD28-LCDR1 as shown in SEQ ID NO: 16, such as SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and CD28-LCDR3 as shown in SEQ ID NO: 18.
特异性结合CD28和PSMA的抗原结合分子或抗CD28抗体，其中：An antigen-binding molecule or anti-CD28 antibody that specifically binds CD28 and PSMA, wherein:
(i)所述CD28-VH包含如SEQ ID NO：19所示的CD28-HCDR1、如SEQ ID NO：20所示的CD28-HCDR2和如SEQ ID NO：21所示的CD28-HCDR3，和所述CD28-VL包含如SEQ ID NO：22所示的CD28-LCDR1、如SEQ ID NO：23所示的CD28-LCDR2和如SEQ ID NO：24所示的CD28-LCDR3；或(i) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 19, CD28-HCDR2 as shown in SEQ ID NO: 20 and CD28-HCDR3 as shown in SEQ ID NO: 21, and The CD28-VL includes CD28-LCDR1 as set forth in SEQ ID NO: 22, CD28-LCDR2 as set forth in SEQ ID NO: 23, and CD28-LCDR3 as set forth in SEQ ID NO: 24; or
(ii)所述CD28-VH包含如SEQ ID NO：13所示的CD28-HCDR1、如SEQ ID NO：14所示的CD28-HCDR2和如SEQ ID NO：15所示的CD28-HCDR3，和所述CD28-VL如SEQ ID NO：16所示的CD28-LCDR1、如SEQ ID NO：62所示的CD28-LCDR2和如SEQ ID NO：18所示的CD28-LCDR3。(ii) The CD28-VH includes CD28-HCDR1 as shown in SEQ ID NO: 13, CD28-HCDR2 as shown in SEQ ID NO: 14 and CD28-HCDR3 as shown in SEQ ID NO: 15, and The CD28-VL is CD28-LCDR1 as shown in SEQ ID NO: 16, CD28-LCDR2 as shown in SEQ ID NO: 62, and CD28-LCDR3 as shown in SEQ ID NO: 18.
在一些实施方案中，如前所述的抗原结合分子或抗CD28抗体，所述CD28-VH和/或所述CD28-VL是鼠源的或人源化的。在一些实施方案中，所述CD28-VH和/或所述CD28-VL是人源化的。In some embodiments, the antigen-binding molecule or anti-CD28 antibody as described above, the CD28-VH and/or the CD28-VL is murine or humanized. In some embodiments, the CD28-VH and/or the CD28-VL are humanized.
在一些实施方案中，所述人源化的CD28-VH的FR1、FR2、FR3和FR4与SEQ ID NO：98的FR1、FR2、FR3和FR4具有至少60％、70％、80％或90％的序列同一性；所述人源化的CD28-VL的FR1、FR2、FR3和FR4与SEQ ID NO：102的FR1、FR2、FR3和FR4具有至少60％、70％、80％或90％的序列同一性。In some embodiments, the FR1, FR2, FR3, and FR4 of the humanized CD28-VH are at least 60%, 70%, 80%, or 90% identical to the FR1, FR2, FR3, and FR4 of SEQ ID NO:98 Sequence identity; FR1, FR2, FR3 and FR4 of the humanized CD28-VL have at least 60%, 70%, 80% or 90% identity with the FR1, FR2, FR3 and FR4 of SEQ ID NO: 102 Sequence identity.
在一些实施方案中，所述人源化的CD28-VH具有来源于IGHV1-46*01的FR1、FR2、FR3和来源于IGHJ6*01的FR4，并且其是未被取代的或具有选自1E、26A、29L、69L、71V、78A和93S组成的组中的一个或多个氨基酸取代；和/或所述人源化的CD28-VL具有来源于IGKV1-33*01的FR1、FR2、FR3和来源于IGKJ4*01的FR4，并且其是未被取代的或具有选自41D、42G、43T、44I和71Y组成的组中的一个或多个氨基酸取代。在一些实施方案中，上述可变区中的氨基酸位置是根据Kabat编号规则定义的。In some embodiments, the humanized CD28-VH has FR1, FR2, FR3 derived from IGHV1-46*01 and FR4 derived from IGHJ6*01, and is unsubstituted or has FR4 derived from IGHJ6*01 , one or more amino acid substitutions in the group consisting of 26A, 29L, 69L, 71V, 78A and 93S; and/or the humanized CD28-VL has FR1, FR2, FR3 derived from IGKV1-33*01 and FR4 derived from IGKJ4*01 and which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44I and 71Y. In some embodiments, the amino acid positions in the variable regions described above are defined according to Kabat numbering rules.
在一些实施方案中，所述人源化的CD28-VH包含SEQ ID NO：98的氨基酸序列或其变体。在一些实施方案中，所述变体为在SEQ ID NO：98的FR区包含选自26A、29L、69L、71V、78A和93S组成的组中的一个或多个氨基酸取代。在一些实施方案中，所述人源化的CD28-VL包含SEQ ID NO：102的氨基酸序列，或其变体。在一些实施方案中，所述变体为在SEQ ID NO：102的FR区包含选自41D、42G、43T、44I和71Y组成的组中的一个或多个氨基酸取代。In some embodiments, the humanized CD28-VH comprises the amino acid sequence of SEQ ID NO: 98 or a variant thereof. In some embodiments, the variant is one or more amino acid substitutions in the FR region of SEQ ID NO: 98 selected from the group consisting of 26A, 29L, 69L, 71V, 78A, and 93S. In some embodiments, the humanized CD28-VL comprises the amino acid sequence of SEQ ID NO: 102, or a variant thereof. In some embodiments, the variant is one or more amino acid substitutions selected from the group consisting of 41D, 42G, 43T, 44I, and 71Y in the FR region of SEQ ID NO: 102.
在一些实施方案中，所述人源化的CD28-VH的FR1、FR2、FR3和FR4与SEQ ID NO：69的FR1、FR2、FR3和FR4具有至少60％、70％、80％或90％的序列同一性；所述人源化的CD28-VL的FR1、FR2、FR3和FR4与SEQ ID NO：80的FR1、FR2、FR3和FR4具有至少60％、70％、80％或90％的序列同一性。In some embodiments, the FR1, FR2, FR3, and FR4 of the humanized CD28-VH are at least 60%, 70%, 80%, or 90% identical to the FR1, FR2, FR3, and FR4 of SEQ ID NO: 69 Sequence identity; FR1, FR2, FR3 and FR4 of the humanized CD28-VL have at least 60%, 70%, 80% or 90% identity with the FR1, FR2, FR3 and FR4 of SEQ ID NO:80 Sequence identity.
在一些实施方案中，所述人源化的CD28-VH具有来源于IGHV1-3*01的FR1、 FR2、FR3和来源于IGHJ1*01的FR4，并且其是未被取代的或具有选自1E、28S、69L、71V、73K和94S组成的组中的一个或多个氨基酸取代；和/或所述人源化的CD28-VL具有来源于IGKV1-12*01的FR1、FR2、FR3和来源于IGKJ4*01的FR4，并且其是未被取代的或具有选自43S和70K组成的组中的一个或多个氨基酸取代。在一些实施方案中，上述可变区中的氨基酸位置是根据Kabat编号规则定义的。In some embodiments, the humanized CD28-VH has FR1 derived from IGHV1-3*01, FR2, FR3 and FR4 derived from IGHJ1*01 and which are unsubstituted or have one or more amino acid substitutions selected from the group consisting of 1E, 28S, 69L, 71V, 73K and 94S; and/or the The humanized CD28-VL has FR1, FR2, FR3 derived from IGKV1-12*01 and FR4 derived from IGKJ4*01, and is unsubstituted or has a group selected from the group consisting of 43S and 70K One or more amino acid substitutions. In some embodiments, the amino acid positions in the variable regions described above are defined according to Kabat numbering rules.
在一些实施方案中，所述人源化的CD28-VH包含SEQ ID NO：69的氨基酸序列或其变体。在一些实施方案中，所述变体为在SEQ ID NO：69的FR区包含选自28S、69L、71V、73K和94S组成的组中的一个或多个氨基酸取代。在一些实施方案中，所述人源化的CD28-VL包含SEQ ID NO：80的氨基酸序列，或其变体。在一些实施方案中，所述变体为在SEQ ID NO：80的FR区包含一个或多个氨基酸取代。In some embodiments, the humanized CD28-VH comprises the amino acid sequence of SEQ ID NO: 69 or a variant thereof. In some embodiments, the variant is one or more amino acid substitutions selected from the group consisting of 28S, 69L, 71V, 73K, and 94S in the FR region of SEQ ID NO: 69. In some embodiments, the humanized CD28-VL comprises the amino acid sequence of SEQ ID NO: 80, or a variant thereof. In some embodiments, the variant is one or more amino acid substitutions in the FR region of SEQ ID NO: 80.
在一些实施方案中，如前任一项所述的抗原结合分子或抗CD28抗体，其中所述CD28-VH的氨基酸序列与SEQ ID NO：95、35、94、96、97或98具有至少90％、95％、96％、97％、98％或99％的序列同一性，和所述CD28-VL的氨基酸序列与SEQ ID NO：101、36、99、100、102、103、104、105、106、107、108、109、110、111、112、113、114或115具有至少90％、95％、96％、97％、98％或99％的序列同一性。在一些实施方案中，所述CD28-VH的氨基酸序列如SEQ ID NO：95、35、94、96、97或98所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：101、36、99、100、102、103、104、105、106、107、108、109、110、111、112、113、114或115所示。In some embodiments, the antigen-binding molecule or anti-CD28 antibody of any one of the above, wherein the amino acid sequence of CD28-VH is at least 90% identical to SEQ ID NO: 95, 35, 94, 96, 97 or 98 , 95%, 96%, 97%, 98% or 99% sequence identity, and the amino acid sequence of CD28-VL is identical to SEQ ID NO: 101, 36, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115 have at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the CD28-VH has an amino acid sequence as set forth in SEQ ID NO: 95, 35, 94, 96, 97 or 98, and the CD28-VL has an amino acid sequence as SEQ ID NO: 101, 36 , 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115.
在一些实施方案中，如前任一项所述的抗原结合分子或抗CD28抗体，所述CD28-VH的氨基酸序列如SEQ ID NO：94所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：99、100、101、103、104、105、106、107、108、109、110、111、112、113、114或115所示，或In some embodiments, the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items, the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 94, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115, or
所述CD28-VH的氨基酸序列如SEQ ID NO：95所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：99、100、101、103、104、105、106、107、108、109、110、111、112、113、114或115所示，或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 95, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109 , 110, 111, 112, 113, 114 or 115, or
所述CD28-VH的氨基酸序列如SEQ ID NO：96所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：99或100所示，或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 96, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 99 or 100, or
所述CD28-VH的氨基酸序列如SEQ ID NO：97所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：99、100或101所示。The amino acid sequence of CD28-VH is shown in SEQ ID NO: 97, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 99, 100 or 101.
在一些实施方案中，如前任一项所述的抗原结合分子或抗CD28抗体，所述CD28-VH的氨基酸序列如SEQ ID NO：95所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：101所示。In some embodiments, the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items, the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 95, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO:101 shown.
在一些实施方案中，如前任一项所述的抗原结合分子或抗CD28抗体，其中所述CD28-VH的氨基酸序列与SEQ ID NO：68、33、63、64、65、66、67或69具 有至少90％、95％、96％、97％、98％或99％的序列同一性，和所述CD28-VL的氨基酸序列与SEQ ID NO：80、34、70、71、72、73、74、75、76、77、78或79具有至少90％、95％、96％、97％、98％或99％的序列同一性。在一些实施方案中，所述CD28-VH的氨基酸序列如SEQ ID NO：68、33、63、64、65、66、67或69所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：80、34、70、71、72、73、74、75、76、77、78或79所示。In some embodiments, the antigen-binding molecule or anti-CD28 antibody of any one of the preceding items, wherein the amino acid sequence of CD28-VH is consistent with SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69 Tool There is at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and the amino acid sequence of CD28-VL is identical to SEQ ID NO: 80, 34, 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79 have a sequence identity of at least 90%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the CD28-VH has an amino acid sequence set forth in SEQ ID NO: 68, 33, 63, 64, 65, 66, 67, or 69, and the CD28-VL has an amino acid sequence set forth in SEQ ID NO. :80, 34, 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79.
在一些实施方案中，如前任一项所述的抗原结合分子或抗CD28抗体，所述CD28-VH的氨基酸序列如SEQ ID NO：63所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：70或71所示，或In some embodiments, the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items, the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 63, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO:70 or 71 shown, or
所述CD28-VH的氨基酸序列如SEQ ID NO：64所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：70、71或72所示，或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 64, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 70, 71 or 72, or
所述CD28-VH的氨基酸序列如SEQ ID NO：65所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：70、71或72所示，或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 65, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 70, 71 or 72, or
所述CD28-VH的氨基酸序列如SEQ ID NO：66所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：70、71、72、74、75、76或79所示，或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 66, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 70, 71, 72, 74, 75, 76 or 79, or
所述CD28-VH的氨基酸序列如SEQ ID NO：67所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：72、74、75、76或79所示，或The amino acid sequence of CD28-VH is shown in SEQ ID NO: 67, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 72, 74, 75, 76 or 79, or
所述CD28-VH的氨基酸序列如SEQ ID NO：68所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：72、73、74、75、76、77、78、79或80所示。The amino acid sequence of CD28-VH is shown in SEQ ID NO: 68, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79 or 80 .
在一些实施方案中，如前任一项所述的抗原结合分子或抗CD28抗体，所述CD28-VH的氨基酸序列如SEQ ID NO：68所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：80所示。In some embodiments, the antigen-binding molecule or anti-CD28 antibody as described in any one of the preceding items, the amino acid sequence of the CD28-VH is as shown in SEQ ID NO: 68, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO:80 is shown.
在一些实施方案中，如前所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule as described above, wherein:
i)所述PSMA-VH包含如SEQ ID NO：164所示的PSMA-HCDR1，如SEQ ID NO：165所示的PSMA-HCDR2，和如SEQ ID NO：166所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：167所示的PSMA-LCDR1，如SEQ ID NO：168所示的PSMA-LCDR2，和如SEQ ID NO：169所示的PSMA-LCDR3；或i) the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 164, PSMA-HCDR2 as shown in SEQ ID NO: 165, and PSMA-HCDR3 as shown in SEQ ID NO: 166; and Said PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 167, PSMA-LCDR2 as shown in SEQ ID NO: 168, and PSMA-LCDR3 as shown in SEQ ID NO: 169; or
ii)所述PSMA-VH包含如SEQ ID NO：170所示的PSMA-HCDR1，如SEQ ID NO：171所示PSMA-HCDR2，和如SEQ ID NO：172所示的PSMA-HCDR3；并且所述PSMA-VL包含如SEQ ID NO：173所示的PSMA-LCDR1，如SEQ ID NO：174所示的PSMA-LCDR2，和如SEQ ID NO：175所示的PSMA-LCDR3。ii) the PSMA-VH includes PSMA-HCDR1 as shown in SEQ ID NO: 170, PSMA-HCDR2 as shown in SEQ ID NO: 171, and PSMA-HCDR3 as shown in SEQ ID NO: 172; and PSMA-VL includes PSMA-LCDR1 as shown in SEQ ID NO: 173, PSMA-LCDR2 as shown in SEQ ID NO: 174, and PSMA-LCDR3 as shown in SEQ ID NO: 175.
在一些实施方案中，如前任一项所述的抗原结合分子，所述PSMA-VH和/或所述PSMA-VL是鼠源的或人源化的。在一些实施方案中，所述PSMA-VH和/或所述PSMA-VL是人源化的。在一些实施方案中，如前任一项所述的抗原结合分子，其中： In some embodiments, the antigen-binding molecule of any one of the preceding items, said PSMA-VH and/or said PSMA-VL is murine or humanized. In some embodiments, the PSMA-VH and/or the PSMA-VL are humanized. In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
i)所述PSMA-VH的氨基酸序列SEQ ID NO：197、199、332、194、195、196或198所示，和所述PSMA-VL的氨基酸序列SEQ ID NO：201、202、333或200所示；或i) The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 197, 199, 332, 194, 195, 196 or 198, and the amino acid sequence of PSMA-VL is SEQ ID NO: 201, 202, 333 or 200 shown; or
ii)所述PSMA-VH的氨基酸序列如SEQ ID NO：204、184或203所示，和所述PSMA-VL的氨基酸序列SEQ ID NO：206、185或205所示。ii) The amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 204, 184 or 203, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 206, 185 or 205.
在一些实施方案中，如前任一项所述的抗原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
i)所述PSMA-VH的氨基酸序列如SEQ ID NO：204所示，和所述PSMA-VL的氨基酸序列SEQ ID NO：206所示；或i) The amino acid sequence of the PSMA-VH is shown in SEQ ID NO: 204, and the amino acid sequence of the PSMA-VL is shown in SEQ ID NO: 206; or
ii)所述PSMA-VH的氨基酸序列SEQ ID NO：197所示，和所述PSMA-VL的氨基酸序列SEQ ID NO：201所示；或ii) The amino acid sequence of PSMA-VH is shown in SEQ ID NO: 197, and the amino acid sequence of PSMA-VL is shown in SEQ ID NO: 201; or
所述PSMA-VH包含SEQ ID NO：199的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：202的氨基酸序列。The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202.
在一些实施方案中，上述可变区和CDR是根据Kabat编号规则定义的。In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering rule.
根据本文实施例所披露的抗体系列81和129与以上所描述的抗体系列97和94具有类似的技术方案范围。The antibody series 81 and 129 disclosed according to the embodiments herein have a similar technical solution scope to the antibody series 97 and 94 described above.
抗原结合分子的结构The structure of an antigen-binding molecule
本披露的双特异性抗原结合分子并不受限于特定的分子结构，只要其具有所期望的抗原结合功能。例如，本文的双特异性抗原结合分子可以是双价(1+1)的、三价(2+1)的或4价(2+2)的。抗原结合分子中的抗原结合模块可以是任意的具有抗原结合活性的抗体片段，其通过肽连接子融合。本披露的肽连接子(例如连接子1至11)可以是任意适宜的肽链，只要抗原结合分子能够展现出期望的抗原结合活性。例如，肽连接子可以是包含1-50或3-20个氨基酸残基的柔性肽。在一些实施方式中，所述的肽连接子各自独立地具有L1-(GGGGS)n-L2的结构，其中，L1是键、A、GS、GGS(SEQ ID NO：399)、GGGS(SEQ ID NO：400)、SGGGGS(SEQ ID NO：294)、GGGTKLTVLGGG(SEQ ID NO：401)，n是0、1、2、3、4、5、6、7、8、9或10，L2是键、G、GG、GGG(SEQ ID NO：295)或GGGG(SEQ ID NO：256)，并且所述肽连接子不是键。在一些实施方案中，所述肽连接子的长度为3-15个氨基酸残基。在一些实施方案中，所述肽连接子各自独立地具有(GGGGS)n的结构，其中n是1、2或3。在一些实施方案中，所述连接子1、连接子2、连接子3和连接子4的氨基酸序列如SEQ ID NO：223所示。The bispecific antigen-binding molecules of the present disclosure are not limited to a specific molecular structure as long as they have the desired antigen-binding function. For example, the bispecific antigen-binding molecules herein may be bivalent (1+1), trivalent (2+1), or tetravalent (2+2). The antigen-binding module in the antigen-binding molecule can be any antibody fragment with antigen-binding activity, which is fused through a peptide linker. The peptide linkers of the present disclosure (eg, linkers 1 to 11) can be any suitable peptide chain as long as the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker can be a flexible peptide containing 1-50 or 3-20 amino acid residues. In some embodiments, the peptide linkers each independently have the structure of L 1 -(GGGGS)nL 2 , wherein L 1 is a bond, A, GS, GGS (SEQ ID NO: 399), GGGS (SEQ ID NO: 400), SGGGGS (SEQ ID NO: 294), GGGTKLTVLGGG (SEQ ID NO: 401), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is bond, G, GG, GGG (SEQ ID NO:295) or GGGG (SEQ ID NO:256), and the peptide linker is not a bond. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, the peptide linkers each independently have the structure (GGGGS)n, where n is 1, 2, or 3. In some embodiments, the amino acid sequences of linker 1, linker 2, linker 3 and linker 4 are as shown in SEQ ID NO: 223.
示例性的，本披露的所述抗原结合分子具有式(a)所示结构的第一链、一条具有式(b)所示结构的第二链、一条具有式(c-1)所示结构的第三链和一条具有式(d-1)所示结构的第四链，Exemplarily, the antigen-binding molecule of the present disclosure has a first chain with a structure represented by formula (a), a second chain with a structure represented by formula (b), and a second chain with a structure represented by formula (c-1). The third chain and a fourth chain having the structure shown in formula (d-1),
(a)[CD28-VH]-[CH1]-[Fc1]，(a)[CD28-VH]-[CH1]-[Fc1],
(b)[CD28-VL]-[CL]， (b)[CD28-VL]-[CL],
(c-1)[PSMA-VH]-[GGGGS]-[Titin链]-[Fc2]，(c-1)[PSMA-VH]-[GGGGS]-[Titin chain]-[Fc2],
(d-1)[PSMA-VL]-[GGGGS]-[Obscurin链]，(d-1)[PSMA-VL]-[GGGGS]-[Obscurin chain],
式(a)、(b)、(c-1)和(d-1)所示的结构是从N端至C端排列的。The structures shown in formulas (a), (b), (c-1) and (d-1) are arranged from the N end to the C end.
示例性的抗原结合分子具有：Exemplary antigen binding molecules include:
一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：239的氨基酸序列的第二链、一条包含SEQ ID NO：249的氨基酸序列的第三链和一条包含SEQ ID NO：250的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 239, a third strand comprising the amino acid sequence of SEQ ID NO: 249 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of the 250 amino acid sequence.
示例性的，所述抗原结合分子包含一条具有式(e-1)所示结构的第一链、一条具有式(f-1)所示结构的第二链、一条具有式(g)所示结构的第三链和一条具有式(h)所示结构的第四链，Exemplarily, the antigen-binding molecule includes a first chain having a structure shown in formula (e-1), a second chain having a structure shown in formula (f-1), and a first chain having a structure shown in formula (g) The third chain of the structure and a fourth chain having the structure shown in formula (h),
(e-1)[CD28-VH]-[GGGGS]-[Titin链]-[Fc1]，(e-1)[CD28-VH]-[GGGGS]-[Titin chain]-[Fc1],
(f-1)[CD28-VL]-[GGGGS]-[Obscurin链]，(f-1)[CD28-VL]-[GGGGS]-[Obscurin chain],
(g)[PSMA-VH]-[CH1]-[Fc2]，(g)[PSMA-VH]-[CH1]-[Fc2],
(h)[PSMA-VL]-[CL]，(h)[PSMA-VL]-[CL],
式(e-1)、(f-1)、(g)和(h)所示的结构是从N端至C端排列的。The structures shown in formulas (e-1), (f-1), (g) and (h) are arranged from the N end to the C end.
示例性的抗原结合分子具有：Exemplary antigen binding molecules include:
一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：272的氨基酸序列的第三链和一条包含SEQ ID NO：273的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 272 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence 273; or
一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：274的氨基酸序列的第三链和一条包含SEQ ID NO：275的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 274 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence of 275.
抗原结合分子的变体Variants of Antigen Binding Molecules
在某些实施方案中，涵盖本文中提供的抗原结合分子的氨基酸序列变体。例如，可以期望改善抗体的结合亲和力和/或其它生物学特性。可以通过将合适的修饰引入编码抗体的核苷酸序列中，或者通过肽合成来制备抗体的氨基酸序列变体。此类修饰包括例如对抗原结合分子的氨基酸序列内的残基的删除、和/或插入、和/或取代。可以进行删除、插入、和取代的任何组合以得到最终的构建体，只要最终的构建体拥有期望的特征，例如抗原结合特性。In certain embodiments, amino acid sequence variants of the antigen-binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions, and/or substitutions of residues within the amino acid sequence of the antigen-binding molecule. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, so long as the final construct possesses the desired characteristics, such as antigen-binding properties.
取代、插入、和删除变体Substitute, insert, and delete variants
在某些实施方案中，提供了具有一处或多处氨基酸取代的抗原结合分子变体。可以在CDR和FR进行取代。保守取代在表2中在“优选的取代”的标题下显示。更实质的变化在表2中在“示例性取代”的标题下提供，并且如下文参照氨基酸侧链类别进一步描述的。可以将氨基酸取代引入感兴趣的抗体中，并且对产物筛选期望的活性，例如保留/改善的抗原结合，降低的免疫原性，或改善的ADCC或CDC。 In certain embodiments, antigen-binding molecule variants having one or more amino acid substitutions are provided. Substitutions can be made in CDR and FR. Conservative substitutions are shown in Table 2 under the heading "Preferred substitutions". More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions" and are described further below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest and the product screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
表2.氨基酸的取代
Table 2. Amino acid substitutions
依照常见的侧链特性，氨基酸可以如下分组:According to common side chain properties, amino acids can be grouped as follows:
(1)疏水性的：正亮氨酸，Met，Ala，Val，Leu，Ile；(1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2)中性，亲水性的：Cys，Ser，Thr，Asn，Gln；(2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3)酸性的：Asp，Glu；(3) Acidic: Asp, Glu;
(4)碱性的：His，Lys，Arg；(4) Basic: His, Lys, Arg;
(5)影响链取向的残基：Gly，Pro；(5) Residues that affect chain orientation: Gly, Pro;
(6)芳香族的：Trp，Tyr，Phe。(6) Aromatic: Trp, Tyr, Phe.
非保守取代会是指用一个类别的成员替换另一个类别的成员。A non-conservative substitution would be the substitution of a member of one class for a member of another class.
一类取代变体涉及取代亲本抗体(例如人源化或人抗体)的一个或多个CDR残基。一般地，经选择用于进一步研究的所得变体相对于亲本抗体会具有某些生物学特性(例如升高的亲和力，降低的免疫原性)的改变(例如改善)，和/或会基本上保留亲本抗体的某些生物学特性。一种例示性的取代变体是亲和力成熟的抗体，可以例如使用基于噬菌体展示的亲和力成熟技术(如本文所述的那些技术)，便利地产生所述抗体。简言之，将一个或多个CDR残基突变，并将变体抗体在噬菌体上展示，并对其筛选特定的生物学活性(例如结合亲和力)。可以对CDR做出改变(例如取代)，例如以改善抗体亲和力。可以对CDR“热点”，即在体细胞 成熟过程期间以高频率经历突变的密码子所编码的残基，和/或接触抗原的残基做出此类改变，同时对所得的变体VH或VL测试结合亲和力。在亲和力成熟的一些实施方案中，通过多种方法(例如易错PCR、链改组、或寡核苷酸指导的诱变)的任一种，将多样性引入所选择用于成熟的可变基因中。然后，创建次级文库。然后，筛选文库以鉴定具有期望的亲和力的任何抗体变体。另一种引入多样性的方法涉及CDR定向的方法，其中将几个CDR残基(例如一次4-6个残基)随机化。可以例如使用丙氨酸扫描诱变或建模来特异性鉴定涉及抗原结合的CDR残基。One type of substitution variant involves the substitution of one or more CDR residues of a parent antibody (eg, a humanized or human antibody). Generally, the resulting variants selected for further study will have alterations (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will be substantially Retains certain biological properties of the parent antibody. One exemplary substitution variant is an affinity matured antibody, which may be conveniently produced, for example, using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity). Changes (eg substitutions) can be made to the CDRs, for example to improve antibody affinity. Can target CDR "hot spots", i.e. in somatic cells Residues encoded by codons that undergo mutations at high frequency during the maturation process, and/or residues contacting the antigen, are made such changes, and the resulting variants VH or VL are tested for binding affinity. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. middle. Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity. Another way to introduce diversity involves methods of CDR orientation, in which several CDR residues (eg 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.
在某些实施方案中，取代、插入或缺失可以在一个或多个CDR内发生，只要此类变化不实质性降低抗体结合抗原的能力。例如，可以对CDR做出保守变化(例如保守取代，如本文中提供的)，其不实质性降低结合亲和力。在上文提供的变体VH和VL序列的某些实施方案中，每个CDR是未改变的，或者含有不超过1、2或3处氨基酸取代。In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs as long as such changes do not substantially reduce the ability of the antibody to bind the antigen. For example, conservative changes (eg, conservative substitutions, as provided herein) can be made to the CDRs that do not substantially reduce binding affinity. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.
一种可用于鉴定抗体中可以作为诱变靶位的残基或区域的方法称作“丙氨酸扫描诱变”。在这种方法中，鉴定一个残基或残基组(例如带电荷的残基，诸如Arg、Asp、His、Lys和Glu)，并且替换为中性或带负电荷的氨基酸(例如，Ala或聚丙氨酸)，以确定该抗体与抗原的相互作用是否受影响。可以在对初始取代显示功能敏感性的氨基酸位置引入进一步的取代。此外，可通过研究抗原-抗体复合物的晶体结构来鉴定抗体与抗原间的接触点。这些接触残基及邻近残基可以作为取代候选物被打靶或消除。可以筛选变体以确定它们是否含有期望的特性。One method that can be used to identify residues or regions in an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis." In this approach, a residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. In addition, the contact points between the antibody and the antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain the desired properties.
氨基酸序列插入包括：在氨基和/或羧基端融合1个残基或长度为100或更多个残基的多肽；和单个或多个氨基酸残基的序列内插入。在末端插入的例子包括具有N端甲硫氨酰基残基的抗体。抗体分子的其它插入变体包括，在抗体的N或C端融合有酶(或延长抗体的血清半衰期的多肽)的融合物。Amino acid sequence insertions include: fusion of 1 residue at the amino and/or carboxyl terminus or polypeptides of 100 or more residues in length; and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions with enzymes (or polypeptides that extend the serum half-life of the antibody) fused to the N- or C-terminus of the antibody.
Fab的改造Fab's transformation
在一个方面，本披露的抗原结合分子中，所述特异性结合PSMA的抗原结合模块和所述特异性结合CD28的抗原结合模块两者之一是经替换的Fab，所述经替换的Fab包含重链可变区、轻链可变区、Titin链和Obscurin链。在经替换的Fab中，Fab原有的CH1和CL被Titin链和Obscurin链所替换。示例性的，Titin链和Obscurin链的序列如表3-1和表3-2所示。In one aspect, in the antigen-binding molecule of the present disclosure, one of the antigen-binding module that specifically binds PSMA and the antigen-binding module that specifically binds CD28 is a replaced Fab, and the replaced Fab comprises Heavy chain variable region, light chain variable region, Titin chain and Obscurin chain. In the replaced Fab, the original CH1 and CL of the Fab are replaced by Titin chains and Obscurin chains. For example, the sequences of Titin chain and Obscurin chain are shown in Table 3-1 and Table 3-2.
表3-1.Titin链的氨基酸序列

Table 3-1. Amino acid sequence of Titin chain

表3-2.Obscurin链的氨基酸序列


Table 3-2.Amino acid sequence of Obscurin chain


Fc区的改造Renovation of Fc area
在一个方面，本披露的抗原结合分子的Fc区包含一个或多个氨基酸取代，所述一个或多个氨基酸取代减少其与Fc受体的结合，例如其与Fcγ受体的结合，并且降低或消除效应子功能。天然IgG Fc区，具体地是IgG1Fc区或IgG4Fc区，可能导致本披露的抗原结合分子靶向表达Fc受体的细胞，而不是表达抗原的细胞。本披露改造的Fc区表现出降低的对Fc受体的结合亲和力和/或降低的效应子功能。在一些实施方案中，改造的Fc区与天然Fc区相比，对Fc受体的结合亲和力下降50％、80％、90％或95％以上。在一些实施方案中，所述的Fc受体是Fcγ受体。在一些实施方案中，所述Fc受体是人Fcγ受体，例如FcγRI、FcγRIIa、FcγRIIB、FcγRIIIa。在一些实施方案中，改造的Fc区与天然Fc区相比，对补体，如C1q的结合亲和力也降低。在一些实施方案中，改造的Fc区与天然Fc区相比，对新生儿Fc受体(FcRn)的结合亲和力不降低。在一些实施例中，改造的Fc区具有降低的效应子功能，所述降低的效应子功能可以包括但不限于以下中的一个或多个：降低的补体依赖性细胞毒性(CDC)、降低的抗体依赖性细胞介导的细胞毒性(ADCC)、降低的抗体依赖性细胞吞噬(ADCP)、减少的细胞因子分泌、减少的免疫复合物介导的抗原呈递细胞的抗原摄取、减少的与NK细胞的结合、减少的与巨噬细胞的结合、减少的与单核细胞的结合、减少的与多形核细胞的结合、减少的直接信号传导诱导性细胞凋亡、降低的树突细胞成熟或减少的T细胞引发。对于IgG1Fc区，在238、265、269、270、297、327和329等位置的氨基酸残基取代可降低的效应子功能。在一些实施方案中，所述Fc区是人IgG1Fc区，并且在234和235位置的氨基酸残基为A，编号依据为EU索引。对于IgG4Fc区，在228等位置的氨基酸残基取代可降低的效应子功能。In one aspect, the Fc region of an antigen-binding molecule of the present disclosure includes one or more amino acid substitutions that reduce its binding to an Fc receptor, e.g., its binding to an Fcγ receptor, and reduce or Eliminate effector functions. The native IgG Fc region, specifically the IgG1 Fc region or the IgG4 Fc region, may cause the antigen-binding molecules of the present disclosure to target cells expressing Fc receptors rather than cells expressing the antigen. The engineered Fc region of the present disclosure exhibits reduced binding affinity for Fc receptors and/or reduced effector function. In some embodiments, the engineered Fc region has a reduced binding affinity for the Fc receptor by more than 50%, 80%, 90%, or 95% compared to the native Fc region. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, such as FcγRI, FcγRIIa, FcγRIIB, FcγRIIIa. In some embodiments, the engineered Fc region also has reduced binding affinity for complement, such as Clq, compared to the native Fc region. In some embodiments, the engineered Fc region has no reduced binding affinity for the neonatal Fc receptor (FcRn) compared to the native Fc region. In some embodiments, the engineered Fc region has reduced effector functions, which may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced Antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced interaction with NK cells binding, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced of T cells. For the IgG 1 Fc region, substitutions of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function. In some embodiments, the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. For the IgG 4 Fc region, amino acid residue substitutions at positions such as 228 may reduce effector function.
抗原结合分子还可包含二硫键改造，例如第一亚基的354C和第二亚基的349C。为增加抗原结合分子的血清半衰期，可以引入252Y、254T和256E的突变。Antigen-binding molecules may also contain disulfide bond modifications, such as 354C in the first subunit and 349C in the second subunit. To increase the serum half-life of antigen-binding molecules, mutations 252Y, 254T, and 256E can be introduced.
当抗原结合分子包含与Fc区的两个亚基融合的不同结合模块，可能导致不期望的同源二聚化。为了提高产率和纯度，因此在本披露的抗原结合分子的Fc区中引入促进异源二聚化的修饰将是有利的。在一些实施方式中，本披露的Fc区包含根据杵臼(knob-into-hole，KIH)技术的改造，该方法涉及在第一亚基的界面处引入凸起结构(knob)以及在第二亚基的界面处引入孔结构(hole)。使得所述凸起结构可以定位在孔结构中，促进异源二聚体的形成并抑制同源二聚体的产生。凸起结构是通过用较大侧链(例如酪氨酸或色氨酸)取代来自第一亚基的界面的小氨基酸侧链而构建的。而孔结构是通过用较小的氨基酸侧链(例如丙氨酸或苏氨酸)取代大氨基酸侧链而在第二亚基的界面中创建的。凸起结构和孔结构通过改变编码多肽的核酸来制备，可选的氨基酸取代如下表所示：When the antigen-binding molecule contains different binding modules fused to the two subunits of the Fc region, undesirable homodimerization may result. To increase yield and purity, it would therefore be advantageous to introduce modifications in the Fc region of the antigen-binding molecules of the present disclosure that promote heterodimerization. In some embodiments, the Fc region of the present disclosure includes modifications according to the knob-into-hole (KIH) technique, which involves introducing a knob at the interface of the first subunit and a knob at the interface of the second subunit. A hole structure (hole) is introduced at the interface of the base. This allows the protruding structure to be positioned in the pore structure, promoting the formation of heterodimers and inhibiting the production of homodimers. The bulge structure is built by replacing small amino acid side chains from the interface of the first subunit with larger side chains, such as tyrosine or tryptophan. The pore structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller amino acid side chains, such as alanine or threonine. The bulge and pore structures are prepared by altering the nucleic acid encoding the polypeptide with optional amino acid substitutions as shown in the table below:
表4.KIH突变组合
Table 4. KIH mutation combinations
除了杵臼技术外，用于修饰重链的CH3结构域以实现异源二聚化的其他技术也是本领域中已知的，例如WO1996027011A1、WO1998050431、EP1870459、WO2007110205、WO2009089004、WO2010129304、WO201190754、WO2011143545、WO2012058768、WO2013157954和WO2013096291。In addition to the pestle and mortar technology, other techniques for modifying the CH3 domain of the heavy chain to achieve heterodimerization are also known in the art, such as WO1996027011A1, WO1998050431, EP1870459, WO2007110205, WO2009089004, WO2010129304, WO201190754, WO2011 143545、WO2012058768 , WO2013157954 and WO2013096291.
Fc区的C末端可以是以氨基酸残基PGK结束的完整C末端；也可以是截短的C末端，例如在所述截短的C末端中去除了一个或两个C末端氨基酸残基。在一个优选的方面中，重链的C末端是以PG结束的缩短的C末端。因此，在一些实施方式中，完整抗体的组合物可以包括去除了所有K447残基和/或G446+K447残基的抗体群体。在一些实施方式中，完整抗体的组合物可以包括没有去除K447残基和/或G446+K447残基的抗体群体。在一些实施方式中，完整抗体的组合物具有带有和不带有K447残基和/或G446+K447残基的抗体混合物的抗体群体。The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; it can also be a truncated C-terminus, for example, one or two C-terminal amino acid residues have been removed from the truncated C-terminus. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending in PG. Thus, in some embodiments, a composition of intact antibodies may include a population of antibodies with all K447 residues and/or G446+K447 residues removed. In some embodiments, the composition of intact antibodies can include a population of antibodies without removal of the K447 residue and/or G446+K447 residues. In some embodiments, the composition of intact antibodies has a population of antibodies with and without a K447 residue and/or an antibody mixture of G446+K447 residues.
重组方法Recombination method
抗原结合分子可以使用重组方法来产生。对于这些方法，提供编码抗原结合分子的一个或更多个分离的核酸。Antigen-binding molecules can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding the antigen-binding molecules are provided.
在天然抗体、天然抗体片段或具有同源二聚体重链的双特异性抗体的情况下，需要两个核酸，一个用于轻链或其片段，一个用于重链或其片段。此类核酸编码包含抗体VL的氨基酸序列和/或包含抗体VH的氨基酸序列(例如抗体的轻链和/或重链)。这些核酸可以在相同的表达载体上或在不同的表达载体上。In the case of native antibodies, native antibody fragments or bispecific antibodies with homodimeric heavy chains, two nucleic acids are required, one for the light chain or fragments thereof and one for the heavy chain or fragments thereof. Such nucleic acids encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (eg, the light chain and/or heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.
在具有异二聚体重链的双特异性抗体的情况下，需要例如四个核酸，一个用于第一轻链，一个用于包含第一异源单体Fc区多肽的第一重链，一个用于第二轻链，并且一个用于包含第二异源单体Fc区多肽的第二重链。这四个核酸可包含在一个或更多个核酸分子或表达载体中，通常这些核酸位于两个或三个表达载体上，即一个载体可包含这些核酸中的多于一个。In the case of a bispecific antibody with a heterodimeric heavy chain, for example four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heterologous monomeric Fc region polypeptide, and one one for the second light chain, and one for the second heavy chain comprising a second heterologous monomeric Fc region polypeptide. These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors, usually these nucleic acids are located on two or three expression vectors, that is, one vector can contain more than one of these nucleic acids.
在一个实施方案中，本披露提供了编码如前所述的抗体的分离的核酸。此类核酸可以独立地编码前述的任一多肽链。在另一方面中，本披露提供了包含此类核酸的一种或多种载体(例如表达载体)。在另一方面中，本披露提供了包含此类核酸的宿主细胞。在一个实施方案中，提供制备抗原结合分子的方法，其中所述方法包括，在适合抗体表达的条件下，培养包含编码所述抗体的核酸的宿主细胞，如上文所提供的，和任选地从宿主细胞(或宿主细胞培养基)回收所述抗体。In one embodiment, the present disclosure provides an isolated nucleic acid encoding an antibody as described above. Such nucleic acids can independently encode any of the aforementioned polypeptide chains. In another aspect, the present disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids. In another aspect, the present disclosure provides host cells comprising such nucleic acids. In one embodiment, a method of preparing an antigen-binding molecule is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally The antibody is recovered from the host cell (or host cell culture medium).
为了重组产生抗原结合分子，将编码蛋白的核酸分离并插入一个或更多个载体中，用于在宿主细胞中进一步克隆和/或表达。此类核酸可以使用常规程序容易 地分离和测序(例如通过使用能够与编码抗体重链和轻链的基因特异性结合的寡核苷酸探针)，或者通过重组方法产生或通过化学合成获得。To recombinantly produce an antigen-binding molecule, the nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be easily can be isolated and sequenced (for example, by using oligonucleotide probes capable of binding specifically to the genes encoding the antibody heavy and light chains), or produced by recombinant methods or obtained by chemical synthesis.
用于克隆或表达编码抗体的载体的适当宿主细胞包括本文描述的原核或真核细胞。例如，抗体可在细菌中产生，特别是当抗体不需要糖基化和Fc效应子功能时。在表达后，抗体可以在可溶级分中从细菌细胞糊状物分离，并且可进一步纯化。Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required for the antibody. After expression, the antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
除了原核生物以外，真核微生物诸如丝状真菌或酵母也是用于编码抗体的载体的合适的克隆或表达宿主，包括真菌和酵母菌株，其糖基化途径已经“人源化”，导致产生具有部分或完全人糖基化模式的抗体。适于表达(糖基化)抗体的合适的宿主细胞也可源自多细胞生物体(无脊椎动物和脊椎动物)；无脊椎动物细胞的例子包括植物和昆虫细胞。已经鉴定了许多杆状病毒株，其可与昆虫细胞联合使用，特别是用于草地贪夜蛾(Spodoptera frugiperda)细胞的转染；还可利用植物细胞培养物作为宿主，例如US5959177、US 6040498、US6420548、US 7125978和US6417429；也可将脊椎动物细胞用作宿主，例如适应于在悬浮液中生长的哺乳动物细胞系。适宜的哺乳动物宿主细胞系的其它例子是经SV40转化的猴肾CVl系(COS-7)；人胚肾系(293或293T细胞)；幼仓鼠肾细胞(BHK)；小鼠塞托利(sertoli)细胞(TM4细胞)；猴肾细胞(CV1)；非洲绿猴肾细胞(VERO-76)；人宫颈癌细胞(HELA)；犬肾细胞(MDCK)；水牛鼠(buffalo rat)肝细胞(BRL3A)；人肺细胞(W138)；人肝细胞(Hep G2)；小鼠乳房肿瘤(MMT 060562)；TRI细胞；MRC 5细胞；和FS4细胞。其它适宜的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞；以及骨髓瘤细胞系，如Y0、NS0和Sp2/0。关于适合产生抗体的某些哺乳动物宿主细胞系的综述参见例如Yazaki，P.和Wu，A.M.，Methods in Molecular Biology，Vol.248，Lo，B.K.C.(编)，Humana Press，Totowa，NJ(2004)，第255-268页。In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of vectors with Antibodies with partially or fully human glycosylation patterns. Suitable host cells for expression of (glycosylated) antibodies may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in combination with insect cells, especially for transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, such as US5959177, US 6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, such as mammalian cell lines adapted to growth in suspension. Other examples of suitable mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); mouse Sertoli ( sertoli) cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat (buffalo rat) liver cells ( BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse breast tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as Y0, NSO, and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production see, for example, Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (Eds.), Humana Press, Totowa, NJ (2004) , pp. 255-268.
诊断与治疗组合物Diagnostic and therapeutic compositions
在某些实施方案中，本披露提供的抗原结合分子可用于检测生物学样品中PSMA和/或CD3的存在。在用于本文时，术语“检测”涵盖定量或定性检测。在某些实施方案中，生物学样品包含细胞或组织，诸如肿瘤组织。In certain embodiments, the present disclosure provides antigen-binding molecules that can be used to detect the presence of PSMA and/or CD3 in a biological sample. As used herein, the term "detection" encompasses either quantitative or qualitative detection. In certain embodiments, a biological sample includes cells or tissue, such as tumor tissue.
在一个实施方案中，提供了在诊断或检测方法中使用的抗原结合分子。在又一方面，提供了检测生物学样品中PSMA和/或CD3的存在的方法。在某些实施方案中，该方法包括在适宜条件下使生物学样品与抗原结合分子接触，并检测是否在检测试剂与抗原之间形成复合物。此类方法可以是体外或体内方法。在一个实施方案中，使用抗原结合分子来选择适合治疗的受试者，例如PSMA和/或CD3是用于选择患者的生物标志物。In one embodiment, antigen binding molecules for use in diagnostic or detection methods are provided. In yet another aspect, a method of detecting the presence of PSMA and/or CD3 in a biological sample is provided. In certain embodiments, the method includes contacting a biological sample with an antigen-binding molecule under appropriate conditions and detecting whether a complex is formed between the detection reagent and the antigen. Such methods may be in vitro or in vivo methods. In one embodiment, antigen binding molecules are used to select subjects suitable for treatment, for example PSMA and/or CD3 are biomarkers used to select patients.
可使用本披露的抗原结合分子来诊断的例示性病症，例如肿瘤或癌症。Exemplary conditions that can be diagnosed using the antigen-binding molecules of the present disclosure are, for example, tumors or cancers.
在某些实施方案中，提供了经标记的抗原结合分子。标记物包括但不限于直 接检测的标记物或模块(诸如荧光、发色、电子致密、化学发光、和放射性标记物)，和间接检测的模块(例如，经由酶反应或分子相互作用间接检测的模块，诸如酶或配体)。In certain embodiments, labeled antigen binding molecules are provided. Markers include, but are not limited to, direct Labels or moieties that detect directly (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties that detect indirectly (e.g., moieties that detect indirectly via enzymatic reactions or molecular interactions, such as enzymes or ligands) body).
在另外的方面，提供包含所述抗原结合分子的药物组合物，例如，用于以下任何治疗方法。在一个方面，药物组合物包含本文提供的任何抗原结合分子和药学上可接受的载体。在另一个方面，药物组合物包含本文提供的任何抗原结合分子和至少一种另外的治疗剂。In a further aspect, pharmaceutical compositions comprising the antigen-binding molecules are provided, eg, for use in any of the following methods of treatment. In one aspect, a pharmaceutical composition includes any of the antigen-binding molecules provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition includes any of the antigen-binding molecules provided herein and at least one additional therapeutic agent.
本披露所述的抗原结合分子的药物组合物通过以下制备：将具有所需纯度的此类抗原结合分子与一种或更多种任选的药学上可接受的载体混合，所述药物组合物为冻干组合物或水溶液的形式。用于体内施用的制剂一般是无菌的。无菌性可容易地实现，例如通过穿过无菌滤膜过滤。Pharmaceutical compositions of antigen-binding molecules of the present disclosure are prepared by mixing such antigen-binding molecules with the desired purity with one or more optional pharmaceutically acceptable carriers, the pharmaceutical compositions In the form of lyophilized compositions or aqueous solutions. Formulations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile membrane.
治疗方法与施用途径Treatment methods and routes of administration
本文提供的任何抗原结合分子可用于治疗方法。Any of the antigen-binding molecules provided herein can be used in therapeutic methods.
在又一个方面，本披露提供抗原结合分子在药物的制造或制备中的用途。在一个实施方案中，所述药物用于治疗增殖性疾病或肿瘤。并且所述药物是以对上述疾病的有效量的形式存在的。在一些实施方式中，所述有效量是单位日剂量或单位周剂量。在一个此类实施方案中，所述用途进一步包括向受试者施用有效量的至少一种另外的治疗剂(例如一种、两种、三种、四种、五种或六种另外的治疗剂)。根据任意以上实施方案的“受试者”可以是人。In yet another aspect, the present disclosure provides use of an antigen-binding molecule in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat proliferative diseases or tumors. And the drug is present in an effective amount for the above-mentioned diseases. In some embodiments, the effective amount is a unit daily dose or a unit weekly dose. In one such embodiment, the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional treatments). agent). A "subject" according to any of the above embodiments may be a human.
在又一个的方面，提供包含所述抗原结合分子的药物组合物，例如，其用于以上任何制药用途或治疗方法。在另一个实施方案中，药物组合物还包含至少一种另外的治疗剂。In yet another aspect, there is provided a pharmaceutical composition comprising the antigen-binding molecule, eg, for use in any of the above pharmaceutical uses or methods of treatment. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.
本披露的抗原结合分子可单独使用或与其他试剂联合用于治疗。例如，本披露的抗原结合分子可与至少一种另外的治疗剂共同施用。在一些实施方案中，所述的另外的治疗剂是特异性结合PSMA和CD3的双特异性抗体或抗PD-1抗体。The antigen-binding molecules of the present disclosure can be used alone or in combination with other agents for treatment. For example, the antigen-binding molecules of the present disclosure can be co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is a bispecific antibody or an anti-PD-1 antibody that specifically binds PSMA and CD3.
本披露的抗原结合分子(和任何另外的治疗剂)可通过任何合适的手段施用，包括肠胃外、肺内和鼻内，并且如果需要局部治疗，则病灶内施用。肠胃外输注包括肌肉内、静脉内、动脉内、腹膜内或皮下施用。给药可以通过任何适当的途径，例如，通过注射，诸如静脉内或皮下注射，这部分取决于施用是短期的还是长期的。本文考虑多种给药时间方案，包括但不限于，单次或在多个时间点多次施用，推注施用和脉冲输注。The antigen-binding molecules of the present disclosure (and any additional therapeutic agents) may be administered by any suitable means, including parenterally, intrapulmonary, and intranasal, and, if local treatment is desired, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.
本披露的抗原结合分子将以符合良好医疗实践(GOOD MEDICAL PRACTICE)的方式配制、给药和施用。在此背景下考虑的因素包括所治疗的具体病症、所治疗的具体哺乳动物、个体患者的临床状况、病症的起因、试剂的递送部位、施用方法、施用时间安排以及医学从业者已知的其他因素。抗原结合分子无需但任选地与目前用于预防或治疗所述病症的一种或更多种试剂一起配制。此类其它试剂 的有效量取决于药物组合物中存在的抗原结合分子的量、病症或治疗的类型以及上文讨论的其它因素。这些通常以与本文所述相同的剂量和施用路径使用，或以本文所述剂量的约1至99％使用，或以任何剂量使用，并通过经验/临床确定为合适的任何途径使用。The antigen-binding molecules of the present disclosure will be formulated, administered, and administered in a manner consistent with GOOD MEDICAL PRACTICE. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and others known to the medical practitioner factor. The antigen-binding molecules need not be, but are optionally, formulated with one or more agents currently used to prevent or treat the disorder. Such other reagents The effective amount depends on the amount of antigen-binding molecule present in the pharmaceutical composition, the type of condition or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and by any route empirically/clinically determined to be appropriate.
为了预防或治疗疾病，本披露的抗原结合分子(当单独使用或与一种或更多种其他另外的治疗剂组合使用时)的适当的剂量将取决于待治疗的疾病的类型，治疗分子的类型，疾病的严重性和病程，是为预防还是治疗目的施用，之前的治疗，患者的临床病史和对治疗分子的响应，和主治医师的判断。治疗分子恰当地以一次或经过一系列治疗施用于患者。取决于疾病的类型和严重性，约1μg/kg至15mg/kg的抗原结合分子可以是用于施用至患者的初始候选剂量，不管例如是通过一次或更多次分开的施用还是通过连续输注。一种典型的每日剂量可能在约1μg/kg至100mg/kg或更多的范围内，这取决于上文提及的因素。相应的，以50kg体重为例，示例性的单位日剂量为50μg-5g。To prevent or treat disease, appropriate dosages of the antigen-binding molecules of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the nature of the therapeutic molecule Type, severity and duration of disease, whether administration is for prophylactic or therapeutic purposes, previous treatments, patient's clinical history and response to therapeutic molecules, and the judgment of the attending physician. The therapeutic molecules are appropriately administered to the patient in one session or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg of the antigen-binding molecule may be an initial candidate dose for administration to the patient, whether, for example, by one or more divided administrations or by continuous infusion . A typical daily dose may range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. Correspondingly, taking a body weight of 50kg as an example, the exemplary unit daily dose is 50μg-5g.
制品Products
在本披露的另一方面中，提供一种制品，所述制品包含可用于治疗、预防和/或诊断上述病症的材料。该制品包含容器和在容器上或与容器联合的标签或包装插页(package insert)。合适的容器包括，例如，瓶子、管形瓶、注射器、IV溶液袋等。容器可以自各种材料诸如玻璃或塑料形成。容器装有单独或与另一种组合物组合有效治疗，预防和/或诊断疾患的组合物，并且可具有无菌的存取口(例如，容器可以是具有由皮下注射针可刺穿的塞子的静脉内溶液袋或管形瓶)。组合物中的至少一种活性试剂是本披露的抗原结合分子。标签或包装插页指示使用该组合物是来治疗选择的病况。此外，制品可以包含：(a)其中装有组合物的第一容器，其中所述组合物包含本披露的抗原结合分子；和(b)其中装有组合物的第二容器，其中所述组合物包含另外的细胞毒性剂或其他方面的治疗剂。本披露的该实施方案中的制品可进一步包含包装插页，所述包装插页指示所述组合物可以用于治疗特定病况。备选地，或另外地，制品可进一步包含第二(或第三)容器，所述第二(或第三)容器包含药学上可接受的缓冲液。从商业和用户立场，它可进一步包括所需的其他材料，包括其他缓冲剂、稀释剂、滤器、针头和注射器。作为一个示例，制品制备成药盒(kit)的形式。In another aspect of the present disclosure, an article of manufacture is provided that contains materials useful in treating, preventing, and/or diagnosing the conditions described above. The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. Containers can be formed from a variety of materials such as glass or plastic. A container containing a composition effective to treat, prevent and/or diagnose a condition, alone or in combination with another composition, and may have a sterile access port (e.g., the container may be a container with a stopper pierceable by a hypodermic needle bag or vial of intravenous solution). At least one active agent in the composition is an antigen-binding molecule of the present disclosure. The label or package insert indicates use of the composition to treat the selected condition. Additionally, an article of manufacture may comprise: (a) a first container having a composition therein, wherein the composition comprises an antigen-binding molecule of the present disclosure; and (b) a second container having a composition therein, wherein the composition The agent contains additional cytotoxic agents or other therapeutic agents. The article of manufacture in this embodiment of the present disclosure may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer. It may further include other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes. As an example, the article of manufacture is prepared in the form of a kit.
实施例与测试例Examples and test cases
以下结合实施例和测试例进一步描述本披露，但这些实施例和测试例并非限制着本披露的范围。本披露实施例和测试例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。 The disclosure is further described below in conjunction with examples and test examples, but these examples and test examples do not limit the scope of the disclosure. Experimental methods without specifying specific conditions in the examples and test examples of this disclosure usually follow conventional conditions, such as Cold Spring Harbor's Antibody Technology Experiment Manual, Molecular Cloning Manual; or conditions recommended by raw material or product manufacturers. Reagents whose specific sources are not indicated are conventional reagents purchased in the market.
实施例1.含有Titin链/Obscurin链的抗原结合分子Example 1. Antigen-binding molecules containing Titin chain/Obscurin chain
本披露的Titin链/Obscurin链可以来源于任意适宜的多肽，包括来源于WO2021139758(通过援引完整收入本文)和CN202110527339.7及将其作为优先权文件的专利(通过援引完整收入本文)中的多肽。构建双特异性抗体，其中CL为WO2021139758中的kappa轻链恒定区(序列参见说明书第[0377]段)，Titin链和Obscurin链的氨基酸序列见表3-1和表3-2，连接子序列包括GGGGS(SEQ ID NO：223)、ASTKG(SEQ ID NO：397)或RTVAS(SEQ ID NO：398)，本实施例中的Fc1、Fc2、CH1的氨基酸序列如下所示。The Titin chain/Obscurin chain of the present disclosure can be derived from any suitable polypeptide, including polypeptides derived from WO2021139758 (incorporated herein by reference in its entirety) and CN202110527339.7 and the patents (incorporated herein by reference in their entirety) which are priority documents. . Construct a bispecific antibody, in which CL is the kappa light chain constant region in WO2021139758 (for the sequence, see paragraph [0377] of the description), the amino acid sequences of the Titin chain and Obscurin chain are shown in Table 3-1 and Table 3-2, and the linker sequence Including GGGGS (SEQ ID NO: 223), ASTKG (SEQ ID NO: 397) or RTVAS (SEQ ID NO: 398), the amino acid sequences of Fc1, Fc2, and CH1 in this example are as follows.
>实施例1-Fc1(knob，SEQ ID NO：394)
>Example 1-Fc1 (knob, SEQ ID NO: 394)
>实施例1-Fc2(hole，SEQ ID NO：222)
>Example 1-Fc2 (hole, SEQ ID NO: 222)
>实施例1-CH1(SEQ ID NO：219)>Example 1-CH1 (SEQ ID NO: 219)
1.1 DI双特异性抗体1.1 DI bispecific antibodies
参照WO2021139758的实施例5，构建抗hNGF和hRANKL的DI双特异性抗体：DI-2至DI-20，其包含如下所述的第一重链、第二重链、第一轻链和第二轻链：Referring to Example 5 of WO2021139758, DI bispecific antibodies against hNGF and hRANKL are constructed: DI-2 to DI-20, which include the first heavy chain, the second heavy chain, the first light chain and the second Light chain:
第一重链：从N端到C端依次为：[VH1-I]-[连接子1]-[Obscurin链]-[Fc2]，The first heavy chain: from N-terminus to C-terminus is: [VH1-I]-[linker 1]-[Obscurin chain]-[Fc2],
第一轻链：从N端到C端依次为：[VL1-I]-[连接子2]-[Titin链]，The first light chain: from N-terminal to C-terminal: [VL1-I]-[Linker 2]-[Titin chain],
第二重链：从N端到C端依次为：[VH2-D]-[CH1]-[Fc1]，和Second heavy chain: from N-terminus to C-terminus: [VH2-D]-[CH1]-[Fc1], and
第二轻链：从N端到C端依次为：[VL2-D]-[CL]；Second light chain: from N-terminus to C-terminus: [VL2-D]-[CL];
其中，VH1-I和VL1-I分别为WO2021139758中I0的重链可变区和轻链可变区，VH2-D和VL2-D分别为WO2021139758中D0的重链可变区和轻链可变区。本实施例中DI双特异性抗体中Obscurin链、Titin链、连接子1、连接子2结构见下表。Among them, VH1-I and VL1-I are respectively the heavy chain variable region and light chain variable region of I0 in WO2021139758, and VH2-D and VL2-D are respectively the heavy chain variable region and light chain variable region of D0 in WO2021139758. district. In this example, the structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the DI bispecific antibody are shown in the table below.
表5.DI双特异性抗体中Obscurin链/Titin链和连接子对应表

Table 5. Correspondence table of Obscurin chain/Titin chain and linker in DI bispecific antibodies

注：表格中Titin链和Obscurin链的编号见表3-1和表3-2。Note: The numbers of Titin chain and Obscurin chain in the table are shown in Table 3-1 and Table 3-2.
采用WO2021139758的测试例4中的方法检测DI-2至DI-20双特异性抗体与其抗原的结合活性。对抗体进行热稳定性研究。研究方法：用PBS将抗体的浓度稀释至5mg/mL，采用高通量微分扫描荧光仪(UNCHAINED，规格型号：Unit)测定其热稳定性。实验结果表明，改造后的双特异性抗体对抗原的结合活性没有显著变化；并且，与DI-2相比，DI-4至DI-8、DI-10至DI-16、DI-20的Tm1(℃)、Tonset(℃)有明显的提升，双特异性抗体的热稳定性更优。The method in Test Example 4 of WO2021139758 was used to detect the binding activity of the DI-2 to DI-20 bispecific antibodies and their antigens. Thermal stability studies of antibodies were performed. Research method: Use PBS to dilute the concentration of the antibody to 5 mg/mL, and use a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit) to measure its thermal stability. The experimental results showed that the antigen-binding activity of the modified bispecific antibody did not change significantly; and, compared with DI-2, the Tm1 of DI-4 to DI-8, DI-10 to DI-16, and DI-20 (℃) and Tonset (℃) are significantly improved, and the thermal stability of bispecific antibodies is better.
表6.DI双特异性抗体的结合活性检测
Table 6. Binding activity detection of DI bispecific antibodies
表7.DI双特异性抗体的热稳定性实验结果
Table 7. Thermal stability experimental results of DI bispecific antibodies
采用10mM乙酸，pH5.5，9％蔗糖的缓冲液配制含DI双特异性抗体的溶液，将溶液置于40℃恒温箱中孵育四周，结束后将抗体浓度浓缩至孵育开始时浓度，观察溶液沉淀情况。实验结果表明，DI-2双特异性抗体组溶液出现沉淀，DI-3至DI-7相比DI-2具有更好的稳定性。Use 10mM acetic acid, pH 5.5, and 9% sucrose buffer to prepare a solution containing DI bispecific antibodies. Place the solution in a 40°C incubator and incubate it for four weeks. After completion, concentrate the antibody concentration to the concentration at the beginning of the incubation and observe the solution. sedimentation conditions. Experimental results show that the solution of the DI-2 bispecific antibody group precipitates, and DI-3 to DI-7 have better stability than DI-2.
表8.DI双特异性抗体的沉淀
Table 8. Precipitation of DI bispecific antibodies
1.2 PL双特异性抗体1.2 PL bispecific antibodies
构建抗hPDL1和hCTLA4的PL双特异性抗体：PL-1至PL-19，其包含如下所述的第一重链、第二重链、第一轻链和第二轻链：PL bispecific antibodies against hPDL1 and hCTLA4 were constructed: PL-1 to PL-19, which comprise the first heavy chain, the second heavy chain, the first light chain and the second light chain as follows:
第一重链：从N端到C端依次为：[VH1-P]-[连接子1]-[Obscurin链]-[Fc1]，The first heavy chain: from N end to C end: [VH1-P]-[Linker 1]-[Obscurin chain]-[Fc1],
第一轻链：从N端到C端依次为：[VL1-P]-[连接子2]-[Titin链]，The first light chain: from N-terminal to C-terminal: [VL1-P]-[Linker 2]-[Titin chain],
第二重链：从N端到C端依次为：[VH2-L]-[CH1]-[Fc2]，和Second heavy chain: from N-terminus to C-terminus: [VH2-L]-[CH1]-[Fc2], and
第二轻链：从N端到C端依次为：[VL2-L]-[CL]；Second light chain: from N-terminus to C-terminus: [VL2-L]-[CL];
其中，VH1-P和VL1-P分别为WO2020177733A1中h1831K抗体的重链可变区和轻链可变区，VH2-L和VL2-L的氨基酸序列如下所示。

Among them, VH1-P and VL1-P are the heavy chain variable region and light chain variable region of the h1831K antibody in WO2020177733A1, respectively. The amino acid sequences of VH2-L and VL2-L are as follows.

本实施例中PL双特异性抗体中Obscurin链、Titin链、连接子1、连接子2结构见下表。The structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the PL bispecific antibody in this example are shown in the table below.
表9.PL双特异性抗体中Obscurin链/Titin链和连接子对应表
Table 9. Correspondence table of Obscurin chain/Titin chain and linker in PL bispecific antibodies
注：表格中Titin链和Obscurin链的编号见表3-1和表3-2。Note: The numbers of Titin chain and Obscurin chain in the table are shown in Table 3-1 and Table 3-2.
参照WO2021139758中测试例4中的ELISA方法检测PL双特异性抗体的结合活性，其中hPDL1、hCTLA4抗原购自：Sino biology。对抗体进行热稳定性研究。方法：用PBS将抗体的浓度稀释至1.4-3mg/mL，采用高通量微分扫描荧光仪(UNCHAINED，规格型号：Unit)测定其热稳定性。实验结果表明，PL双特异性抗体对抗原仍具有良好的结合活性；并且，与PL-1相比，PL-2至PL-19的Tm1(℃)、Tagg 266(℃)、Tonset(℃)有明显的提升，双特异性抗体的热稳定性更优。The binding activity of the PL bispecific antibody was detected with reference to the ELISA method in Test Example 4 of WO2021139758, in which hPDL1 and hCTLA4 antigens were purchased from: Sino biology. Thermal stability studies of antibodies were performed. Method: Use PBS to dilute the concentration of the antibody to 1.4-3mg/mL, and use a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit) to measure its thermal stability. The experimental results show that the PL bispecific antibody still has good binding activity to the antigen; and, compared with PL-1, the Tm1 (℃), Tagg 266 (℃), Tonset (℃) of PL-2 to PL-19 There is a significant improvement, and the thermal stability of bispecific antibodies is better.
表10.PL双特异性抗体的结合活性检测

Table 10. Binding activity detection of PL bispecific antibodies

表11.PL双特异性抗体的热稳定性实验结果
Table 11. Thermal stability experimental results of PL bispecific antibodies
1.3 HJ双特异性抗体1.3 HJ bispecific antibodies
构建抗hIL5和hTSLP的HJ双特异性抗体：HJ-3至HJ11，其包含如下所述的第一重链、第二重链、第一轻链和第二轻链：HJ bispecific antibodies against hIL5 and hTSLP were constructed: HJ-3 to HJ11, which comprise the first heavy chain, the second heavy chain, the first light chain and the second light chain as follows:
第一重链：从N端到C端依次为：[VH1-H]-[连接子1]-[Titin链]-[Fc1]，The first heavy chain: from N-terminal to C-terminal is: [VH1-H]-[Linker 1]-[Titin chain]-[Fc1],
第一轻链：从N端到C端依次为：[VL1-H]-[连接子2]-[Obscurin链]，The first light chain: from N end to C end: [VL1-H]-[Linker 2]-[Obscurin chain],
第二重链：从N端到C端依次为：[VH2-J]-[CH1]-[Fc2]，和Second heavy chain: from N terminus to C terminus: [VH2-J]-[CH1]-[Fc2], and
第二轻链：从N端到C端依次为：[VL2-J]-[CL]；Second light chain: from N-terminus to C-terminus: [VL2-J]-[CL];
其中，VH1-H和VL1-H分别为WO2021139758中H0的重链可变区和轻链可变区，VH2-J和VL2-J分别为WO2021139758中J1的重链可变区和轻链可变区。本实施例中HJ双特异性抗体中Obscurin链、Titin链、连接子1、连接子2结构见下表。Among them, VH1-H and VL1-H are respectively the heavy chain variable region and light chain variable region of H0 in WO2021139758, and VH2-J and VL2-J are respectively the heavy chain variable region and light chain variable region of J1 in WO2021139758. district. The structures of the Obscurin chain, Titin chain, linker 1, and linker 2 in the HJ bispecific antibody in this example are shown in the table below.
表12.HJ双特异性抗体中Obscurin链/Titin链和连接子对应表

Table 12. Correspondence table of Obscurin chain/Titin chain and linker in HJ bispecific antibody

参照WO2021139758中测试例4中的方法检测HJ双特异性抗体的抗原结合活性。对抗体的热稳定性进行研究，方法：用10mM乙酸pH5.5、9％蔗糖的缓冲液配制HJ双特异性抗体稀释溶液，然后通过超滤浓缩的方法将双特异性抗体浓缩，获得不同浓度的HJ双特异性抗体溶液(HJ双特异性抗体的浓度见表13-2)，然后将浓缩溶液置于40℃恒温箱中孵育，第0天(也即40℃孵育开始前，D0)，第7天(40℃孵育第7天，D7)，第14天(40℃孵育第14天，D14)，第21天(40℃孵育第21天，D21)和第28天(40℃孵育第28天，D28)检测样品的SEC纯度，40℃孵育28天后，马上取样检测样品CE-SDS纯度。实验结果表明，本披露构建的HJ双特异性抗体对抗原的结合活性没有显著变化；并且，与HJ-3相比，HJ-5至HJ-11双特异性抗体的热稳定性更优。The antigen-binding activity of the HJ bispecific antibody was detected with reference to the method in Test Example 4 in WO2021139758. To study the thermal stability of the antibody, the method is: prepare the HJ bispecific antibody dilution solution with a buffer solution of 10mM acetic acid pH 5.5 and 9% sucrose, and then concentrate the bispecific antibody through ultrafiltration concentration to obtain different concentrations. HJ bispecific antibody solution (see Table 13-2 for the concentration of HJ bispecific antibody), and then place the concentrated solution in a 40°C incubator for incubation. On day 0 (that is, before the 40°C incubation starts, D0), Day 7 (day 7 of incubation at 40°C, D7), day 14 (day 14 of incubation at 40°C, D14), day 21 (day 21 of incubation at 40°C, D21) and day 28 (day 21 of incubation at 40°C, D21) 28 days (D28)) Test the SEC purity of the sample. After incubation at 40°C for 28 days, take a sample immediately to test the CE-SDS purity of the sample. Experimental results show that the antigen-binding activity of the HJ bispecific antibodies constructed in the present disclosure does not change significantly; and, compared with HJ-3, the thermal stability of the HJ-5 to HJ-11 bispecific antibodies is better.
表13-1.HJ双特异性抗体的结合活性检测
Table 13-1. Binding activity detection of HJ bispecific antibodies
表13-2.HJ双特异性抗体加速稳定性实验结果
Table 13-2. HJ bispecific antibody accelerated stability test results
实施例2.抗人CD28杂交瘤抗体的筛选和鉴定 Example 2. Screening and identification of anti-human CD28 hybridoma antibodies
2.1细胞株构建2.1 Cell line construction
构建CHO-hPSMA,CHO-APC-hPSMA，CHO-hCD28,HEK293-hCD28，CHO-APC-hPSMA-PDL1和Jurkat-PD1细胞株，相关蛋白序列如下：

Construct CHO-hPSMA, CHO-APC-hPSMA, CHO-hCD28, HEK293-hCD28, CHO-APC-hPSMA-PDL1 and Jurkat-PD1 cell lines. The relevant protein sequences are as follows:

将编码人PSMA或人CD28全长基因克隆到哺乳动物细胞表达载体pCDH上，用pVSV-G、pCMV-dR8.91和pCDH-hPSMA三种质粒共同转染HEK293T细胞(ATCC,CRL-11268)包装病毒。转染48小时后，收集病毒感染HEK293(ATCC,CRL-1573)，CHO-K1(ATCC,CCL-61)或CHO-APC细胞(Promega,JA9441)，通过加压筛选两周后，进行细胞亚克隆，经过FACS检测获得高表达PSMA或CD28的CHO-hPSMA,CHO-APC-hPSMA，CHO-hCD28和HEK293-hCD28细胞株。Clone the full-length gene encoding human PSMA or human CD28 into the mammalian cell expression vector pCDH, and co-transfect HEK293T cells (ATCC, CRL-11268) with three plasmids: pVSV-G, pCMV-dR8.91 and pCDH-hPSMA for packaging. Virus. 48 hours after transfection, virus-infected HEK293 (ATCC, CRL-1573), CHO-K1 (ATCC, CCL-61) or CHO-APC cells (Promega, JA9441) were collected. After two weeks of pressure screening, the cells were subdivided. After cloning, CHO-hPSMA, CHO-APC-hPSMA, CHO-hCD28 and HEK293-hCD28 cell lines with high expression of PSMA or CD28 were obtained through FACS detection.
将编码人PDL1或人PD1全长基因克隆到哺乳动物细胞表达载体pCDH上，用pVSV-G、pCMV-dR8.91和pCDH-hPSMA三种质粒共同转染HEK293T细胞(CRL-11268)包装病毒，转染48小时后，收集病毒感染前文所述CHO-APC-hPSMA或Jurkat(ATCC,TIB-152)细胞，通过加压筛选，进行细胞分选获得高表达PDL1或PD1的CHO-APC-hPSMA-PDL1和Jurkat-PD1的细胞株。The gene encoding human PDL1 or human PD1 full-length was cloned into the mammalian cell expression vector pCDH, and HEK293T cells were co-transfected with three plasmids: pVSV-G, pCMV-dR8.91 and pCDH-hPSMA ( CRL-11268) packaged virus, 48 hours after transfection, collect the virus to infect CHO-APC-hPSMA or Jurkat (ATCC, TIB-152) cells as mentioned above, and perform cell sorting through pressure screening to obtain high expression of PDL1 or PD1 CHO-APC-hPSMA-PDL1 and Jurkat-PD1 cell lines.
2.2抗CD28杂交瘤抗体的筛选和鉴定2.2 Screening and identification of anti-CD28 hybridoma antibodies
本披露通过杂交瘤技术制备了针对人CD28的单克隆抗体，所得抗体以较高的亲和力与人CD28特异性结合，并且可以与食蟹猴CD28有交叉结合，与细胞表面的人CD28和食蟹猴CD28均有较好的结合活性。This disclosure uses hybridoma technology to prepare monoclonal antibodies against human CD28. The resulting antibodies specifically bind to human CD28 with higher affinity, and can cross-bind with cynomolgus CD28 and bind to human CD28 and cynomolgus monkeys on the cell surface. CD28 has good binding activity.
以人CD28-his,人CD28-Fc,CHO-hCD28细胞或HEK293-hCD28细胞为免疫原。蛋白初次免疫50μg，加强免疫每次25μg，以Gold Adjuvant(Sigma Cat No.T2684)与ThermoAlum(Thermo Cat No.77161)为佐剂交叉免疫。细胞免疫按照每次5E6进行免疫。经过初次免疫和7次加强免疫后选择血清中抗体滴度高小鼠进行脾细胞融合。免疫原序列如下：

Use human CD28-his, human CD28-Fc, CHO-hCD28 cells or HEK293-hCD28 cells as immunogens. The initial immunization with protein is 50 μg, and the booster immunization is 25 μg each time. Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No. 77161) is an adjuvant for cross-immunization. Cellular immunity was immunized according to 5E6 each time. After the initial immunization and seven boosting immunizations, mice with high serum antibody titers were selected for spleen cell fusion. The immunogen sequence is as follows:

融合后根据杂交瘤细胞生长密度，对杂交瘤培养上清进行检测。筛选出和细胞表面的人CD28有较好的结合活性，且具有良好激活功能的杂交瘤。收集单克隆杂交瘤细胞，用NucleoZol(MN)提取RNA(按照试剂盒说明书步骤)，并进行反转录(PrimeScriptTM Reverse Transcriptase，Takara,cat#2680A)。将反转录得到的cDNA采用小鼠Ig-Primer Set(Novagen，TB326Rev.B 0503)进行PCR扩增后测序，获得杂交瘤细胞株中抗体的的CDR和可变区的氨基酸序列。After fusion, the hybridoma culture supernatant was detected according to the hybridoma cell growth density. Hybridomas with good binding activity to human CD28 on the cell surface and good activation function were screened out. Monoclonal hybridoma cells were collected, RNA was extracted with NucleoZol (MN) (according to the kit instructions), and reverse transcription was performed (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and then sequenced to obtain the amino acid sequence of the CDR and variable region of the antibody in the hybridoma cell line.
表14.抗CD28抗体的CDR



注：下划线部分为根据Kabat编号规则获得的CDR区。Table 14. CDRs of anti-CD28 antibodies



Note: The underlined part is the CDR area obtained according to Kabat numbering rules.
鼠源抗CD28抗体的可变区序列分别与SEQ ID NO：144和SEQ ID NO：145所示的重链和轻链恒定区组合获得嵌合抗体。示例性的，Chi81表示包含m81鼠源重链可变区、轻链可变区和所述SEQ ID NO：144和SEQ ID NO：145所示的重链和轻链恒定区的嵌合抗体。Chi94、Chi97和Chi129依此类推。The variable region sequence of the mouse anti-CD28 antibody was combined with the heavy chain and light chain constant regions shown in SEQ ID NO: 144 and SEQ ID NO: 145 to obtain a chimeric antibody. Exemplarily, Chi81 represents a chimeric antibody comprising the m81 murine heavy chain variable region, the light chain variable region, and the heavy chain and light chain constant regions shown in SEQ ID NO: 144 and SEQ ID NO: 145. Chi94, Chi97 and Chi129 and so on.
实施例3.抗CD28单克隆抗体的人源化Example 3. Humanization of anti-CD28 monoclonal antibodies
鼠源抗体的人源化根据本领域许多文献公示的方法进行。简言之，在所获得 的鼠源抗体VH/VL CDR典型结构的基础上，从人源种系数据库中搜索轻链可变区(VL)和重链可变区(VH)的同源序列，按FR的同源性由高到低排序，选取FR同源性最高的种系作为模板，将鼠源抗体的CDR区移植到人源模板上，对可变区的某些氨基酸突变，并将鼠源抗体的恒定区替换为人恒定区，得到最终的人源化抗体。Humanization of murine antibodies is carried out according to methods published in many documents in this field. In short, after obtaining Based on the typical structure of the mouse antibody VH/VL CDR, the homologous sequences of the light chain variable region (VL) and heavy chain variable region (VH) were searched from the human germline database, and the homology of the FR was Sort from high to low, select the germline with the highest FR homology as the template, transplant the CDR region of the mouse antibody to the human template, mutate certain amino acids in the variable region, and replace the constant region of the mouse antibody with Replaced with human constant regions, resulting in the final humanized antibody.
3-1.m81抗体的人源化3-1. Humanization of m81 antibody
m81抗体的人源化抗体选择IGKV1-12*01的FR1、FR2、FR3，和IGKJ4*01的FR4作为轻链框架区模板；选择IGHV1-46*01的FR1、FR2、FR3和IGHJ1*01的FR4作为重链框架区模板。任选地，对人源化抗体的轻链可变区上第43、54和/或73位的氨基酸残基进行取代；和/或对人源化抗体的重链可变区上第1、69、71、73、78、94、100a、100b和/或100c位的氨基酸残基进行取代。For the humanized m81 antibody, FR1, FR2, and FR3 of IGKV1-12*01 and FR4 of IGKJ4*01 were selected as the light chain framework region template; FR1, FR2, FR3 of IGHV1-46*01, and IGHJ1*01 were selected. FR4 serves as the heavy chain framework region template. Optionally, the amino acid residues at positions 43, 54 and/or 73 of the light chain variable region of the humanized antibody are substituted; and/or the amino acid residues at positions 1, 54 and/or 73 of the heavy chain variable region of the humanized antibody are substituted. The amino acid residues at positions 69, 71, 73, 78, 94, 100a, 100b and/or 100c are substituted.
表15.人源化抗体81的氨基酸取代

注：R71V表示依照Kabat编号系统，将71位R突变为V；100a表示按照kabat编号规则的
100A位，其他类推。Table 15. Amino acid substitutions of humanized antibody 81

Note: R71V means that according to the Kabat numbering system, R at position 71 is mutated to V; 100a means that according to the kabat numbering rules
100A bit, and so on.
m81的人源化抗体的CDR如下：The CDRs of the humanized antibody of m81 are as follows:
表16.人源化抗体81的CDR
Table 16. CDRs of humanized antibody 81
人源化抗体h81可变区序列如下：



注：单下划线部分为CDR，双下划线处为氨基酸取代位点，其余部分为
FR。The sequence of the variable region of humanized antibody h81 is as follows:



Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are
fr.
3-2.m94抗体的人源化3-2. Humanization of m94 antibody
m94的人源化抗体选择IGKV1-12*01的FR1、FR2、FR3，和IGKJ4*01的FR4作为轻链框架区模板；选择IGHV1-3*01的FR1、FR2、FR3和IGHJ1*01的FR4作为重链框架区模板。任选地，对人源化抗体的轻链可变区上第43、50、51、53、54、55和/或70位的氨基酸残基进行取代；和/或对人源化抗体的重链可变区上第1、28、66、69、71、73和/或94位的氨基酸残基进行取代。The humanized antibody of m94 selects FR1, FR2, FR3 of IGKV1-12*01, and FR4 of IGKJ4*01 as the light chain framework region template; selects FR1, FR2, FR3 of IGHV1-3*01 and FR4 of IGHJ1*01 As a heavy chain framework region template. Optionally, the amino acid residues at positions 43, 50, 51, 53, 54, 55 and/or 70 of the light chain variable region of the humanized antibody are substituted; and/or the heavy amino acid residues of the humanized antibody are substituted. The amino acid residues at positions 1, 28, 66, 69, 71, 73 and/or 94 in the variable region of the chain are substituted.
表17.人源化抗体94的氨基酸取代

注：R94S表示依照Kabat编号系统，将94位R突变为S。Table 17. Amino acid substitutions of humanized antibody 94

Note: R94S means that R at position 94 is mutated to S according to the Kabat numbering system.
m94的人源化抗体的CDR区序列如下： The CDR region sequence of the humanized antibody of m94 is as follows:
表18.人源化抗体94的CDR
Table 18. CDRs of humanized antibody 94
人源化抗体h94可变区序列如下：



注：单下划线部分为CDR，双下划线处为氨基酸取代位点，其余部分为
FR。The sequence of the humanized antibody h94 variable region is as follows:



Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are
fr.
3-3.m97的人源化3-3 Humanization of m97
鼠源抗体97的人源化抗体选择IGKV1-33*01的FR1、FR2、FR3，和IGKJ4*01的FR4作为轻链框架区模板；选择IGHV1-46*01的FR1、FR2、FR3和IGHJ6*01的FR4作为重链框架区模板。任选地，对人源化抗体的轻链可变区上第41、42、43、44、50、51、52、53、54、55、56和/或71位的氨基酸残基进行取代；和/或对人源化抗体的重链可变区上第1、26、29、69、71、78和/或93位的氨基酸残基进行取代。For the humanized antibody of mouse antibody 97, FR1, FR2, FR3 of IGKV1-33*01 and FR4 of IGKJ4*01 were selected as the light chain framework region template; FR1, FR2, FR3 and IGHJ6* of IGHV1-46*01 were selected. FR4 of 01 serves as the heavy chain framework region template. Optionally, the amino acid residues at positions 41, 42, 43, 44, 50, 51, 52, 53, 54, 55, 56 and/or 71 on the light chain variable region of the humanized antibody are substituted; and/or substitution of amino acid residues at positions 1, 26, 29, 69, 71, 78 and/or 93 of the heavy chain variable region of the humanized antibody.
表19.人源化抗体97的氨基酸取代


注：F71Y表示依照Kabat编号系统，将71位F突变回Y。Table 19. Amino acid substitutions of humanized antibody 97


Note: F71Y means that F at position 71 is mutated back to Y according to the Kabat numbering system.
m97人源化抗体的CDR区如下：The CDR regions of the m97 humanized antibody are as follows:
表20.人源化抗体97的CDR
Table 20. CDRs of humanized antibody 97
人源化抗体h97可变区序列如下：



注：单下划线部分为CDR，双下划线处为氨基酸取代位点，其余部分为
FR。The sequence of the variable region of humanized antibody h97 is as follows:



Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are
fr.
3-4.m129的人源化3-4.Humanization of m129
鼠源抗体m129的人源化抗体选择IGKV1-33*01的FR1、FR2、FR3，和IGKJ4*01的FR4作为轻链框架区模板；选择IGHV1-46*01的FR1、FR2、FR3和IGHJ6*01的FR4作为重链框架区模板。任选地，对人源化抗体的轻链可变区上第41、42、43、44、50、53、54、55和/或73位的氨基酸残基进行取代；和/或对人 源化抗体的重链可变区上第1、25、30、34、52、71和/或78位的氨基酸残基进行取代。The humanized antibody of mouse antibody m129 selects FR1, FR2, FR3 of IGKV1-33*01, and FR4 of IGKJ4*01 as the light chain framework region template; selects FR1, FR2, FR3 and IGHJ6* of IGHV1-46*01 FR4 of 01 serves as the heavy chain framework region template. Optionally, the amino acid residues at positions 41, 42, 43, 44, 50, 53, 54, 55 and/or 73 of the light chain variable region of the humanized antibody are substituted; and/or human The amino acid residues at positions 1, 25, 30, 34, 52, 71 and/or 78 of the heavy chain variable region of the humanized antibody are substituted.
表21.人源抗体129的氨基酸取代

注：P44V表示依照Kabat编号系统，将44位P突变为V。Table 21. Amino acid substitutions of human antibody 129

Note: P44V means that P at position 44 is mutated to V according to the Kabat numbering system.
m129的人源化抗体的CDR如下：The CDRs of the humanized antibody to m129 are as follows:
表22.人源化抗体129的CDR
Table 22. CDRs of humanized antibody 129
人源化抗体129的可变区序列如下：



The variable region sequence of humanized antibody 129 is as follows:



将抗CD28人源化抗体的重链可变区和轻链可变区分别与重链恒定区hIgG1:CH1-Fc和轻链恒定区CL重组，获得全长的人源化抗体。
The heavy chain variable region and light chain variable region of the anti-CD28 humanized antibody were recombined with the heavy chain constant region hIgG1:CH1-Fc and the light chain constant region CL, respectively, to obtain a full-length humanized antibody.
本披露抗CD28的人源化抗体如下: The humanized antibodies against CD28 of the present disclosure are as follows:
表23-1. 81系列的人源化抗体
Table 23-1. 81 series of humanized antibodies
表23-2. 94系列的人源化抗体
Table 23-2. 94 series of humanized antibodies
表23-3. 97系列的人源化抗体
Table 23-3. 97 Series Humanized Antibodies
表23-4. 129系列的人源化抗体
Table 23-4. 129 series of humanized antibodies
表23-5. 129系列的人源化抗体
Table 23-5. 129 series of humanized antibodies
示例性的，抗CD28人源化抗体的全长序列如下：


Exemplarily, the full-length sequence of the anti-CD28 humanized antibody is as follows:


本披露使用的抗CD28抗体的对照抗体如下：Control antibodies for anti-CD28 antibodies used in this disclosure are as follows:
REGN-hIgG1参考专利US20190389951A1构建，其序列如下：

REGN-hIgG1 was constructed with reference to patent US20190389951A1, and its sequence is as follows:

CD28超级激动剂TGN1412参考专利US07585960B2制备，具体序列如下：
CD28 super agonist TGN1412 was prepared with reference to patent US07585960B2. The specific sequence is as follows:
实施例4.抗人PSMA杂交瘤抗体的筛选和鉴定Example 4. Screening and identification of anti-human PSMA hybridoma antibodies
本披露通过杂交瘤技术制备了针对人PSMA的单克隆抗体。所得抗体与人PSMA以较高的亲和力特异性结合，并且可以与食蟹猴PSMA发生交叉反应；所得抗体与细胞表面的人PSMA和食蟹猴PSMA有较好的结合活性。The present disclosure prepares monoclonal antibodies against human PSMA through hybridoma technology. The obtained antibody specifically binds to human PSMA with high affinity and can cross-react with cynomolgus monkey PSMA; the obtained antibody has good binding activity to human PSMA and cynomolgus monkey PSMA on the cell surface.
将人PSMA-ECD-his、LnCap细胞(ATCC)或CHOK1-hPSMA细胞作为免疫试剂，或者将人PSMA-ECD-his和LnCap细胞作为交叉免疫试剂，Gold Adjuvant(Sigma Cat No.T2684)与ThermoAlum(Thermo Cat No.77161)作为佐剂交叉免疫小鼠。经过初次免疫和7次加强免疫后选择血清中抗体滴度高的小鼠(滴度>307.2K)进行脾细胞融合。融合后根据杂交瘤细胞生长密度，对杂交瘤培养上清进行检测。筛选得到活性好的单克隆杂交瘤细胞株。分别收集对数生长期杂交瘤细胞，用NucleoZol(MN)提取RNA(按照试剂盒说明书步骤)，并进行反转录(PrimeScriptTM Reverse Transcriptase，Takara,cat#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen，TB326 Rev.B 0503)进行PCR扩增后测序。筛选的单克隆杂交瘤细胞株的CDR和可变区的氨基酸序列如下： Use human PSMA-ECD-his, LnCap cells (ATCC) or CHOK1-hPSMA cells as immune reagents, or use human PSMA-ECD-his and LnCap cells as cross-immunization reagents, Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No. 77161) was used as an adjuvant to cross-immunize mice. After the initial immunization and 7 boosted immunizations, mice with high antibody titers in their serum (titers >307.2K) were selected for spleen cell fusion. After fusion, the hybridoma culture supernatant was detected according to the hybridoma cell growth density. Screen and obtain monoclonal hybridoma cell lines with good activity. Hybridoma cells in the logarithmic growth phase were collected respectively, RNA was extracted with NucleoZol (MN) (according to the instructions of the kit), and reverse transcription was performed (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was PCR amplified and sequenced using mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503). The amino acid sequences of the CDR and variable regions of the screened monoclonal hybridoma cell lines are as follows:
表24.PSMA抗体CDR
Table 24. PSMA antibody CDRs
>13鼠源重链可变区：
>13 murine heavy chain variable regions:
>13鼠源轻链可变区：
>13 murine light chain variable regions:
>15鼠源重链可变区：
>15 murine heavy chain variable regions:
>15鼠源轻链可变区：
>15 murine light chain variable regions:
>19鼠源重链可变区：
>19 murine heavy chain variable regions:
>19鼠源轻链可变区：
>19 murine light chain variable regions:
>31鼠源重链可变区：
>31 mouse heavy chain variable region:
>31鼠源轻链可变区：

注：下划线标记区为根据Kabat编号规则获得的CDR区。>31 mouse light chain variable region:

Note: The underlined area is the CDR area obtained according to the Kabat numbering rule.
鼠源抗PSMA抗体的可变区序列与SEQ ID NO：144和SEQ ID NO：145所示的重链和轻链恒定区组合获得嵌合抗体。示例性的，Chi13表示包含13鼠源重链可变区、轻链可变区和所述SEQ ID NO：144和SEQ ID NO：145所示的重链和轻链恒定区的嵌合抗体。Chi15、Chi19和Chi31以此类推。

The variable region sequence of the murine anti-PSMA antibody was combined with the heavy chain and light chain constant regions shown in SEQ ID NO: 144 and SEQ ID NO: 145 to obtain a chimeric antibody. Exemplarily, Chi13 represents a chimeric antibody comprising a 13 murine heavy chain variable region, a light chain variable region, and the heavy chain and light chain constant regions shown in SEQ ID NO: 144 and SEQ ID NO: 145. Chi15, Chi19 and Chi31 and so on.

实施例5：抗人PSMA单克隆抗体的人源化设计Example 5: Humanized design of anti-human PSMA monoclonal antibodies
鼠源单克隆抗体人源化根据本领域许多文献公示的方法进行。简言之，在所获得的鼠源抗体VH/VL CDR典型结构的基础上，从人源种系数据库中搜索轻链可变区(VL)和重链可变区(VH)的同源序列，将鼠源抗体的CDR区移植到人源模板上，并对VL和VH的部分残基进行突变，将鼠源抗体的恒定区替换为人恒定区，得到最终的人源化分子。Humanization of murine monoclonal antibodies is carried out according to methods published in many documents in this field. Briefly, based on the obtained typical structure of mouse antibody VH/VL CDR, the homologous sequences of the light chain variable region (VL) and heavy chain variable region (VH) were searched from the human germline database. , transplant the CDR region of the mouse antibody onto the human template, mutate some residues of VL and VH, and replace the constant region of the mouse antibody with the human constant region to obtain the final humanized molecule.
5-1.抗体13的人源化5-1. Humanization of antibody 13
鼠源抗体13的人源化抗体选择IGHV2-26*01的FR1、FR2、FR3，和IGHJ6*01的FR4作为重链框架区模板；选择IGKV1-39*01的FR1、FR2、FR3和IGKJ4*01的FR4作为轻链框架区模板。任选地，对人源化抗体的重链可变区上第1、30、44、49、73和/或89位的氨基酸进行取代；和/或对人源化抗体的轻链可变区上第41、42、43、44和/或71位的氨基酸进行取代。For the humanized antibody of mouse antibody 13, FR1, FR2, FR3 of IGHV2-26*01 and FR4 of IGHJ6*01 were selected as the heavy chain framework region template; FR1, FR2, FR3 and IGKJ4* of IGKV1-39*01 were selected. FR4 of 01 serves as the light chain framework region template. Optionally, the amino acids at positions 1, 30, 44, 49, 73 and/or 89 of the heavy chain variable region of the humanized antibody are substituted; and/or the light chain variable region of the humanized antibody is substituted. The amino acid at positions 41, 42, 43, 44 and/or 71 is substituted.
表25-1.人源化抗体13的氨基酸取代
Table 25-1. Amino acid substitutions of humanized antibody 13
抗体可变区的序列如下：


注：下划线部分为CDR，其余部分为FR。The sequence of the antibody variable region is as follows:


Note: The underlined part is CDR, and the remaining parts are FR.
5-2.抗体15的人源化5-2. Humanization of antibody 15
鼠源抗体15的人源化抗体选择IGHV1-3*01的FR1、FR2、FR3，和IGHJ1*01的FR4作为重链框架区模板；选择IGKV1-27*01的FR1、FR2、FR3和IGKJ2*01的FR4作为轻链框架区模板。任选地，对人源化抗体的重链可变区上第1、2、28、44、48、67、69、71、73和/或76位的氨基酸进行取代；和/或对人源化抗体的轻链可变区上第43、60和/或100位的氨基酸进行取代。For the humanized antibody of mouse antibody 15, FR1, FR2, FR3 of IGHV1-3*01 and FR4 of IGHJ1*01 were selected as the heavy chain framework region template; FR1, FR2, FR3 and IGKJ2* of IGKV1-27*01 were selected. FR4 of 01 serves as the light chain framework region template. Optionally, the amino acids at positions 1, 2, 28, 44, 48, 67, 69, 71, 73 and/or 76 of the heavy chain variable region of the humanized antibody are substituted; and/or the human The amino acids at positions 43, 60 and/or 100 of the light chain variable region of the antibody are substituted.
表25-2.人源化抗体15的氨基酸取代

Table 25-2. Amino acid substitutions of humanized antibody 15

抗体可变区的序列如下：


注：下划线部分为CDR，其余部分为FR。The sequence of the antibody variable region is as follows:


Note: The underlined part is CDR, and the remaining parts are FR.
5-3.抗体19的人源化5-3. Humanization of antibody 19
鼠源抗体19的人源化抗体选择IGHV3-7*01的FR1、FR2、FR3，和IGHJ6*01的FR4作为重链框架区模板；选择IGKV1-39*01的FR1、FR2、FR3和IGKJ4*01的FR4作为轻链框架区模板。任选地，对人源化抗体的重链可变区上第3位的氨基酸进行取代；和/或对人源化抗体的轻链可变区上第71位的氨基酸进行取代。For the humanized antibody of mouse antibody 19, FR1, FR2, FR3 of IGHV3-7*01 and FR4 of IGHJ6*01 were selected as the heavy chain framework region template; FR1, FR2, FR3 and IGKJ4* of IGKV1-39*01 were selected. FR4 of 01 serves as the light chain framework region template. Optionally, the amino acid at position 3 on the heavy chain variable region of the humanized antibody is substituted; and/or the amino acid at position 71 on the light chain variable region of the humanized antibody is substituted.
表25-3.人源化抗体19的氨基酸取代
Table 25-3. Amino acid substitutions of humanized antibody 19
抗体可变区的序列如下：


注：下划线部分为CDR，其余部分为FR。The sequence of the antibody variable region is as follows:


Note: The underlined part is CDR, and the remaining parts are FR.
5-4.抗体31的人源化5-4. Humanization of antibody 31
鼠源抗体31的人源化抗体选择IGHV3-7*01的FR1、FR2、FR3，和IGHJ1*01的FR4作为重链框架区模板；选择IGKV1-39*01的FR1、FR2、FR3和IGKJ4*01的FR4作为轻链框架区模板。任选地，对人源化抗体的重链可变区上第94位的氨基酸进行取代；和/或对人源化抗体的轻链可变区上第43、45、48和/或70位的氨基酸进行取代。For the humanized antibody of mouse antibody 31, FR1, FR2, FR3 of IGHV3-7*01 and FR4 of IGHJ1*01 were selected as the heavy chain framework region template; FR1, FR2, FR3 and IGKJ4* of IGKV1-39*01 were selected. FR4 of 01 serves as the light chain framework region template. Optionally, the amino acid at position 94 on the heavy chain variable region of the humanized antibody is substituted; and/or the amino acid at position 43, 45, 48 and/or 70 on the light chain variable region of the humanized antibody is substituted. amino acids are substituted.
表25-4.人源化抗体31的氨基酸取代
Table 25-4. Amino acid substitutions of humanized antibody 31
抗体可变区的序列如下：


注：下划线部分为CDR，其余部分为FR。The sequence of the antibody variable region is as follows:


Note: The underlined part is CDR, and the remaining parts are FR.
实施例6.抗PSMA/CD28双特异性抗体的制备Example 6. Preparation of anti-PSMA/CD28 bispecific antibodies
本披露PSMA/CD28双特异性抗体的PSMA臂可以来源于任意适宜的抗PSMA抗体。示例性的，本披露中的双特异性抗体中的抗PSMA臂可以为实施例4中的任意抗PSMA抗体的可变区，或者US20190389951A1专利中披露的PSMA臂的可变区，其中本披露使用的US20190389951A1中的PSMA臂的可变区及CDR序列如下：The PSMA arms of the PSMA/CD28 bispecific antibodies of the present disclosure can be derived from any suitable anti-PSMA antibody. Exemplarily, the anti-PSMA arm in the bispecific antibody in the present disclosure can be the variable region of any anti-PSMA antibody in Example 4, or the variable region of the PSMA arm disclosed in the US20190389951A1 patent, in which the present disclosure uses The variable region and CDR sequences of the PSMA arm in US20190389951A1 are as follows:
bs16429D的PSMA重链可变区(也称h20VH)：
PSMA heavy chain variable region of bs16429D (also known as h20VH):
bs16429D的PSMA轻链可变区(也称h20VL)：
PSMA light chain variable region of bs16429D (also known as h20VL):
表26.h20的PSMA的CDR
Table 26. CDR of PSMA for h20
本披露的PSMA-CD28双特异性抗体分子的结构如下：The structure of the disclosed PSMA-CD28 bispecific antibody molecule is as follows:
式1为非对称结构分子，包含完整分子共四条链，四条链均不相同，具体如下：Formula 1 is an asymmetric structure molecule, including a complete molecule with a total of four chains. The four chains are all different, as follows:
链1：[CD28-VH]-[IgG1(CH1)]-[IgG1Fc(Knob)]；Chain 1: [CD28-VH]-[IgG1(CH1)]-[IgG1Fc(Knob)];
链2：[CD28-VL]-[CL]；Chain 2: [CD28-VL]-[CL];
链3：[PSMA-VH]-[连接子1a]-[Titin]-[IgG1Fc(Hole)]；和Chain 3: [PSMA-VH]-[Linker 1a]-[Titin]-[IgG1Fc(Hole)]; and
链4：[PSMA-VL]-[连接子1a]-[Obscurin]；Chain 4: [PSMA-VL]-[linker 1a]-[Obscurin];
其示意图如图1A所示(其中：Ob代表Obscurin)。The schematic diagram is shown in Figure 1A (where: Ob represents Obscurin).
式2为非对称结构分子，包含完整分子共四条链，四条链均不相同，具体如下：Formula 2 is an asymmetric structure molecule, including a complete molecule with a total of four chains. The four chains are all different, as follows:
链1：[CD28-VH]-[连接子1a]-[Titin]-[IgG1Fc(Knob)]；Chain 1: [CD28-VH]-[linker 1a]-[Titin]-[IgG1Fc(Knob)];
链2：[CD28-VL]-[连接子1a]-[Obscurin]；Chain 2: [CD28-VL]-[linker 1a]-[Obscurin];
链3：[PSMA-VH]-[IgG1(CH1)]-[IgG1Fc(Hole)]；Chain 3: [PSMA-VH]-[IgG1(CH1)]-[IgG1Fc(Hole)];
链4：[PSMA-VL]-[CL]；Chain 4: [PSMA-VL]-[CL];
其示意图如图1B所示(Ob代表Obscurin)。Its schematic diagram is shown in Figure 1B (Ob represents Obscurin).
相关序列如下：

The relevant sequences are as follows:

表27.本披露的PSMA-CD28双特异性抗体

Table 27. PSMA-CD28 bispecific antibodies of the present disclosure

本披露示例性使用式1构建的双特异性抗体的序列如下：The sequence of the bispecific antibody constructed using Formula 1 is as follows:
1.Chi81-F11.Chi81-F1
>Chi81-F1链1：

>Chi81-F1 chain 1:

>Chi81-F1链2：
>Chi81-F1 chain 2:
>Chi81-F1链3；
>Chi81-F1 chain 3;
>Chi81-F1链4：
>Chi81-F1 chain 4:
2. 81H1L1-F12. 81H1L1-F1
>81H1L1-F1链1：

>81H1L1-F1 chain 1:

>81H1L1-F1链2：
>81H1L1-F1 chain 2:
>81H1L1-F1链3(同Chi81-F1链3，SEQ ID NO：228)>81H1L1-F1 chain 3 (same as Chi81-F1 chain 3, SEQ ID NO: 228)
>81H1L1-F1链4(同Chi81-F1链4，SEQ ID NO：229)>81H1L1-F1 chain 4 (same as Chi81-F1 chain 4, SEQ ID NO: 229)
3. 81H1L3-F13. 81H1L3-F1
>81H1L3-F1链1(同81H1L1-F1链1，SEQ ID NO：230)>81H1L3-F1 chain 1 (same as 81H1L1-F1 chain 1, SEQ ID NO: 230)
>81H1L3-F1链2：
>81H1L3-F1 chain 2:
>81H1L3-F1链3(同Chi81-F1链3，SEQ ID NO：228)>81H1L3-F1 chain 3 (same as Chi81-F1 chain 3, SEQ ID NO: 228)
>81H1L3-F1链4(同Chi81-F1链4，SEQ ID NO：229)>81H1L3-F1 chain 4 (same as Chi81-F1 chain 4, SEQ ID NO: 229)
4.Chi94-F14.Chi94-F1
>Chi94-F1链1：
>Chi94-F1 chain 1:
>Chi94-F1链2：

>Chi94-F1 chain 2:

>Chi94-F1链3：(同Chi81-F1链3，SEQ ID NO：228)>Chi94-F1 chain 3: (Same as Chi81-F1 chain 3, SEQ ID NO: 228)
>Chi94-F1链4：(同Chi81-F1链4，SEQ ID NO：229)>Chi94-F1 chain 4: (Same as Chi81-F1 chain 4, SEQ ID NO: 229)
5. 94H3L2-F15. 94H3L2-F1
>94H3L2-F1链1：
>94H3L2-F1 chain 1:
>94H3L2-F1链2：
>94H3L2-F1 chain 2:
>94H3L2-F1链3：(同Chi81-F1链3，SEQ ID NO：228)>94H3L2-F1 chain 3: (Same as Chi81-F1 chain 3, SEQ ID NO: 228)
>94H3L2-F1链4：(同Chi81-F1链4，SEQ ID NO：229)>94H3L2-F1 chain 4: (Same as Chi81-F1 chain 4, SEQ ID NO: 229)
6. 94H6L9-F16. 94H6L9-F1
>94H6L9-F1链1：

>94H6L9-F1 chain 1:

>94H6L9-F1链2：
>94H6L9-F1 chain 2:
>94H6L9-F1链3：(同Chi81-F1链3，SEQ ID NO：228)>94H6L9-F1 chain 3: (Same as Chi81-F1 chain 3, SEQ ID NO: 228)
>94H6L9-F1链4：(同Chi81-F1链4，SEQ ID NO：229)>94H6L9-F1 chain 4: (Same as Chi81-F1 chain 4, SEQ ID NO: 229)
7. 94H6L12-F17. 94H6L12-F1
>94H6L12-F1链1(同94H6L9-F1链1，SEQ ID NO：237)>94H6L12-F1 chain 1 (same as 94H6L9-F1 chain 1, SEQ ID NO: 237)
>94H6L12-F1链2：
>94H6L12-F1 chain 2:
>94H6L12-F1链3：(同Chi81-F1链3，SEQ ID NO：228)>94H6L12-F1 chain 3: (Same as Chi81-F1 chain 3, SEQ ID NO: 228)
>94H6L12-F1链4：(同Chi81-F1链4，SEQ ID NO：229)>94H6L12-F1 chain 4: (Same as Chi81-F1 chain 4, SEQ ID NO: 229)
8. 97H2L3-F18. 97H2L3-F1
>97H2L3-F1链1：
>97H2L3-F1 chain 1:
>97H2L3-F1链2：
>97H2L3-F1 chain 2:
>97H2L3-F1链3：(同Chi81-F1链3，SEQ ID NO：228)>97H2L3-F1 chain 3: (Same as Chi81-F1 chain 3, SEQ ID NO: 228)
>97H2L3-F1链4：(同Chi81-F1链4，SEQ ID NO：229)>97H2L3-F1 chain 4: (Same as Chi81-F1 chain 4, SEQ ID NO: 229)
9. 97H2L1-4-F19. 97H2L1-4-F1
>97H2L1-4-F1链1：(同97H2L3-F1链1，SEQ ID NO：240)>97H2L1-4-F1 chain 1: (Same as 97H2L3-F1 chain 1, SEQ ID NO: 240)
>97H2L1-4-F1链2：
>97H2L1-4-F1 chain 2:
>97H2L1-4-F1链3：(同Chi81-F1链3，SEQ ID NO：228)>97H2L1-4-F1 chain 3: (Same as Chi81-F1 chain 3, SEQ ID NO: 228)
>97H2L1-4-F1链4：(同Chi81-F1链4，SEQ ID NO：229)>97H2L1-4-F1 chain 4: (Same as Chi81-F1 chain 4, SEQ ID NO: 229)
10.Chi129-F110.Chi129-F1
>Chi129-F1链1：
>Chi129-F1 chain 1:
>Chi129-F1链2：

>Chi129-F1 chain 2:

>Chi129-F1链3：(同Chi81-F1链3，SEQ ID NO：228)>Chi129-F1 chain 3: (Same as Chi81-F1 chain 3, SEQ ID NO: 228)
>Chi129-F1链4：(同Chi81-F1链4，SEQ ID NO：229)>Chi129-F1 chain 4: (Same as Chi81-F1 chain 4, SEQ ID NO: 229)
11. 129H4L1-F111. 129H4L1-F1
>129H4L1-F1链1：
>129H4L1-F1 chain 1:
>129H4L1-F1链2：
>129H4L1-F1 chain 2:
>129H4L1-F1链3：(同Chi81-F1链3，SEQ ID NO：228)>129H4L1-F1 chain 3: (Same as Chi81-F1 chain 3, SEQ ID NO: 228)
>129H4L1-F1链4：(同Chi81-F1链4，SEQ ID NO：229)>129H4L1-F1 chain 4: (Same as Chi81-F1 chain 4, SEQ ID NO: 229)
12. 81H1L3-15-F112. 81H1L3-15-F1
>81H1L3-15-F1链1(同81H1L1-F1链1，SEQ ID NO：230)>81H1L3-15-F1 chain 1 (same as 81H1L1-F1 chain 1, SEQ ID NO: 230)
>81H1L3-15-F1链2(同81H1L3-F1链2，SEQ ID NO：232)>81H1L3-15-F1 chain 2 (same as 81H1L3-F1 chain 2, SEQ ID NO: 232)
>81H1L3-15-F1链3：

>81H1L3-15-F1 chain 3:

>81H1L3-15-F1链4：
>81H1L3-15-F1 chain 4:
13. 81H1L3-19-F113. 81H1L3-19-F1
>81H1L3-19-F1链1：(同81H1L1-F1链1，SEQ ID NO：230)>81H1L3-19-F1 chain 1: (Same as 81H1L1-F1 chain 1, SEQ ID NO: 230)
>81H1L3-19-F1链2：(同81H1L3-F1链2，SEQ ID NO：232)>81H1L3-19-F1 chain 2: (Same as 81H1L3-F1 chain 2, SEQ ID NO: 232)
>81H1L3-19-F1链3：
>81H1L3-19-F1 chain 3:
>81H1L3-19-F1链4：
>81H1L3-19-F1 chain 4:
14. 81H3L3-15-F114. 81H3L3-15-F1
>81H3L3-15-F1链1：

>81H3L3-15-F1 chain 1:

>81H3L3-15-F1链2(同81H1L3-F1链2，SEQ ID NO：232)>81H3L3-15-F1 chain 2 (same as 81H1L3-F1 chain 2, SEQ ID NO: 232)
>81H3L3-15-F1链3(同81H1L3-15-F1链3，SEQ ID NO：247)>81H3L3-15-F1 chain 3 (same as 81H1L3-15-F1 chain 3, SEQ ID NO: 247)
>81H3L3-15-F1链4(81H1L3-15-F1链4，SEQ ID NO：248)>81H3L3-15-F1 chain 4 (81H1L3-15-F1 chain 4, SEQ ID NO: 248)
15. 81H3L3-19-F115. 81H3L3-19-F1
>81H3L3-19-F1链1：(同81H3L3-15-F1链1，SEQ ID NO：251)>81H3L3-19-F1 chain 1: (Same as 81H3L3-15-F1 chain 1, SEQ ID NO: 251)
>81H3L3-19-F1链2：(同81H1L3-F1链2，SEQ ID NO：232)>81H3L3-19-F1 chain 2: (Same as 81H1L3-F1 chain 2, SEQ ID NO: 232)
>81H3L3-19-F1链3：(同81H1L3-19-F1链3，SEQ ID NO：249)>81H3L3-19-F1 chain 3: (Same as 81H1L3-19-F1 chain 3, SEQ ID NO: 249)
>81H3L3-19-F1链4：(同81H1L3-19-F1链4，SEQ ID NO：250)>81H3L3-19-F1 chain 4: (Same as 81H1L3-19-F1 chain 4, SEQ ID NO: 250)
16. 81H3L3-13-F116. 81H3L3-13-F1
>81H3L3-13-F1链1：(同81H3L3-15-F1链1，SEQ ID NO：251)>81H3L3-13-F1 chain 1: (Same as 81H3L3-15-F1 chain 1, SEQ ID NO: 251)
>81H3L3-13-F1链2：(同81H1L3-F1链2，SEQ ID NO：232)>81H3L3-13-F1 chain 2: (Same as 81H1L3-F1 chain 2, SEQ ID NO: 232)
>81H3L3-13-F1链3：
>81H3L3-13-F1 chain 3:
>81H3L3-13-F1链4：
>81H3L3-13-F1 chain 4:
17. 81H3L3-31-F117. 81H3L3-31-F1
>81H3L3-31-F1链1：((同81H3L3-15-F1链1，SEQ ID NO：251)>81H3L3-31-F1 chain 1: ((Same as 81H3L3-15-F1 chain 1, SEQ ID NO: 251)
>81H3L3-31-F1链2：(同81H1L3-F1链2，SEQ ID NO：232)>81H3L3-31-F1 chain 2: (Same as 81H1L3-F1 chain 2, SEQ ID NO: 232)
>81H3L3-31-F1链3：
>81H3L3-31-F1 chain 3:
>81H3L3-31-F1链4：
>81H3L3-31-F1 chain 4:
18. 94H6L12-15-F118. 94H6L12-15-F1
>94H6L12-15-F1链1：(同94H6L9-F1链1，SEQ ID NO：237)>94H6L12-15-F1 chain 1: (Same as 94H6L9-F1 chain 1, SEQ ID NO: 237)
>94H6L12-15-F1链2：(同94H6L12-F1链2，SEQ ID NO：239)>94H6L12-15-F1 chain 2: (Same as 94H6L12-F1 chain 2, SEQ ID NO: 239)
>94H6L12-15-F1链3：(同81H1L3-15-F1链3，SEQ ID NO：247)>94H6L12-15-F1 chain 3: (Same as 81H1L3-15-F1 chain 3, SEQ ID NO: 247)
>94H6L12-15-F1链4：(81H1L3-15-F1链4，SEQ ID NO：248)>94H6L12-15-F1 chain 4: (81H1L3-15-F1 chain 4, SEQ ID NO: 248)
19. 94H6L12-19-F119. 94H6L12-19-F1
>94H6L12-19-F1链1：(同94H6L9-F1链1，SEQ ID NO：237)>94H6L12-19-F1 chain 1: (Same as 94H6L9-F1 chain 1, SEQ ID NO: 237)
>94H6L12-19-F1链2：(同94H6L12-F1链2，SEQ ID NO：239)>94H6L12-19-F1 chain 2: (Same as 94H6L12-F1 chain 2, SEQ ID NO: 239)
>94H6L12-19-F1链3：(同81H1L3-19-F1链3，SEQ ID NO：249)>94H6L12-19-F1 chain 3: (Same as 81H1L3-19-F1 chain 3, SEQ ID NO: 249)
>94H6L12-19-F1链4：(同81H1L3-19-F1链4，SEQ ID NO：250)>94H6L12-19-F1 chain 4: (Same as 81H1L3-19-F1 chain 4, SEQ ID NO: 250)
20. 94H6L12-13-F120. 94H6L12-13-F1
>94H6L12-13-F1链1：(同94H6L9-F1链1，SEQ ID NO：237) >94H6L12-13-F1 chain 1: (Same as 94H6L9-F1 chain 1, SEQ ID NO: 237)
>94H6L12-13-F1链2：(同94H6L12-F1链2，SEQ ID NO：239)>94H6L12-13-F1 chain 2: (Same as 94H6L12-F1 chain 2, SEQ ID NO: 239)
>94H6L12-13-F1链3：(同81H3L3-13-F1链3，SEQ ID NO：252)>94H6L12-13-F1 chain 3: (Same as 81H3L3-13-F1 chain 3, SEQ ID NO: 252)
>94H6L12-13-F1链4：(同81H3L3-13-F1链4，SEQ ID NO：253)>94H6L12-13-F1 chain 4: (Same as 81H3L3-13-F1 chain 4, SEQ ID NO: 253)
21. 94H6L12-31-F121. 94H6L12-31-F1
>94H6L12-31-F1链1：(同94H6L9-F1链1，SEQ ID NO：237)>94H6L12-31-F1 chain 1: (Same as 94H6L9-F1 chain 1, SEQ ID NO: 237)
>94H6L12-31-F1链2：(同94H6L12-F1链2，SEQ ID NO：239)>94H6L12-31-F1 chain 2: (Same as 94H6L12-F1 chain 2, SEQ ID NO: 239)
>94H6L12-31-F1链3：(同81H3L3-31-F1链3，SEQ ID NO：254)>94H6L12-31-F1 chain 3: (Same as 81H3L3-31-F1 chain 3, SEQ ID NO: 254)
>94H6L12-31-F1链4：(同81H3L3-31-F1链4，SEQ ID NO：255)>94H6L12-31-F1 chain 4: (Same as 81H3L3-31-F1 chain 4, SEQ ID NO: 255)
22. 97H2L1-4-15-F122. 97H2L1-4-15-F1
>97H2L1-4-15-F1链1：(同97H2L3-F1链1，SEQ ID NO：240)>97H2L1-4-15-F1 chain 1: (Same as 97H2L3-F1 chain 1, SEQ ID NO: 240)
>97H2L1-4-15-F1链2：(同97H2L1-4-F1链2，SEQ ID NO：242)>97H2L1-4-15-F1 chain 2: (Same as 97H2L1-4-F1 chain 2, SEQ ID NO: 242)
>97H2L1-4-15-F1链3：(同81H1L3-15-F1链3，SEQ ID NO：247)>97H2L1-4-15-F1 chain 3: (Same as 81H1L3-15-F1 chain 3, SEQ ID NO: 247)
>97H2L1-4-15-F1链4(81H1L3-15-F1链4，SEQ ID NO：248)>97H2L1-4-15-F1 chain 4 (81H1L3-15-F1 chain 4, SEQ ID NO: 248)
23. 97H2L1-4-19-F123. 97H2L1-4-19-F1
>97H2L1-4-19-F1链1：(同97H2L1-4-15-F1链1，SEQ ID NO：240)>97H2L1-4-19-F1 chain 1: (Same as 97H2L1-4-15-F1 chain 1, SEQ ID NO: 240)
>97H2L1-4-19-F1链2：(同97H2L1-4-15-F1链2，SEQ ID NO：242)>97H2L1-4-19-F1 chain 2: (Same as 97H2L1-4-15-F1 chain 2, SEQ ID NO: 242)
>97H2L1-4-19-F1链3：(同81H1L3-19-F1链3，SEQ ID NO：249)>97H2L1-4-19-F1 chain 3: (Same as 81H1L3-19-F1 chain 3, SEQ ID NO: 249)
>97H2L1-4-19-F1链4：(同81H1L3-19-F1链4，SEQ ID NO：250)>97H2L1-4-19-F1 chain 4: (Same as 81H1L3-19-F1 chain 4, SEQ ID NO: 250)
24. 97H2L1-4-13-F124. 97H2L1-4-13-F1
>97H2L1-4-13-F1链1：(同97H2L1-4-15-F1链1，SEQ ID NO：240)>97H2L1-4-13-F1 chain 1: (Same as 97H2L1-4-15-F1 chain 1, SEQ ID NO: 240)
>97H2L1-4-13-F1链2：(同97H2L1-4-15-F1链2，SEQ ID NO：242)>97H2L1-4-13-F1 chain 2: (Same as 97H2L1-4-15-F1 chain 2, SEQ ID NO: 242)
>97H2L1-4-13-F1链3：(同81H3L3-13-F1链3，SEQ ID NO：252)>97H2L1-4-13-F1 chain 3: (Same as 81H3L3-13-F1 chain 3, SEQ ID NO: 252)
>97H2L1-4-13-F1链4:(同81H3L3-13-F1链4，SEQ ID NO：253)>97H2L1-4-13-F1 chain 4: (Same as 81H3L3-13-F1 chain 4, SEQ ID NO: 253)
25. 97H2L1-4-31-F125. 97H2L1-4-31-F1
>97H2L1-4-31-F1链1：(同97H2L1-4-15-F1链1，SEQ ID NO：240)>97H2L1-4-31-F1 chain 1: (Same as 97H2L1-4-15-F1 chain 1, SEQ ID NO: 240)
>97H2L1-4-31-F1链2：(同97H2L1-4-15-F1链2，SEQ ID NO：242)>97H2L1-4-31-F1 chain 2: (Same as 97H2L1-4-15-F1 chain 2, SEQ ID NO: 242)
>97H2L1-4-31-F1链3：(同81H3L3-31-F1链3，SEQ ID NO：254)>97H2L1-4-31-F1 chain 3: (Same as 81H3L3-31-F1 chain 3, SEQ ID NO: 254)
>97H2L1-4-31-F1链4：(同81H3L3-31-F1链4，SEQ ID NO：255)>97H2L1-4-31-F1 chain 4: (Same as 81H3L3-31-F1 chain 4, SEQ ID NO: 255)
示例性的，使用式2构建的PSMA/CD28双特异性抗体的序列如下：Exemplarily, the sequence of the PSMA/CD28 bispecific antibody constructed using Formula 2 is as follows:
26. 81H1L3-F2 26. 81H1L3-F2
>81H1L3-F2链1：
>81H1L3-F2 chain 1:
>81H1L3-F2链2：
>81H1L3-F2 chain 2:
>81H1L3-F2链3：
>81H1L3-F2 chain 3:
>81H1L3-F2链4：
>81H1L3-F2 chain 4:
27. 97H2L3-F2 27. 97H2L3-F2
>97H2L3-F2链1：
>97H2L3-F2 chain 1:
>97H2L3-F2链2：
>97H2L3-F2 chain 2:
>97H2L3-F2链3：(同81H1L3-F2链3，SEQ ID NO：259)>97H2L3-F2 chain 3: (Same as 81H1L3-F2 chain 3, SEQ ID NO: 259)
>97H2L3-F2链4：(同81H1L3-F2链4，SEQ ID NO：260)>97H2L3-F2 chain 4: (Same as 81H1L3-F2 chain 4, SEQ ID NO: 260)
28. 97H2L1-4-F228. 97H2L1-4-F2
>97H2L1-4-F2链1：(同97H2L3-F2链1，SEQ ID NO：261)>97H2L1-4-F2 chain 1: (Same as 97H2L3-F2 chain 1, SEQ ID NO: 261)
>97H2L1-4-F2链2：
>97H2L1-4-F2 chain 2:
>97H2L1-4-F2链3：(同81H1L3-F2链3，SEQ ID NO：259)>97H2L1-4-F2 chain 3: (Same as 81H1L3-F2 chain 3, SEQ ID NO: 259)
>97H2L1-4-F2链4：(同81H1L3-F2链4，SEQ ID NO：260)>97H2L1-4-F2 chain 4: (Same as 81H1L3-F2 chain 4, SEQ ID NO: 260)
29. 97H1L3-F229. 97H1L3-F2
>97H1L3-F2链1：

>97H1L3-F2 chain 1:

>97H1L3-F2链2：
>97H1L3-F2 chain 2:
>97H1L3-F2链3：(同81H1L3-F2链3，SEQ ID NO：259)>97H1L3-F2 chain 3: (Same as 81H1L3-F2 chain 3, SEQ ID NO: 259)
>97H1L3-F2链4：(同81H1L3-F2链4，SEQ ID NO：260)>97H1L3-F2 chain 4: (Same as 81H1L3-F2 chain 4, SEQ ID NO: 260)
30.Chi129-F230.Chi129-F2
>Chi129-F2链1：
>Chi129-F2 chain 1:
>Chi129-F2链2：
>Chi129-F2 chain 2:
>Chi129-F2链3：(同81H1L3-F2链3，SEQ ID NO：259)>Chi129-F2 chain 3: (Same as 81H1L3-F2 chain 3, SEQ ID NO: 259)
>Chi129-F2链4：(同81H1L3-F2链4，SEQ ID NO：260) >Chi129-F2 chain 4: (Same as 81H1L3-F2 chain 4, SEQ ID NO: 260)
31. 129H4L1-F231. 129H4L1-F2
>129H4L1-F2链1：
>129H4L1-F2 chain 1:
>129H4L1-F2链2：
>129H4L1-F2 chain 2:
>129H4L1-F2链3：(同81H1L3-F2链3，SEQ ID NO：259)>129H4L1-F2 chain 3: (Same as 81H1L3-F2 chain 3, SEQ ID NO: 259)
>129H4L1-F2链4：(同81H1L3-F2链4，SEQ ID NO：260)>129H4L1-F2 chain 4: (Same as 81H1L3-F2 chain 4, SEQ ID NO: 260)
32. 129H7L5-F232. 129H7L5-F2
>129H7L5-F2链1：
>129H7L5-F2 chain 1:
>129H7L5-F2链2：

>129H7L5-F2 chain 2:

>129H7L5-F2链3：(同81H1L3-F2链3，SEQ ID NO：259)>129H7L5-F2 chain 3: (Same as 81H1L3-F2 chain 3, SEQ ID NO: 259)
>129H7L5-F2链4：(同81H1L3-F2链4，SEQ ID NO：260)>129H7L5-F2 chain 4: (Same as 81H1L3-F2 chain 4, SEQ ID NO: 260)
33. 97H2L3-15-F233. 97H2L3-15-F2
>97H2L3-15-F2链1：(同97H2L3-F2链1，SEQ ID NO：261)>97H2L3-15-F2 chain 1: (Same as 97H2L3-F2 chain 1, SEQ ID NO: 261)
>97H2L3-15-F2链2：(同97H2L3-F2链2，SEQ ID NO：262)>97H2L3-15-F2 chain 2: (Same as 97H2L3-F2 chain 2, SEQ ID NO: 262)
>97H2L3-15-F2链3：
>97H2L3-15-F2 chain 3:
>97H2L3-15-F2链4：
>97H2L3-15-F2 chain 4:
34. 97H2L3-19-F234. 97H2L3-19-F2
>97H2L3-19-F2链1：(同97H2L3-F2链1，SEQ ID NO：261)>97H2L3-19-F2 chain 1: (Same as 97H2L3-F2 chain 1, SEQ ID NO: 261)
>97H2L3-19-F2链2：(同97H2L3-F2链2，SEQ ID NO：262)>97H2L3-19-F2 chain 2: (Same as 97H2L3-F2 chain 2, SEQ ID NO: 262)
>97H2L3-19-F2链3：

>97H2L3-19-F2 chain 3:

>97H2L3-19-F2链4：
>97H2L3-19-F2 chain 4:
35. 97H2L3-13-F235. 97H2L3-13-F2
>97H2L3-13-F2链1(同97H2L3-F2链1，SEQ ID NO：261)>97H2L3-13-F2 chain 1 (same as 97H2L3-F2 chain 1, SEQ ID NO: 261)
>97H2L3-13-F2链2：(同97H2L3-F2链2，SEQ ID NO：262)>97H2L3-13-F2 chain 2: (Same as 97H2L3-F2 chain 2, SEQ ID NO: 262)
>97H2L3-13-F2链3：
>97H2L3-13-F2 chain 3:
>97H2L3-13-F2链4：
>97H2L3-13-F2 chain 4:
36. 97H2L3-31-F236. 97H2L3-31-F2
>97H2L3-31-F2链1：(同97H2L3-F2链1，SEQ ID NO：261) >97H2L3-31-F2 chain 1: (Same as 97H2L3-F2 chain 1, SEQ ID NO: 261)
>97H2L3-31-F2链2：(同97H2L3-F2链2，SEQ ID NO：262)>97H2L3-31-F2 chain 2: (Same as 97H2L3-F2 chain 2, SEQ ID NO: 262)
>97H2L3-31-F2链3：
>97H2L3-31-F2 chain 3:
>97H2L3-31-F2链4：
>97H2L3-31-F2 chain 4:
37. 129H4L1-15-F237. 129H4L1-15-F2
>129H4L1-15-F2链1：(同129H4L1-F2链1，SEQ ID NO：268)>129H4L1-15-F2 chain 1: (Same as 129H4L1-F2 chain 1, SEQ ID NO: 268)
>129H4L1-15-F2链2：(同129H4L1-F2链2，SEQ ID NO：269)>129H4L1-15-F2 chain 2: (Same as 129H4L1-F2 chain 2, SEQ ID NO: 269)
>129H4L1-15-F2链3：(同97H2L3-15-F2链3，SEQ ID NO：272)>129H4L1-15-F2 chain 3: (Same as 97H2L3-15-F2 chain 3, SEQ ID NO: 272)
>129H4L1-15-F2链4：(同97H2L3-15-F2链4，SEQ ID NO：273)>129H4L1-15-F2 chain 4: (Same as 97H2L3-15-F2 chain 4, SEQ ID NO: 273)
38. 129H4L1-19-F238. 129H4L1-19-F2
>129H4L1-19-F2链1：(同129H4L1-F2链1，SEQ ID NO：268)>129H4L1-19-F2 chain 1: (Same as 129H4L1-F2 chain 1, SEQ ID NO: 268)
>129H4L1-19-F2链2：(同129H4L1-F2链2，SEQ ID NO：269)>129H4L1-19-F2 chain 2: (Same as 129H4L1-F2 chain 2, SEQ ID NO: 269)
>129H4L1-19-F2链3：(同97H2L3-19-F2链3，SEQ ID NO：274)>129H4L1-19-F2 chain 3: (Same as 97H2L3-19-F2 chain 3, SEQ ID NO: 274)
>129H4L1-19-F2链4：(同97H2L3-19-F2链4，SEQ ID NO：275)>129H4L1-19-F2 chain 4: (Same as 97H2L3-19-F2 chain 4, SEQ ID NO: 275)
39. 129H4L1-13-F239. 129H4L1-13-F2
>129H4L1-13-F2链1：(同129H4L1-F2链1，SEQ ID NO：268)>129H4L1-13-F2 chain 1: (Same as 129H4L1-F2 chain 1, SEQ ID NO: 268)
>129H4L1-13-F2链2：(同129H4L1-F2链2，SEQ ID NO：269)>129H4L1-13-F2 chain 2: (Same as 129H4L1-F2 chain 2, SEQ ID NO: 269)
>129H4L1-13-F2链3：(同97H2L3-13-F2链3，SEQ ID NO：276)>129H4L1-13-F2 chain 3: (Same as 97H2L3-13-F2 chain 3, SEQ ID NO: 276)
>129H4L1-13-F2链4：(同97H2L3-13-F2链4，SEQ ID NO：277) >129H4L1-13-F2 chain 4: (Same as 97H2L3-13-F2 chain 4, SEQ ID NO: 277)
40. 129H4L1-31-F240. 129H4L1-31-F2
>129H4L1-31-F2链1：(同129H4L1-F2链1，SEQ ID NO：268)>129H4L1-31-F2 chain 1: (Same as 129H4L1-F2 chain 1, SEQ ID NO: 268)
>129H4L1-31-F2链2：(同129H4L1-F2链2，SEQ ID NO：269)>129H4L1-31-F2 chain 2: (Same as 129H4L1-F2 chain 2, SEQ ID NO: 269)
>129H4L1-31-F2链3：(同97H2L3-31-F2链3，SEQ ID NO：278)>129H4L1-31-F2 chain 3: (Same as 97H2L3-31-F2 chain 3, SEQ ID NO: 278)
>129H4L1-31-F2链4：(同97H2L3-31-F2链4，SEQ ID NO：279)>129H4L1-31-F2 chain 4: (Same as 97H2L3-31-F2 chain 4, SEQ ID NO: 279)
注：双下划线标记区为Titin或Obscurin序列，斜体为恒定区。Note: The double underlined region is the Titin or Obscurin sequence, and the italicized region is the constant region.
本披露的PSMA/CD28双特异性抗体的对照分子为REGN5678V，参考US20190389951A1中bs16429D(又称REGN-5678)构建，将其Fc换成本披露IgG1Fc(Knob)和IgG1Fc(Hole)，得到REGN5678V，其具有共同轻链的结构。具体序列如下：The control molecule of the disclosed PSMA/CD28 bispecific antibody is REGN5678V. It is constructed with reference to bs16429D (also known as REGN-5678) in US20190389951A1. Its Fc is replaced with the disclosed IgG1 Fc (Knob) and IgG 1 Fc (Hole) to obtain REGN5678V. , which share the structure of a common light chain. The specific sequence is as follows:
>REGN5678V第一链：
>REGN5678V first chain:
>REGN5678V第二链：
>REGN5678V second chain:
>REGN5678V第三链：

>REGN5678V third chain:

实施例7：抗PSMA/CD3双特异性抗体的制备Example 7: Preparation of anti-PSMA/CD3 bispecific antibodies
本披露的CD3结合分子可以来源于任意适宜的抗体。具体的，本披露的实施例采用的是S107E。CD3 binding molecules of the present disclosure can be derived from any suitable antibody. Specifically, the embodiment of the present disclosure adopts S107E.
表28.S107E的CDR
Table 28. CDR of S107E
S107E可变区序列如下：
The S107E variable region sequence is as follows:
本披露中的PSMA/CD3双特异性抗体具有以下式1和式5的分子结构：The PSMA/CD3 bispecific antibody in the present disclosure has the following molecular structures of Formula 1 and Formula 5:
式1为非对称结构分子，包含：Formula 1 is an asymmetric structure molecule, including:
链1：VH(anti-PSMA)-IgG1(CH1)-IgG1Fc(Knob,L234A/L235A/S354C/T366W)；Chain 1: VH (anti-PSMA)-IgG 1 (CH1)-IgG 1 Fc (Knob, L234A/L235A/S354C/T366W);
链2：VL(anti-PSMA)-CL；Chain 2: VL(anti-PSMA)-CL;
链3(Ob-hole)：VH(S107E)-连接子1a-Obscurin-IgG1Fc(Hole,L234A/L235A/Y349C/T366S/L368A/Y407V)；和Chain 3 (Ob-hole): VH(S107E)-linker 1a-Obscurin-IgG 1 Fc(Hole,L234A/L235A/Y349C/T366S/L368A/Y407V); and
链4(VL-Titin)：VL(S107E)-连接子1a-Titin。Chain 4 (VL-Titin): VL(S107E)-linker 1a-Titin.
其示意图如图1C所示。Its schematic diagram is shown in Figure 1C.
>IgG1(CH1)(SEQ ID NO：219)
>IgG 1 (CH1)(SEQ ID NO: 219)
>CL(SEQ ID NO：145)
>CL(SEQ ID NO:145)
>IgG1Fc(Knob,L234A/L235A/S354C/T366W)(SEQ ID NO：220)

>IgG 1 Fc(Knob,L234A/L235A/S354C/T366W)(SEQ ID NO: 220)

>Ob-hole(SEQ ID NO：291)
>Ob-hole(SEQ ID NO: 291)
>VL-Titin：VL(S107E)-连接子1a-Titin(SEQ ID NO：292)
>VL-Titin: VL(S107E)-linker 1a-Titin (SEQ ID NO: 292)
>连接子1a(SEQ ID NO：223)

注：单下划线标记区为根据Kabat编号规则获得的CD3结合结构域的CDR区，
双下划线标记区为Titin或Obscurin序列，斜体为恒定区。下同。>Linker 1a (SEQ ID NO: 223)

Note: The single underlined region is the CDR region of the CD3 binding domain obtained according to Kabat numbering rules.
The double underlined region indicates the Titin or Obscurin sequence, and the italicized region indicates the constant region. The same below.
式5为非对称结构分子，包含 Formula 5 is an asymmetric structure molecule, including
链1：VH(anti-PSMA)-连接子2a-VL(anti-PSMA)-连接子3a-VH(S107E)-连接子2a-VL(S107E)-连接子4a-IgG1Fc(Hole,L234A/L235A/Y349C/T366S/L368A/Y407V)；和Chain 1: VH(anti-PSMA)-linker 2a-VL(anti-PSMA)-linker 3a-VH(S107E)-linker 2a-VL(S107E)-linker 4a-IgG 1 Fc(Hole,L234A /L235A/Y349C/T366S/L368A/Y407V); and
链3(Fc(Knob))：IgG1Fc(Knob,L234A/L235A/S354C/T366W)，其示意图如图1D所示。Chain 3 (Fc(Knob)): IgG 1 Fc(Knob, L234A/L235A/S354C/T366W), its schematic diagram is shown in Figure 1D.
>IgG1Fc(Knob,L234A/L235A/S354C/T366W)(SEQ ID NO：220)>IgG 1 Fc(Knob,L234A/L235A/S354C/T366W)(SEQ ID NO: 220)
>IgG1Fc(Hole,Y349C/T366S/L368A/Y407V)(SEQ ID NO：221)>IgG 1 Fc(Hole,Y349C/T366S/L368A/Y407V)(SEQ ID NO: 221)
>连接子2a:(SEQ ID NO：293)>Connector 2a: (SEQ ID NO: 293)
>连接子3a:(SEQ ID NO：294)>Connector 3a: (SEQ ID NO: 294)
>连接子4a:(SEQ ID NO：295)>Connector 4a: (SEQ ID NO: 295)
基于15和19的人源化抗体的氨基酸序列，分别构建了如下所示的双特异性抗体。Based on the amino acid sequences of humanized antibodies 15 and 19, the bispecific antibodies shown below were constructed respectively.
表29.本披露的PSMA-CD3双特异性抗体
Table 29. PSMA-CD3 bispecific antibodies of the present disclosure
其中，15-F5-1表示该分子采用抗体15及其人源化抗体的可变区作为PSMA结合域，采用式5作为分子结构。hu15H4L2-F5-1表示该链采用hu15H4作为重链可变区，hu15L2作为轻链可变区，以此类推。具体氨基酸序列如下：


Among them, 15-F5-1 means that the molecule uses the variable region of antibody 15 and its humanized antibody as the PSMA binding domain, and uses formula 5 as the molecular structure. hu15H4L2-F5-1 means that the chain uses hu15H4 as the heavy chain variable region, hu15L2 as the light chain variable region, and so on. The specific amino acid sequence is as follows:


此外，本披露也使用了PSMA-CD3双特异性抗体CC-1，参考专利WO2017121905A1双抗制备，其具有IgG-scFv结构，完整分子包含两条第一链和两条第二链，具体如下：In addition, this disclosure also uses the PSMA-CD3 bispecific antibody CC-1, which is prepared with reference to the patent WO2017121905A1 dual antibody. It has an IgG-scFv structure, and the complete molecule contains two first chains and two second chains, as follows:
链1：[PSMA-VH]-[IgG恒定区]-[连接子1b]-[CD3-VL]-[连接子2a]-[CD3-VH]；Chain 1: [PSMA-VH]-[IgG constant region]-[linker 1b]-[CD3-VL]-[linker 2a]-[CD3-VH];
链2:[PSMA-VL]-CL；Chain 2:[PSMA-VL]-CL;
具体序列如下：

The specific sequence is as follows:

表30.CC1的序列


Table 30. Sequence of CC1


IgG恒定区：
IgG constant region:
CL：SEQ ID NO：145CL：SEQ ID NO：145
连接子1b：SGConnector 1b: SG
连接子2a:GGGGSGGGGSGGGGSLinker 2a:GGGGSGGGGSGGGGS
SEQ ID NO：293SEQ ID NO: 293
本披露所用的PSMA/CD3双特异性抗体的对照分子为Aca-Mab，其氨基酸序列如WO2017134158A1中的SEQ ID NO：382所示。The control molecule of the PSMA/CD3 bispecific antibody used in this disclosure is Aca-Mab, and its amino acid sequence is shown in SEQ ID NO: 382 in WO2017134158A1.
本披露还涉及PSMA/CD28双特异性抗体与抗PD-1抗体的联用，使用的抗PD-1抗体参考专利WO2020156509A1制备，具体序列如下： This disclosure also involves the combination of PSMA/CD28 bispecific antibody and anti-PD-1 antibody. The anti-PD-1 antibody used is prepared with reference to patent WO2020156509A1. The specific sequence is as follows:
表31.抗PD-1抗体的CDR区
Table 31. CDR regions of anti-PD-1 antibodies
>PD-1VH
>PD-1VH
>PD-1VL：
>PD-1VL:
>PD-1重链恒定区：
>PD-1 heavy chain constant region:
>PD-1轻链恒定区：
>PD-1 light chain constant region:
>PD-1重链：

>PD-1 heavy chain:

>PD-1轻链：
>PD-1 light chain:
此外，本披露的测试例中还使用了抗CD3抗体TNB383B-H，参考WO2018052503A1制备，具体序列如下：In addition, the anti-CD3 antibody TNB383B-H was also used in the test examples disclosed herein, which was prepared with reference to WO2018052503A1. The specific sequence is as follows:
>TNB383B-H重链：
>TNB383B-H heavy chain:
>TNB383B-H轻链：
>TNB383B-H light chain:
测试例test case
测试例1.CD28单抗对T细胞的激活作用Test example 1. Activation effect of CD28 monoclonal antibody on T cells
为测试本披露CD28抗体的共刺激作用，本测试例用Jurkat-NFAT lum(Promega,J133A)细胞检测CD28单抗在溶液中对Jurkat细胞的激活。方法如下： In order to test the costimulatory effect of the CD28 antibody of the present disclosure, this test example uses Jurkat-NFAT lum (Promega, J133A) cells to detect the activation of Jurkat cells by the CD28 monoclonal antibody in solution. Methods as below:
Jurkat-NFAT lum细胞在完全培养基中传代培养，将抗体用1640+10％FBS的培养基梯度稀释，50μL每孔加入96孔酶标板中。离心收集Jurkat-NFAT lum细胞集，使用1640+10％FBS的培养基重悬计数，调整细胞数为1E6细胞/mL，向加有抗体的96孔酶标板中每孔加入50μL(5E4/孔)细胞；37℃放置6小时后，每孔加入50μL firefly-glo荧光素酶报告基因检测试剂(美仑生物Cat.#MA0519-2)，室温放置8分钟后，将液体转入白板中，用多功能酶标仪(BMG Cat.#PHERAstar)读板。结果见下表32-1至表32-4。Jurkat-NFAT lum cells were subcultured in complete culture medium. The antibody was serially diluted with 1640+10% FBS culture medium, and 50 μL was added to each well into a 96-well microplate. Collect the Jurkat-NFAT lum cell set by centrifugation, resuspend and count using 1640+10% FBS culture medium, adjust the number of cells to 1E6 cells/mL, and add 50 μL (5E4/well) to each well of a 96-well enzyme plate with antibodies. ) cells; after being placed at 37°C for 6 hours, add 50 μL of firefly-glo luciferase reporter gene detection reagent (Meilun Biotech Cat.#MA0519-2) to each well. After being placed at room temperature for 8 minutes, transfer the liquid to a white plate and use Read the plate with a multifunctional microplate reader (BMG Cat.#PHERAstar). The results are shown in Table 32-1 to Table 32-4 below.
表32-1.人源化抗体81的T细胞激活作用(生物发光值)
Table 32-1. T cell activation effect of humanized antibody 81 (bioluminescence value)
表32-2.抗体94的T细胞激活作用(生物发光值)

Table 32-2. T cell activation effect of antibody 94 (bioluminescence value)

表32-3.人源化抗体97的T细胞激活作用(生物发光值)

Table 32-3. T cell activation effect of humanized antibody 97 (bioluminescence value)

表32-4.人源化抗体129的T细胞激活作用(生物发光值)
Table 32-4. T cell activation effect of humanized antibody 129 (bioluminescence value)
结果显示，81，94，97和129的所有人源化抗体对T细胞NFAT信号通路的激活程度都远小于超级激动剂TGN1412。The results showed that all humanized antibodies 81, 94, 97 and 129 activated the T cell NFAT signaling pathway to a much smaller extent than the super agonist TGN1412.
测试例2.CD28单抗联合CD3单抗对T细胞的激活作用Test example 2. Activation effect of CD28 monoclonal antibody combined with CD3 monoclonal antibody on T cells
为测试本披露CD28抗体的共刺激作用，本测试例用Jurkat-IL2 lum细胞(Promega，J129A)检测CD28和CD3单抗(TNB383B-H)在溶液中对Jurkat-IL2 lum细胞的激活。方法如下：In order to test the co-stimulatory effect of the CD28 antibody of the present disclosure, this test example uses Jurkat-IL2 lum cells (Promega, J129A) to detect the activation of Jurkat-IL2 lum cells by CD28 and CD3 monoclonal antibodies (TNB383B-H) in solution. Methods as below:
Jurkat-IL2 lum细胞用1640完全培养基培养，每2天传代一次。实验时，将细胞吹打均匀，离心，去除上清，用PBS重悬细胞，离心。再次用1640完全培养基重悬细胞，并计数，将细胞密度调整为3E4细胞/50μL/孔。用2μg/mL Anti-hu(H+L)包被到高吸附细胞培养板上，细胞培养箱包被2小时；弃上清，加入30ng/mL TNB383B-H，细胞培养箱包被0.5～1小时；弃上清，加入稀释好的CD28人源化抗体(从3μg/mL开始，3倍稀释成8个浓度)，细胞培养箱包被1小时；弃上清， 加入Jurkat-IL2-lum细胞，密度调整为3E4细胞/50μL/孔，培养过夜读板：加入50μL/孔firefly-glo荧光素酶报告基因检测试剂(美仑生物Cat.#MA0519-2)，室温避光孵育10分钟后PheraStar进行lumin的信号读取。Jurkat-IL2 lum cells were cultured in 1640 complete medium and passaged every 2 days. During the experiment, pipet the cells evenly, centrifuge, remove the supernatant, resuspend the cells in PBS, and centrifuge. Resuspend the cells in 1640 complete medium again, count, and adjust the cell density to 3E4 cells/50 μL/well. Coat the high-adsorption cell culture plate with 2 μg/mL Anti-hu (H+L), and cover the cell culture box for 2 hours; discard the supernatant, add 30ng/mL TNB383B-H, and cover the cell culture box for 0.5 to 1 hour; Discard the supernatant, add diluted CD28 humanized antibody (starting from 3 μg/mL, dilute 3 times to 8 concentrations), and cover the cell culture box for 1 hour; discard the supernatant, Add Jurkat-IL2-lum cells, adjust the density to 3E4 cells/50μL/well, culture overnight and read the plate: add 50μL/well firefly-glo luciferase reporter gene detection reagent (Meilun Biotech Cat.#MA0519-2), room temperature After incubation in the dark for 10 minutes, PheraStar reads the lumin signal.
结果显示，TNB383B-H单用时IL2激活信号较弱，与CD28单抗联用可以增强IL2激活信号，81，94，97，129的所有抗体均能激活T细胞的IL2信号通路。The results show that the IL2 activation signal of TNB383B-H is weak when used alone, but the IL2 activation signal can be enhanced when combined with CD28 monoclonal antibody. All antibodies of 81, 94, 97, and 129 can activate the IL2 signaling pathway of T cells.
表33.联用CD3单抗后各CD28抗体对T细胞IL2信号通路的激活结果

Table 33. Activation results of each CD28 antibody on T cell IL2 signaling pathway after combined with CD3 monoclonal antibody

测试例3.抗CD28人源化单抗与CD28抗原的结合活性Test Example 3. Binding activity of anti-CD28 humanized monoclonal antibody to CD28 antigen
为测试本披露CD28靶向性人源化抗体结合CD28抗原的能力，本测试例用ELISA法检测CD28人源化抗体与人CD28抗原(购自SinoBiological，11524-HCCH)或猴CD28抗原(购自SinoBiological，90182-C08H)的结合活性。具体如下：In order to test the ability of the disclosed CD28-targeted humanized antibody to bind to CD28 antigen, this test example uses ELISA to detect the combination of CD28 humanized antibody with human CD28 antigen (purchased from SinoBiological, 11524-HCCH) or monkey CD28 antigen (purchased from SinoBiological, 90182-C08H) binding activity. details as follows:
将CD28抗原按照0.5μg/孔包被于96孔板，放置于4℃过夜。弃去上清后用5％脱脂牛奶封闭2小时。用1％BSA配置不同浓度的各人源化抗体，起始浓度为200nM，5倍稀释8个浓度。将封闭后的96孔板洗两次后加入各人源化抗体，4℃孵育1小时，洗四次后用抗人IgG Fc-HRP二抗(1:10000)在4℃孵育1小时， TMB显色后用0.1M硫酸终止反应。ELISA结果如下表34和35所示。CD28 antigen was coated on a 96-well plate at 0.5 μg/well and placed at 4°C overnight. Discard the supernatant and block with 5% skim milk for 2 hours. Use 1% BSA to prepare different concentrations of each humanized antibody. The starting concentration is 200nM and diluted 5 times to 8 concentrations. Wash the blocked 96-well plate twice, add each humanized antibody, and incubate at 4°C for 1 hour. Wash four times and incubate with anti-human IgG Fc-HRP secondary antibody (1:10000) at 4°C for 1 hour. After TMB color development, the reaction was terminated with 0.1M sulfuric acid. The ELISA results are shown in Tables 34 and 35 below.
表34.CD28人源化单抗与人CD28抗原的结合作用
Table 34. Binding effect of CD28 humanized monoclonal antibody and human CD28 antigen
表35.CD28人源化单抗与猴CD28抗原的结合作用
Table 35. Binding effect of CD28 humanized monoclonal antibody and monkey CD28 antigen
结果显示，本披露CD28抗体与人和猴CD28均可特异性结合。The results show that the CD28 antibody of the present disclosure can specifically bind to both human and monkey CD28.
测试例4.抗PSMA抗体对PSMA的结合活性Test Example 4. Binding activity of anti-PSMA antibody to PSMA
为测试本披露PSMA靶向性人源化抗体结合PSMA抗原的能力，本测试例用ELISA法检测PSMA人源化抗体与人PSMA抗原(购自R&D SYSTEM，4234-ZN-010)的结合。具体如下：In order to test the ability of the disclosed PSMA-targeted humanized antibody to bind to PSMA antigen, this test example uses ELISA to detect the binding of PSMA humanized antibody to human PSMA antigen (purchased from R&D SYSTEM, 4234-ZN-010). details as follows:
将PSMA抗原并按照1μg/孔包被于96孔板，放置于4℃过夜。弃去上清后用5％脱脂牛奶封闭2小时。用1％BSA配置不同浓度的各人源化抗体，起始浓度为10μg/mL，4倍稀释12个浓度。将封闭后的96孔板洗两次后加入配置的PSMA抗原，4℃孵育1h，洗四次后用抗人IgG Fc-HRP二抗(1:10000)在4℃孵育1h，用TMB显色后用0.1M硫酸终止反应。ELISA结果如下表36所示。Coat PSMA antigen on a 96-well plate at 1 μg/well and place it at 4°C overnight. Discard the supernatant and block with 5% skim milk for 2 hours. Use 1% BSA to prepare different concentrations of each humanized antibody. The starting concentration is 10 μg/mL and diluted 4 times to 12 concentrations. Wash the blocked 96-well plate twice, add the prepared PSMA antigen, and incubate it at 4°C for 1 hour. After washing four times, incubate it with anti-human IgG Fc-HRP secondary antibody (1:10000) for 1 hour at 4°C, and use TMB to develop color. The reaction was terminated with 0.1M sulfuric acid. The ELISA results are shown in Table 36 below.
表36.CD28抗体与人PSMA抗原的ELISA结合作用
Table 36. ELISA binding effect of CD28 antibody and human PSMA antigen
注：Top表示最大吸光度。Note: Top represents the maximum absorbance.
结果显示，本披露所筛选的PSMA人源化抗体及嵌合抗体与人PSMA抗原具有良好结合作用。The results show that the PSMA humanized antibodies and chimeric antibodies screened in this disclosure have good binding effect with human PSMA antigen.
测试例5.抗体对表达PSMA的细胞的结合活性Test Example 5. Binding activity of antibodies to cells expressing PSMA
本测试例用流式细胞术方法检测了抗体与过表达人PSMA的稳转细胞株 CHO-hPSMA的结合活性。This test example uses flow cytometry to detect antibodies and stably transfected cell lines overexpressing human PSMA. Binding activity of CHO-hPSMA.
实验一：采用10％FBS的F12培养基培养CHO-hPSMA。接种了细胞的培养基放置于37℃，5％CO2培养箱中培养2天，按每孔细胞数1×105个将细胞加至细胞板中，离心洗涤。梯度稀释抗体，将抗体溶液按100μL/孔加入细胞板中，4℃孵育1小时后洗涤。向细胞板中加入100μL/孔的AF488-Goat Anti-human IgG Fc荧光二抗稀释液(1:400)，4℃孵育1个小时后洗涤。向细胞板中加入100μL/孔的PBS后读板。抗体的细胞结合活性如下表37-1所示。Experiment 1: Cultivate CHO-hPSMA in F12 medium with 10% FBS. The culture medium in which cells were seeded was placed in a 37°C, 5% CO 2 incubator for 2 days. The cells were added to the cell plate at a cell number of 1×10 5 per well, and centrifuged and washed. Gradually dilute the antibody, add 100 μL/well of the antibody solution to the cell plate, incubate at 4°C for 1 hour and then wash. Add 100 μL/well of AF488-Goat Anti-human IgG Fc fluorescent secondary antibody dilution (1:400) to the cell plate, incubate at 4°C for 1 hour and then wash. Add 100 μL/well of PBS to the cell plate and read the plate. The cell-binding activity of the antibody is shown in Table 37-1 below.
表37-1.CD28抗体与CHO-hPSMA的结合活性
Table 37-1. Binding activity of CD28 antibody to CHO-hPSMA
实验二：采用与实验一相同的方法培养细胞并将其接种至细胞板中。然后梯度稀释抗体，将抗体溶液按100μL/孔加入细胞板中，4℃孵育1小时后洗涤。向细胞板中加入100μL/孔的HRP-Goat Anti-human IgG(H+L)二抗稀释液(1:8000)，4℃孵育1个小时后离心洗涤。用TMB显色后用0.1M硫酸终止反应，并检测OD450值。抗体的细胞结合活性如下表37-2所示。Experiment 2: Use the same method as Experiment 1 to culture cells and seed them into cell plates. Then gradiently dilute the antibody, add the antibody solution to the cell plate at 100 μL/well, incubate at 4°C for 1 hour and then wash. Add 100 μL/well of HRP-Goat Anti-human IgG (H+L) secondary antibody dilution (1:8000) to the cell plate, incubate at 4°C for 1 hour, and then centrifuge and wash. After color development with TMB, the reaction was terminated with 0.1M sulfuric acid, and the OD450 value was detected. The cell-binding activity of the antibody is shown in Table 37-2 below.
表37-2.抗体与CHO-hPSMA的结合活性(EC50，μg/mL)
Table 37-2. Binding activity of antibodies to CHO-hPSMA (EC 50 , μg/mL)
实验三：采用实验一的方法检测双特异性抗体的结合活性。抗体在各浓度条件下与细胞的结合活性如下表37-3所示。Experiment 3: Use the method of Experiment 1 to detect the binding activity of bispecific antibodies. The binding activity of the antibody to cells under various concentration conditions is shown in Table 37-3 below.
表37-3.PSMA/CD3双特异性抗体与CHO-hPSMA的结合活性(吸光值)
Table 37-3. Binding activity of PSMA/CD3 bispecific antibody to CHO-hPSMA (absorbance value)
实验五：用Protein A生物传感芯片亲和捕获抗体，然后于芯片表面流经人PSMA，用Biacore T200仪器在25℃条件下实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用Glycine 1.5将生物传感芯片洗净再生。数据拟合模型采用1:1Model。其中人PSMA抗原购于Acro(货号为PSA-H52H3)；hCD3D&CD3E复合物抗原购于Sino Biological(货号为CT038-H2508H)。各抗体结合及解离情况如下表37-4。Experiment 5: Use the Protein A biosensor chip to affinity capture the antibody, then flow human PSMA on the chip surface, and use the Biacore T200 instrument to detect the reaction signal in real time at 25°C to obtain the binding and dissociation curves. After the dissociation of each experimental cycle is completed, the biosensor chip is washed and regenerated with Glycine 1.5. The data fitting model uses 1:1Model. The human PSMA antigen was purchased from Acro (catalog number: PSA-H52H3); the hCD3D&CD3E complex antigen was purchased from Sino Biological (catalog number: CT038-H2508H). The binding and dissociation conditions of each antibody are shown in Table 37-4.
表37-4.PSMA/CD3双特异性抗体对人PSMA的结合活性
Table 37-4. Binding activity of PSMA/CD3 bispecific antibody to human PSMA
结果显示，本披露的抗PSMA抗体和双特异性抗体与人PSMA和表达PSMA的细胞均具有良好结合作用，强于Aca-mab。The results show that the anti-PSMA antibodies and bispecific antibodies disclosed in the present disclosure have good binding effects on human PSMA and PSMA-expressing cells, which are stronger than Aca-mab.
测试例6.本披露的PSMA/CD28双特异性抗体与CD28抗原的结合Test Example 6. Binding of PSMA/CD28 bispecific antibodies of the present disclosure to CD28 antigen
为测试本披露PSMA/CD28双特异性抗体结合CD28抗原的能力，本测试例用ELISA法检测PSMA/CD28双特异性抗体与人或猴CD28抗原(购自SinoBiological，11524-HCCH；90182-C08H)的结合。具体如下：In order to test the ability of the disclosed PSMA/CD28 bispecific antibody to bind to CD28 antigen, this test example uses ELISA to detect the PSMA/CD28 bispecific antibody and human or monkey CD28 antigen (purchased from SinoBiological, 11524-HCCH; 90182-C08H) combination. details as follows:
将CD28抗原并按照5μg/孔包被于96孔板，放置于4℃过夜。弃去上清后用5％脱脂牛奶封闭2小时。用1％BSA配置不同浓度的各抗体，起始浓度为200nM，5倍稀释8个浓度。将封闭后的96孔板洗两次后加入配置各抗体，4℃孵育1小时，洗四次后用anti human IgG Fc-HRP二抗二抗(1:10000)在4℃孵育1小时，用TMB显色后用0.1M硫酸终止反应。ELISA结果如下表38-1和38-2所示。CD28 antigen was coated on a 96-well plate at 5 μg/well and placed at 4°C overnight. Discard the supernatant and block with 5% skim milk for 2 hours. Use 1% BSA to prepare each antibody at different concentrations. The starting concentration is 200nM and diluted 5 times to 8 concentrations. Wash the blocked 96-well plate twice, add each antibody, and incubate at 4°C for 1 hour. After washing four times, use anti-human IgG Fc-HRP secondary antibody (1:10000) to incubate at 4°C for 1 hour. After TMB color development, the reaction was terminated with 0.1M sulfuric acid. The ELISA results are shown in Tables 38-1 and 38-2 below.
表38-1.PSMA/CD28双特异性抗体与人CD28的结合作用(吸光值)
Table 38-1. Binding effect of PSMA/CD28 bispecific antibody and human CD28 (absorbance value)
表38-2 PSMA/CD28双特异性抗体与猴CD28的结合作用
Table 38-2 Binding effect of PSMA/CD28 bispecific antibody and monkey CD28
结果显示，本披露PSMA/CD28抗体与人或猴CD28抗原均有较好结合作用。 The results show that the disclosed PSMA/CD28 antibody has a good binding effect on both human and monkey CD28 antigens.
测试例7.本披露的双特异性抗体的细胞结合活性Test Example 7. Cell-binding activity of bispecific antibodies of the present disclosure
为测试本披露PSMA-CD28双特异性抗体与PSMA及CD28的结合能力，本测试例用流式细胞术方法检测了双特异性抗体与过表达人PSMA的稳转细胞株CHO-hPSMA和过表达人CD28的稳转细胞株CHO-hCD28细胞的结合能力。具体如下：In order to test the binding ability of the disclosed PSMA-CD28 bispecific antibody to PSMA and CD28, this test example used flow cytometry to detect the bispecific antibody and the stably transfected cell line CHO-hPSMA overexpressing human PSMA and overexpressing human PSMA. Binding ability of CHO-hCD28 cells, a stably transduced cell line of human CD28. details as follows:
上述细胞培养于10％FBS的F12培养基中，放置于37℃，5％CO2培养箱中，培养2天，按每孔细胞数1×105个将细胞加至细胞板中，300g离心5分钟，1％BSA洗一次。抗体梯度稀释8个浓度，按每孔100μL加入细胞板中，4℃孵育1小时，1％BSA洗一次，每孔加入100μL AF488-Goat Anti-human IgG Fc荧光二抗(Invitrogen,A-11013)稀释液(1:400)，4℃孵育1个小时。1％BSA洗板三次，每孔加入100μL PBS读板。各抗体与CHO-hCD28细胞结合结果如下表39-1至表39-4所示。The above cells were cultured in F12 medium with 10% FBS, placed in a 37°C, 5% CO 2 incubator, and cultured for 2 days. The cells were added to the cell plate at a cell number of 1× 105 per well, and centrifuged at 300g. Wash once for 5 minutes with 1% BSA. The antibody was diluted to 8 concentrations in a gradient, and 100 μL per well was added to the cell plate, incubated at 4°C for 1 hour, washed once with 1% BSA, and 100 μL of AF488-Goat Anti-human IgG Fc fluorescent secondary antibody (Invitrogen, A-11013) was added to each well. dilution (1:400) and incubate at 4°C for 1 hour. Wash the plate three times with 1% BSA, add 100 μL PBS to each well and read the plate. The binding results of each antibody to CHO-hCD28 cells are shown in Table 39-1 to Table 39-4 below.
表39-1.PSMA-CD28双特异性抗体与CHO-hPSMA细胞的结合
Table 39-1. Binding of PSMA-CD28 bispecific antibody to CHO-hPSMA cells
表39-2.PSMA-CD28双特异性抗体与CHO-hPSMA细胞的结合

Table 39-2. Binding of PSMA-CD28 bispecific antibody to CHO-hPSMA cells

表39-3.PSMA-CD28双特异性抗体与CHO-hCD28细胞的结合(荧光值)
Table 39-3. Binding of PSMA-CD28 bispecific antibody to CHO-hCD28 cells (fluorescence value)
表39-4.PSMA-CD28双特异性抗体抗与CHO-hCD28细胞的结合(荧光值)
Table 39-4. Binding of PSMA-CD28 bispecific antibody to CHO-hCD28 cells (fluorescence value)
结果显示，本披露PSMA-CD28双特异性抗体与表达PSMA和CD28的细胞均有较好结合作用。The results show that the PSMA-CD28 bispecific antibody of the present disclosure has a good binding effect on cells expressing PSMA and CD28.
测试例8.PSMA/CD28双特异性抗体与PSMA/CD3双特异性抗体与PSMA共结合Test Example 8. PSMA/CD28 bispecific antibody and PSMA/CD3 bispecific antibody co-bind with PSMA
为测试本披露PSMA-CD28双特异性抗体与PSMA-CD3双特异性抗体共同结 合PSMA抗原的结合能力，本测试例用流式细胞术方法检测了双特异性抗体与过表达人PSMA的稳转细胞株CHO-hPSMA结合能力。具体如下：To test the disclosed PSMA-CD28 bispecific antibodies co-binding with PSMA-CD3 bispecific antibodies Combined with the binding ability of PSMA antigen, this test example uses flow cytometry to detect the binding ability of bispecific antibodies to the stably transduced cell line CHO-hPSMA that overexpresses human PSMA. details as follows:
CHO-hPSMA细胞培养于10％FBS的F12培养基中，放置于37℃，5％CO2培养箱中，培养2天，按每孔细胞数1×105个将细胞加至细胞板中，300g离心5分钟，1％BSA洗一次。PSMA/CD28双特异性抗体稀释到40μg/mL，按每孔100μL加入细胞板中，4℃孵育1小时，1％BSA洗三次。预先用生物素标记(DO JINDO，LK03)PSMA-CD3双特异性抗体15-F5-1和19-F1，并梯度稀释8个浓度，按每孔100μL加入细胞板中以重悬细胞，4℃孵育1小时，1％BSA洗一次，每孔加入100μL SA-PE荧光二抗(Biolegend，740452)稀释液，4℃孵育1个小时。1％BSA洗板三次，每孔加入100μL PBS读板。PSMA/CD28双特异性抗体预先与细胞结合后，PSMA/CD3双特异性抗体在各浓度条件下与CHO-hPSMA细胞结合情况与如下表40所示。CHO-hPSMA cells were cultured in F12 medium with 10% FBS, placed in a 37°C, 5% CO 2 incubator, and cultured for 2 days. The cells were added to the cell plate at a rate of 1×10 5 cells per well. Centrifuge at 300g for 5 minutes and wash once with 1% BSA. PSMA/CD28 bispecific antibody was diluted to 40 μg/mL, 100 μL per well was added to the cell plate, incubated at 4°C for 1 hour, and washed three times with 1% BSA. PSMA-CD3 bispecific antibodies 15-F5-1 and 19-F1 were pre-labeled with biotin (DO JINDO, LK03) and serially diluted to 8 concentrations. Add 100 μL per well to the cell plate to resuspend the cells at 4°C. Incubate for 1 hour, wash once with 1% BSA, add 100 μL of SA-PE fluorescent secondary antibody (Biolegend, 740452) dilution to each well, and incubate at 4°C for 1 hour. Wash the plate three times with 1% BSA, add 100 μL PBS to each well and read the plate. After the PSMA/CD28 bispecific antibody is pre-bound to the cells, the binding of the PSMA/CD3 bispecific antibody to CHO-hPSMA cells under various concentration conditions is shown in Table 40 below.
表40.PSMA-CD3双抗与CHO-hPSMA细胞的结合


注：NC表示未加PSMA/CD28双特异性抗体预处理。Table 40. Binding of PSMA-CD3 double antibody to CHO-hPSMA cells


Note: NC means no pretreatment with PSMA/CD28 bispecific antibody.
结果表明，对于PSMA/CD3双特异性抗体15-F5-1，其PSMA靶向区域为人源化抗体15可变区，因此对于采用相同表位的15作为PSMA/CD28双特异性抗体PSMA靶向区域时，预先孵育饱和剂量PSMA/CD28双特异性抗体会影响后续PSMA/CD3双特异性抗体与PSMA的结合；但对于采用不同表位的19或31作为PSMA/CD28双特异性抗体PSMA靶向区域时，PSMA/CD28双特异性抗体与PSMA/CD3双特异性抗体可共同结合到CHO-hPSMA细胞上。The results show that for the PSMA/CD3 bispecific antibody 15-F5-1, the PSMA targeting region is the humanized antibody 15 variable region, so for 15 using the same epitope as the PSMA/CD28 bispecific antibody PSMA targeting region, pre-incubation with a saturating dose of PSMA/CD28 bispecific antibody will affect the subsequent binding of PSMA/CD3 bispecific antibody to PSMA; but for 19 or 31 using different epitopes as PSMA/CD28 bispecific antibody PSMA targeting region, the PSMA/CD28 bispecific antibody and the PSMA/CD3 bispecific antibody can jointly bind to CHO-hPSMA cells.
对于PSMA/CD3双特异性抗19-F1，其PSMA靶向区域为人源化抗体19，对于采用相同或相似表位的19或31作为PSMA/CD28双特异性抗体PSMA靶向区域时，预先孵育饱和剂量PSMA/CD28双特异性抗体会影响后续PSMA/CD3双特异性抗体与PSMA的结合；但对于采用不同表位的15作为PSMA/CD28双特异性抗体PSMA靶向区域时，PSMA/CD28双特异性抗体与PSMA/CD3双特异性抗体可共同结合到CHO-hPSMA细胞上。For PSMA/CD3 bispecific anti-19-F1, the PSMA targeting region is humanized antibody 19. When using the same or similar epitope 19 or 31 as the PSMA/CD28 bispecific antibody PSMA targeting region, pre-incubation The saturating dose of PSMA/CD28 bispecific antibody will affect the subsequent binding of PSMA/CD3 bispecific antibody to PSMA; but when using 15 with different epitopes as the PSMA target region of the PSMA/CD28 bispecific antibody, the PSMA/CD28 bispecific antibody Specific antibodies and PSMA/CD3 bispecific antibodies can bind to CHO-hPSMA cells together.
测试例9.本披露的双特异性抗体的Biacore结合作用Test Example 9. Biacore Binding Effect of Bispecific Antibodies of the Present Disclosure
为测试本披露PSMA/CD28双特异性抗体与CD28的结合能力，用Protein A生物传感芯片亲和捕获抗体，然后于芯片表面流经hCD28-his抗原，用Biacore T200仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用Glycine 1.5将生物传感芯片洗净再生。数据拟合模型采用1:1Model。各抗体结合及解离情况如下表41-1和41-2所示。In order to test the binding ability of the disclosed PSMA/CD28 bispecific antibody to CD28, the Protein A biosensor chip was used to affinity capture the antibody, and then the hCD28-his antigen was flowed on the chip surface, and the Biacore T200 instrument was used to detect the reaction signal in real time to obtain the binding and dissociation curves. After the dissociation of each experimental cycle is completed, the biosensor chip is washed and regenerated with Glycine 1.5. The data fitting model uses 1:1Model. The binding and dissociation conditions of each antibody are shown in Tables 41-1 and 41-2 below.
表41-1.双特异性抗体的BIACORE结合结果
Table 41-1. BIACORE binding results of bispecific antibodies
表41-2.双特异性抗体的BIACORE结合结果
Table 41-2. BIACORE binding results of bispecific antibodies
结果显示，本披露双特异性抗体与CD28抗原均具有良好结合作用。The results show that the bispecific antibody of the present disclosure has good binding effect with CD28 antigen.
测试例10.双特异性抗体的激活作用Test Example 10. Activation of bispecific antibodies
为测试本披露PSMA/CD28双特异性抗体对T细胞的激活作用，本测试例检测了双特异性抗体在T细胞存在第一激活信号的情况下对Jurkat-IL2lum细胞的激活作用。用没有抗性的培养基稀释CHO-APC-hPSMA细胞到2E5/mL，加入到细胞培养板中，100μL/孔。37℃CO2培养箱中过夜。用补充了10％FBS的培养基稀释抗体到起始浓度5μg/mL，4倍稀释，弃掉细胞板中的培养基，然后加入50μL/孔稀释好的抗体，37℃CO2培养箱孵育1小时。用无抗生素的1640培养基(Hyclone,SH30027.01)稀释Jurkat-IL2lum细胞到5E5/mL；用排枪吸掉细胞板中的抗体，加入100μL/孔稀释好的Jurkat-IL2Lum细胞。37℃,CO2培养箱孵育5小时。加入50μL/孔荧光素酶底物(美天仑，MA0519-2)，室温孵育10分钟，然后转移100μL的反应液到白色不透明底板中，上机读数。各双抗对Jurkat-IL2lum激活作用见下表42。In order to test the activation effect of the PSMA/CD28 bispecific antibody of the present disclosure on T cells, this test example detects the activation effect of the bispecific antibody on Jurkat-IL2lum cells in the presence of the first activation signal of T cells. Dilute CHO-APC-hPSMA cells with non-resistant medium to 2E5/mL and add to the cell culture plate at 100 μL/well. Place in a 37 °C CO 2 incubator overnight. Dilute the antibody to a starting concentration of 5 μg/mL with culture medium supplemented with 10% FBS, dilute 4 times, discard the culture medium in the cell plate, then add 50 μL/well of the diluted antibody, and incubate in a 37°C CO 2 incubator for 1 Hour. Dilute Jurkat-IL2lum cells to 5E5/mL with antibiotic-free 1640 medium (Hyclone, SH30027.01); use a volley gun to suck off the antibodies in the cell plate, and add 100 μL/well of diluted Jurkat-IL2lum cells. Incubate in a 37°C, CO2 incubator for 5 hours. Add 50 μL/well luciferase substrate (Miltenren, MA0519-2), incubate at room temperature for 10 minutes, then transfer 100 μL of the reaction solution to a white opaque bottom plate, and read on the machine. The effects of each dual antibody on Jurkat-IL2lum activation are shown in Table 42 below.
表42.双特异性抗体对Jurkat-IL2lum细胞的激活作用
Table 42. Activation effect of bispecific antibodies on Jurkat-IL2lum cells
结果显示，本披露双特异性抗体在第一信号存在情况下能有效刺激T细胞IL-2信号通路。The results show that the bispecific antibody of the present disclosure can effectively stimulate the T cell IL-2 signaling pathway in the presence of the first signal.
测试例11.双特异性抗体的体外细胞毒性Test Example 11. In vitro cytotoxicity of bispecific antibodies
本测试例研究了本披露的双特异性抗体在T细胞第一刺激信号存在情况下的 杀伤作用。用表达PSMA的细胞系CHO-APC-hPSMA细胞的作为靶细胞来检测本披露的双特异性抗体的靶点特异性细胞毒活性。This test case studies the performance of the bispecific antibody of the present disclosure in the presence of the first T cell stimulation signal. Killing effect. The PSMA-expressing cell line CHO-APC-hPSMA cells were used as target cells to detect the target-specific cytotoxic activity of the bispecific antibodies of the present disclosure.
CHO-APC-hPSMA细胞培养在补充了10％FBS(ThermoFisher Scientific，10099-141)的DMEM/F12(美仑生物，PWL005)完全培养基中。冻存PBMC(妙顺生物)复苏后用补充了10％FBS的1640(Gibco，11875119)完全培养基重悬，置于T75培养瓶培养4小时(密度为2E6cells/mL)。PBMC收集离心后用补充了4％FBS无酚红1640(Gibco，11835-030)重悬，再次离心后重悬、计数，调整细胞数为7.5E5细胞/mL，每孔加入50μL。CHO-APC-hPSMA细胞消化，1000rpm离心3分钟，重悬，计数，调整细胞数为1.5E5细胞/mL，每孔加入50μL，确保E:T比值为5:1。抗体用无酚红1640+4％FBS培养基稀释至起始浓度50nM，5倍稀释，每孔加入50μL。处理好的细胞放在37℃，5％CO2的培养箱培养72小时。检测前，在只有靶细胞的其中两个孔中吸出15μL培养基，然后加入15μL裂解液(10X)，裂解45分钟。45分钟后取出培养板，1000rpm离心3分钟，吸取50μL上清于新的96孔板中，并加入50μL CytoToxReagent(Promega，G1780)，室温孵育0.5小时后加入50μL终止液，用FlexStation 3检测吸收光(490nm)。CHO-APC-hPSMA cells were cultured in DMEM/F12 (Meilun Biotech, PWL005) complete medium supplemented with 10% FBS (ThermoFisher Scientific, 10099-141). After resuscitation, the frozen PBMC (Miaoshun Biotech) were resuspended in 1640 (Gibco, 11875119) complete medium supplemented with 10% FBS, and placed in a T75 culture flask for 4 hours (density: 2E6 cells/mL). PBMC were collected and centrifuged and resuspended in phenol red-free 1640 (Gibco, 11835-030) supplemented with 4% FBS. They were centrifuged again and resuspended and counted. The cell number was adjusted to 7.5E5 cells/mL and 50 μL was added to each well. Digest CHO-APC-hPSMA cells, centrifuge at 1000 rpm for 3 minutes, resuspend, count, adjust the number of cells to 1.5E5 cells/mL, add 50 μL to each well to ensure that the E:T ratio is 5:1. The antibody was diluted to a starting concentration of 50 nM with phenol red-free 1640+4% FBS culture medium, diluted 5 times, and added 50 μL to each well. The treated cells were cultured in a 37°C, 5% CO2 incubator for 72 hours. Before detection, aspirate 15 μL of culture medium from two wells containing only target cells, then add 15 μL of lysis solution (10X) and lyse for 45 minutes. After 45 minutes, take out the culture plate, centrifuge at 1000 rpm for 3 minutes, pipet 50 μL of supernatant into a new 96-well plate, and add 50 μL of CytoTox Reagent (Promega, G1780), incubate at room temperature for 0.5 hours, add 50 μL of stop solution, and use FlexStation 3 to detect absorbance (490 nm).
靶细胞完全裂解的值减去靶细胞的背景值作为100％裂解值，即最大LDH释放，在计算之前所有组别减去仅PBMC组的背景值，同浓度下的杀伤百分比为：The value of complete lysis of target cells minus the background value of target cells is taken as the 100% lysis value, that is, the maximum LDH release. Before calculation, all groups subtract the background value of only the PBMC group. The killing percentage at the same concentration is:
裂解率％＝100×检测孔OD490/LDH最大释放量孔OD490Cleavage rate% = 100×detection hole OD490/LDH maximum release hole OD490
双特异性抗体的杀伤作用如下表43。The killing effect of bispecific antibodies is shown in Table 43.
表43.PSMA-CD28双特异性抗体的细胞杀伤作用
Table 43. Cell killing effect of PSMA-CD28 bispecific antibody
结果显示，在TCR第一激活信号存在时，本披露双特异性抗体对表达PSMA的细胞表现出较强肿瘤杀伤作用。The results showed that in the presence of the first TCR activation signal, the bispecific antibody of the present disclosure showed a strong tumor killing effect on PSMA-expressing cells.
测试例12.PSMA/CD28双特异性抗体与PSMA/CD3双特异性抗体的联合激活作用Test Example 12. Joint activation of PSMA/CD28 bispecific antibody and PSMA/CD3 bispecific antibody
为测试本披露PSMA/CD28双特异性抗体与PSMA/CD3双抗联用对T细胞的激活作用，本测试例检测了PSMA/CD28与PSMA/CD3双抗联用对Jurkat-IL2lum细胞的激活作用。In order to test the activation effect of the disclosed PSMA/CD28 bispecific antibody and PSMA/CD3 dual antibody in combination with T cells, this test example detects the activation effect of PSMA/CD28 in combination with PSMA/CD3 dual antibody on Jurkat-IL2lum cells. .
计数仪计数CHO-hPSMA细胞，用没有抗性的培养基稀释到2E5/mL，加入到细胞培养板中，100μL/孔。37℃CO2培养箱中过夜。用补充了10％FBS的培养基稀释PSMA/CD28抗体到4X起始浓度20μg/mL，4倍稀释。同时用补充了10％FBS的培养基稀释PSMA/CD3双特异性抗体15-F5-1和15-F5-2到80ng/mL，19-F1稀 释到1250ng/mL。弃掉细胞板中的培养基，然后各加入50μL/孔稀释好的PSMA/CD28双抗和PSMA/CD3双抗，37℃CO2培养箱孵育1小时。用无抗生素的1640培养基(Hyclone,SH30027.01)稀释Jurkat-IL2lum细胞到5E5/mL；吸掉细胞板中的抗体，加入100μL/孔稀释好的Jurkat-IL2Lum细胞。37℃,CO2培养箱孵育5小时。加入50μL/孔荧光素酶底物(美天仑，MA0519-2)，室温孵育10分钟，然后转移100μL的反应液到白色不透明底板中，上机读数。各双抗联用对Jurkat-IL2lum激活作用见下表44。Count CHO-hPSMA cells with a counter, dilute them to 2E5/mL with non-resistant medium, and add 100 μL/well to the cell culture plate. Place in a 37 °C CO 2 incubator overnight. Dilute the PSMA/CD28 antibody to 4X the starting concentration of 20 μg/mL in medium supplemented with 10% FBS, 4-fold dilution. At the same time, PSMA/CD3 bispecific antibodies 15-F5-1 and 15-F5-2 were diluted to 80ng/mL with medium supplemented with 10% FBS, and 19-F1 was diluted Released to 1250ng/mL. Discard the culture medium in the cell plate, then add 50 μL/well of diluted PSMA/CD28 double antibody and PSMA/CD3 double antibody, and incubate in a 37°C CO 2 incubator for 1 hour. Dilute Jurkat-IL2lum cells to 5E5/mL with antibiotic-free 1640 medium (Hyclone, SH30027.01); aspirate the antibodies in the cell plate and add 100 μL/well of diluted Jurkat-IL2lum cells. Incubate in a 37°C, CO2 incubator for 5 hours. Add 50 μL/well luciferase substrate (Miltenren, MA0519-2), incubate at room temperature for 10 minutes, then transfer 100 μL of the reaction solution to a white opaque bottom plate, and read on the machine. The activation effects of each dual antibody combination on Jurkat-IL2lum are shown in Table 44 below.
表44.双特异性抗体联用对Jurkat-IL2lum细胞的激活作用
Table 44. Activation effect of bispecific antibody combination on Jurkat-IL2lum cells
结果显示，本披露PSMA/CD28双特异性抗体与PSMA/CD3双特异性抗体联用可显著增强对T细胞的激活作用。The results show that the combination of the disclosed PSMA/CD28 bispecific antibody and the PSMA/CD3 bispecific antibody can significantly enhance the activation of T cells.
测试例13.双特异性抗体联用的体外细胞毒性(固定PSMA/CD3双特异性抗体的剂量)Test Example 13. In vitro cytotoxicity of bispecific antibody combination (fixed dose of PSMA/CD3 bispecific antibody)
本测试例检测了PSMA/CD28双特异性抗体与PSMA/CD3双特异性抗体联用介导的杀伤作用。This test example detects the killing effect mediated by the combination of PSMA/CD28 bispecific antibody and PSMA/CD3 bispecific antibody.
CHO-huPSMA细胞培养在补充了10％FBS(ThermoFisher Scientific，10099-141)的DMEM/F12(美仑生物，PWL005)完全培养基中。冻存PBMC(妙顺生物)复苏后用补充了10％FBS的1640(Gibco，11875119)完全培养基重悬，置于T75培养瓶培养4小时(密度为2E6细胞/mL)。PBMC收集离心后用补充了4％FBS无酚红1640培养基(Gibco，11835-030)重悬，再次离心后重悬、计数，调整细胞数为1.5E6细胞/mL，每孔加入50μL。CHO-huPSMA细胞消化，1000rpm离心3分钟，重悬，计数，调整细胞数为1.5E5细胞/mL，每孔加入50μL，确保E:T比值为10:1。抗体用无酚红1640+4％FBS培养基稀释，其中PSMA-CD3双抗CC-1配置成16pM，同时配置PSMA/CD28双特异性抗体，起始浓度为100nM，5倍稀 释，9个剂量点。每孔加入PSMA/CD3双抗及PSMA-CD28各25μL，处理好的细胞放在37℃，5％CO2的培养箱培养72小时。检测前，在只有靶细胞的其中两个孔中吸出15μL培养基，然后加入试剂盒提供的15μL裂解液(10X)，裂解45分钟。45分钟后取出培养板，1000rpm离心3分钟，吸取50μL上清于新的96孔板(#3590)中，并加入50μL CytoToxReagent(Promega，G1780)，室温孵育0.5小时后加入50μL终止液，用FlexStation 3检测吸收光(490nm)。CHO-huPSMA cells were cultured in DMEM/F12 (Meilun Biotechnology, PWL005) complete medium supplemented with 10% FBS (ThermoFisher Scientific, 10099-141). After resuscitation, the frozen PBMC (Miaoshun Biotech) were resuspended in 1640 (Gibco, 11875119) complete medium supplemented with 10% FBS, and placed in a T75 culture flask for 4 hours (density: 2E6 cells/mL). PBMC were collected, centrifuged, and resuspended in phenol red-free 1640 medium (Gibco, 11835-030) supplemented with 4% FBS, centrifuged again, resuspended, counted, and the cell number was adjusted to 1.5E6 cells/mL, and 50 μL was added to each well. Digest CHO-huPSMA cells, centrifuge at 1000 rpm for 3 minutes, resuspend, count, adjust the number of cells to 1.5E5 cells/mL, add 50 μL to each well, and ensure that the E:T ratio is 10:1. The antibody was diluted with phenol red-free 1640+4% FBS culture medium, in which the PSMA-CD3 dual anti-CC-1 was configured at 16pM, and the PSMA/CD28 bispecific antibody was configured at the same time. The starting concentration was 100nM, 5 times diluted. release, 9 dose points. Add 25 μL of PSMA/CD3 double antibody and PSMA-CD28 to each well, and culture the treated cells in a 37°C, 5% CO 2 incubator for 72 hours. Before detection, aspirate 15 μL of culture medium from two wells containing only target cells, then add 15 μL of lysis solution (10X) provided by the kit, and lyse for 45 minutes. After 45 minutes, take out the culture plate, centrifuge at 1000 rpm for 3 minutes, pipet 50 μL of supernatant into a new 96-well plate (#3590), and add 50 μL of CytoTox Reagent (Promega, G1780), incubate at room temperature for 0.5 hours, add 50 μL of stop solution, and use FlexStation 3 to detect absorbance (490 nm).
靶细胞完全裂解的值减去靶细胞的背景值作为100％裂解值，即LDH最大释放，在计算之前所有组别减去PBMC only组的背景值，同浓度下的杀伤百分比为：The value of complete lysis of the target cells minus the background value of the target cells is taken as the 100% lysis value, that is, the maximum release of LDH. Before calculation, the background value of the PBMC only group is subtracted from all groups. The killing percentage at the same concentration is:
裂解率％＝100×检测孔OD490/LDH最大释放量孔OD490。Cleavage rate% = 100×detection hole OD490/LDH maximum release hole OD490.
各抗体联用的杀伤作用见图2。The killing effect of each antibody combination is shown in Figure 2.
结果显示，本披露双特异性PSMA/CD28双特异性抗体单独无法诱导明显杀伤作用，这与该试验体系不存在第一信号相关。在PSMA/CD28双特异性抗体低剂量条件下，PSMA/CD3双特异性抗体由于剂量较低而无明显杀伤作用，但增强PSMA/CD28双特异性抗体的剂量能在低剂量PSMA-CD3双特异性抗体存在时发挥对PSMA表达细胞的肿瘤杀伤作用。The results show that the disclosed bispecific PSMA/CD28 bispecific antibody alone cannot induce significant killing effect, which is related to the absence of a first signal in the test system. Under the condition of low dose of PSMA/CD28 bispecific antibody, PSMA/CD3 bispecific antibody has no obvious killing effect due to the low dose. However, the dose of enhanced PSMA/CD28 bispecific antibody can improve the performance of PSMA/CD3 bispecific antibody at low dose. In the presence of specific antibodies, it exerts a tumor-killing effect on PSMA-expressing cells.
测试例14.双特异性抗体联用的体外细胞毒性(固定PSMA/CD28双特异性抗体剂量)Test Example 14. In vitro cytotoxicity of bispecific antibody combination (fixed PSMA/CD28 bispecific antibody dose)
本测试例检测了PSMA/CD28双特异性抗体与PSMA/CD3双特异性抗体联用介导的杀伤作用。按照测试例13所述方法培养细胞。冻存PBMC(妙顺生物)复苏后用补充了10％FBS的1640(Gibco，11875119)完全培养基重悬，置于T75培养瓶培养4小时(密度为2E6细胞/mL)。PBMC收集离心后用补充了4％FBS无酚红1640(Gibco，11835-030)重悬，再次离心后重悬、计数，调整细胞数为1.5E6细胞/mL，每孔加入50μL。CHO-huPSMA细胞消化，1000rpm离心3分钟，重悬，计数，调整细胞数为1.5E5细胞/mL，每孔加入50μL，确保E:T比值为10:1。抗体用无酚红1640+4％FBS培养基稀释，其中PSMA/CD28抗体稀释至4nM，同时将PSMA-CD3双抗配置至起始浓度4nM，5倍稀释，9个剂量点；每孔加入PSMA/CD3及PSMA/CD28各25μL，处理好的细胞放在37℃，5％CO2的培养箱培养72小时。检测前，在只有靶细胞的其中两个孔中吸出15μL培养基，然后加入试剂盒提供的15μL裂解液(10X)，裂解45分钟。45分钟后取出培养板，1000rpm离心3分钟，吸取50μL上清于新的96孔板中，并加入50μL CytoToxReagent(Promega，G1780)，室温孵育0.5小时后加入50μL终止液，用FlexStation3检测吸收光(490nm)。This test example detects the killing effect mediated by the combination of PSMA/CD28 bispecific antibody and PSMA/CD3 bispecific antibody. Cells were cultured according to the method described in Test Example 13. After resuscitation, the frozen PBMC (Miaoshun Biotech) were resuspended in 1640 (Gibco, 11875119) complete medium supplemented with 10% FBS, and placed in a T75 culture flask for 4 hours (density: 2E6 cells/mL). PBMC were collected and centrifuged and resuspended in phenol red-free 1640 (Gibco, 11835-030) supplemented with 4% FBS. They were centrifuged again and resuspended and counted. The cell number was adjusted to 1.5E6 cells/mL and 50 μL was added to each well. Digest CHO-huPSMA cells, centrifuge at 1000 rpm for 3 minutes, resuspend, count, adjust the number of cells to 1.5E5 cells/mL, add 50 μL to each well, and ensure that the E:T ratio is 10:1. The antibody was diluted with phenol red-free 1640+4% FBS culture medium, in which the PSMA/CD28 antibody was diluted to 4nM. At the same time, the PSMA-CD3 double antibody was configured to a starting concentration of 4nM, 5-fold dilution, and 9 dosage points; PSMA was added to each well. /CD3 and PSMA/CD28, 25 μL each, and the treated cells were cultured in a 37°C, 5% CO 2 incubator for 72 hours. Before detection, aspirate 15 μL of culture medium from two wells containing only target cells, then add 15 μL of lysis solution (10X) provided by the kit, and lyse for 45 minutes. After 45 minutes, take out the culture plate, centrifuge at 1000 rpm for 3 minutes, pipet 50 μL of supernatant into a new 96-well plate, and add 50 μL of CytoTox Reagent (Promega, G1780), incubate at room temperature for 0.5 hours, add 50 μL of stop solution, and use FlexStation3 to detect the absorbance (490nm).
靶细胞完全裂解的值减去靶细胞的背景值作为100％裂解值，即LDH最大释放，在计算之前所有组别减去仅PBMC组的背景值，同浓度下的杀伤百分比为：The value of complete lysis of target cells minus the background value of target cells is taken as the 100% lysis value, that is, the maximum release of LDH. Before calculation, all groups subtract the background value of only the PBMC group. The killing percentage at the same concentration is:
裂解率％＝100×检测孔OD490/LDH最大释放量孔OD490。 Cleavage rate% = 100×detection hole OD490/LDH maximum release hole OD490.
各抗体联用的杀伤作用见下表45-1至表45-4。The killing effect of each antibody combination is shown in Table 45-1 to Table 45-4 below.
表45-1.固定浓度的PSMA/CD28双抗对PSMA/CD3双抗CC-1细胞杀伤作用的影响
Table 45-1. Effect of fixed concentration of PSMA/CD28 dual antibody on the killing effect of PSMA/CD3 dual antibody on CC-1 cells
表45-2.固定浓度的PSMA/CD28双抗对PSMA/CD3双抗CC1的细胞杀伤作用的影响
Table 45-2. Effect of fixed concentration of PSMA/CD28 dual antibody on the cell killing effect of PSMA/CD3 dual antibody CC1
表45-3.固定浓度的PSMA/CD28双抗对PSMA/CD3双特异性抗体15-F5-1的细胞杀伤作用的影响
Table 45-3. Effect of fixed concentration of PSMA/CD28 bisantibody on the cell killing effect of PSMA/CD3 bispecific antibody 15-F5-1
表45-4.固定浓度的PSMA/CD28双抗对PSMA/CD3双特异性抗体19-F1的细胞杀伤作用的影响

Table 45-4. Effect of fixed concentration of PSMA/CD28 bisantibody on the cell killing effect of PSMA/CD3 bispecific antibody 19-F1

结果显示，本披露PSMA/CD28双特异性抗体能协同低剂量PSMA/CD3双特异性抗体发挥对表达PSMA细胞更强的杀伤作用。The results show that the disclosed PSMA/CD28 bispecific antibody can cooperate with a low-dose PSMA/CD3 bispecific antibody to exert a stronger killing effect on PSMA-expressing cells.
测试例15.双特异性抗体的细胞因子释放水平Test Example 15. Cytokine release levels of bispecific antibodies
本测试例检测了在PSMA/CD3双抗、PSMA/CD28双抗和CHO-huPSMA细胞的共同作用下，PBMC中细胞因子IL-6及IFN-γ分泌的变化。This test example detects the changes in the secretion of cytokines IL-6 and IFN-γ in PBMC under the combined action of PSMA/CD3 double antibodies, PSMA/CD28 double antibodies and CHO-huPSMA cells.
将测试例13中收集的抗体及细胞混合培养上清冻存于-20℃冰箱的上清，常温解冻，震荡混匀，样品使用1640+4％FBS培养基稀释20倍备用。使用无菌水溶解IL-6及IFN-γ标准品粉末，使用1640+4％FBS培养基稀释标准品至需要的浓度。取出要检测的细胞因子的试剂盒，包括人IL6试剂盒(CISBIO，62HIL06PEG)和人IFN-γ试剂盒(CISBIO，62HIFNGPEG)，平衡试剂盒至常温，使用检测缓冲液稀释试剂盒中的两个检测抗体。取16μL细胞上清(原液用于IL-6检测，20倍稀释用于IFNγ检测)及IL-6或IFNγ标准品于96孔板中，加入4μL稀释好的对应的检测抗体；震荡混匀，1000rpm离心1分钟，常温孵育过夜。取出孵育过夜的样品，1000rpm离心1分钟，使用PHERAstar多功能酶标仪读取665nm和620nm的吸收值。结果见图3和图4。The antibody and cell mixed culture supernatant collected in Test Example 13 was frozen and stored in a -20°C refrigerator, thawed at room temperature, shaken and mixed, and the sample was diluted 20 times with 1640+4% FBS culture medium for later use. Use sterile water to dissolve IL-6 and IFN-γ standard powder, and use 1640+4% FBS culture medium to dilute the standards to the required concentration. Take out the kits for the cytokines to be detected, including human IL6 kit (CISBIO, 62HIL06PEG) and human IFN-γ kit (CISBIO, 62HIFNGPEG), balance the kit to room temperature, and use the detection buffer to dilute the two kits Detect antibodies. Take 16 μL of cell supernatant (original solution is used for IL-6 detection, 20-fold dilution is used for IFNγ detection) and IL-6 or IFNγ standard in a 96-well plate, add 4 μL of the diluted corresponding detection antibody; shake and mix. Centrifuge at 1000 rpm for 1 minute and incubate at room temperature overnight. Take out the sample that was incubated overnight, centrifuge at 1000 rpm for 1 minute, and use a PHERAstar multifunctional microplate reader to read the absorbance values at 665 nm and 620 nm. The results are shown in Figures 3 and 4.
结果显示，本披露的PSMA/CD28双特异性抗体与PSMA/CD3双特异性抗体联用后诱导的PBMC的细胞因子IL6和IFNγ的释放水平相对较低。The results show that the combination of the disclosed PSMA/CD28 bispecific antibody and the PSMA/CD3 bispecific antibody induces relatively low levels of release of cytokines IL6 and IFNγ from PBMC.
测试例16.双特异性抗体与PD-1单抗联用的激活作用Test Example 16. Activation effect of bispecific antibody combined with PD-1 monoclonal antibody
由于PD1/PDL1信号通路会阻断CD28的共刺激信号，为测试本披露PSMA/CD28双特异性抗体与PD1/PDL1联用在T细胞存在第一信号条件下的激活作用，本测试例检测了双特异性抗体与PD1单抗联用对Jurkat-PD1细胞的激活作用。Since the PD1/PDL1 signaling pathway blocks the costimulatory signal of CD28, in order to test the activation effect of the disclosed PSMA/CD28 bispecific antibody in combination with PD1/PDL1 in the presence of the first signal of T cells, this test example detected Activation effect of bispecific antibody combined with PD1 monoclonal antibody on Jurkat-PD1 cells.
CHO-APC-hPSMA-PDL1用无抗性1640培养基稀释到4E5/mL，25μL每孔铺入96孔板；Jurkat-PD1用无抗性1640培养基稀释到4E6/mL，25μL每孔铺入96孔板。PD1单抗HRP01511用无抗性1640培养基稀释到20μLg/mL，每孔加入25μl(终浓度为5μg/mL)；PSMA/CD28双抗用无抗性1640培养基稀释到40μg/mL，每孔25μL(终浓度为10μg/mL)，1：10梯度稀释。在细胞悬液中加入稀释的抗体。终体积100μL，37℃，5％CO2，继续培养72小时。取出细胞培养板离心(300×g，10分钟)；取出16μL上清转移至96孔检测板；按照IL2检测说明书(Cisbio,62HIL02PEG)稀释检测抗体，每孔加入4μL，总体积20μL，贴紧封板膜防止液体挥发。室温放置3小时后上机检测。各PSMA/CD28双抗单用或联用PD1单抗对Jurkat-PD1细胞IL2刺激情况如下表46。 CHO-APC-hPSMA-PDL1 was diluted with non-resistant 1640 medium to 4E5/mL, and 25 μL was spread into each well of a 96-well plate; Jurkat-PD1 was diluted with non-resistant 1640 medium to 4E6/mL, and 25 μL was spread into each well. 96-well plates. PD1 monoclonal antibody HRP01511 was diluted to 20 μLg/mL with non-resistant 1640 medium, and 25 μl was added to each well (final concentration was 5 μg/mL); PSMA/CD28 double antibody was diluted to 40 μg/mL with non-resistant 1640 medium, and added to each well. 25μL (final concentration is 10μg/mL), 1:10 gradient dilution. Add diluted antibodies to the cell suspension. The final volume was 100 μL, and the culture was continued for 72 hours at 37°C, 5% CO 2 . Take out the cell culture plate and centrifuge (300 × g, 10 minutes); take out 16 μL of supernatant and transfer it to a 96-well detection plate; dilute the detection antibody according to the IL2 detection instructions (Cisbio, 62HIL02PEG), add 4 μL to each well, the total volume is 20 μL, and seal tightly The membrane prevents liquid from evaporating. Leave it at room temperature for 3 hours and then put it on the machine for testing. The stimulation of IL2 in Jurkat-PD1 cells by each PSMA/CD28 dual antibody alone or in combination with PD1 monoclonal antibody is shown in Table 46.
表46.PSMA/CD28双特异性抗体联用PD-1抗体后的IL-2刺激作用
Table 46. IL-2 stimulating effect of PSMA/CD28 bispecific antibody combined with PD-1 antibody
结果显示，PD1/PDL1信号通路的存在会抑制本披露双特异性抗体对T细胞激活作用，而本披露双特异性抗体与PD1抗体联用能有效刺激T细胞。The results show that the existence of the PD1/PDL1 signaling pathway will inhibit the activation of T cells by the bispecific antibody of the disclosure, and the combination of the bispecific antibody of the disclosure and the PD1 antibody can effectively stimulate T cells.
体内活性生物学评价Biological evaluation of in vivo activity
测试例17.本披露的双特异性抗体在22RV1皮下移植瘤模型中的药效Test Example 17. The efficacy of the bispecific antibody of the present disclosure in the 22RV1 subcutaneous transplant tumor model
本披露使用人前列腺癌22RV1细胞人PBMC NOG小鼠异种移植瘤模型，对PSMA-CD3联用PSMA/CD28双抗在抗肿瘤活性方面进行评价。This disclosure uses the human prostate cancer 22RV1 cell human PBMC NOG mouse xenograft tumor model to evaluate the anti-tumor activity of PSMA-CD3 combined with PSMA/CD28 dual antibodies.
NOG小鼠，雄性，4-5周龄，购自北京维通利华上海分公司。NOG mice, male, 4-5 weeks old, were purchased from Beijing Vitong Lever Shanghai Branch.
人前列腺癌22RV1细胞，购于ATCC。Human prostate cancer 22RV1 cells were purchased from ATCC.
将22RV1细胞1×106细胞/只/200μL(含50％matrigel)接种于NOG小鼠右肋部皮下，次日将复苏后状态良好的人PBMC以5×106细胞/100μL注射到NOG小鼠腹腔，当荷瘤小鼠肿瘤体积达到130mm3左右时，去除体积过大过小、体重过小的进行分组给药。将分组当天定义为该实验Day0(第零天)，Day0开始腹腔注射各抗体，每周2次，共给药5次。每周2次监测肿瘤体积、动物重量并记录数据。当实验动物肿瘤体积超过2000mm3或多数肿瘤出现破溃或体重下降20％时，将荷瘤动物进行安乐死作为实验终点。22RV1 cells 1×10 6 cells/mouse/200 μL (containing 50% matrigel) were inoculated subcutaneously into the right flank of NOG mice. The next day, human PBMC in good condition after recovery were injected into NOG mice at 5×10 6 cells/100 μL. In the abdominal cavity of mice, when the tumor volume of tumor-bearing mice reaches about 130 mm 3 , the tumors that are too large, too small, or those with too small body weight will be removed and administered in groups. The day of grouping was defined as day 0 (day zero) of the experiment. On day 0, each antibody was injected intraperitoneally, twice a week, for a total of 5 times. Tumor volume and animal weight were monitored twice a week and data were recorded. When the tumor volume of the experimental animals exceeds 2000 mm 3 or most tumors appear to be ruptured or the body weight decreases by 20%, the tumor-bearing animals will be euthanized as the end point of the experiment.
肿瘤体积(V)计算公式为：V＝1/2×a×b2其中a、b分别表示长、宽。The calculation formula of tumor volume (V) is: V=1/2×a×b 2 , where a and b represent length and width respectively.
相对肿瘤增殖率T/C(％)＝(T-T0)/(C-C0)×100％，其中T、C为实验结束时治疗组和对照组的肿瘤体积；T0、C0为实验开始时的肿瘤体积。Relative tumor proliferation rate T/C (%) = (T-T0)/(C-C0)×100%, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are at the beginning of the experiment tumor volume.
抑瘤率TGI(％)＝1-T/C(％)。Tumor inhibition rate TGI(%)=1-T/C(%).
表47.PSMA/CD28与PSMA/CD3双特异性抗体15-F5-1联用的抗肿瘤活性
Table 47. Anti-tumor activity of PSMA/CD28 in combination with PSMA/CD3 bispecific antibody 15-F5-1
结果显示，本披露PSMA/CD28双抗能有效提高PSMA/CD3双抗的药效。实 验期间未观测到药物相关动物死亡及其他明显药物相关毒副反应。The results show that the disclosed PSMA/CD28 dual antibodies can effectively improve the efficacy of PSMA/CD3 dual antibodies. Reality During the trial, no drug-related animal deaths or other obvious drug-related toxic and side effects were observed.
测试例18.本披露的双特异性抗体在22RV1皮下移植瘤模型中的药效Test Example 18. The efficacy of the bispecific antibody of the present disclosure in the 22RV1 subcutaneous transplant tumor model
本披露使用人前列腺癌22RV1细胞人PBMC NCG小鼠异种移植瘤模型，对PSMA-CD3联用PSMA/CD28双抗在抗肿瘤活性方面进行评价。This disclosure uses the human prostate cancer 22RV1 cell human PBMC NCG mouse xenograft tumor model to evaluate the anti-tumor activity of PSMA-CD3 combined with PSMA/CD28 dual antibodies.
NOG小鼠，雄性，4-5周龄，购自北京维通利华上海分公司。NOG mice, male, 4-5 weeks old, were purchased from Beijing Vitong Lever Shanghai Branch.
人前列腺癌22RV1细胞，购于ATCC。Human prostate cancer 22RV1 cells were purchased from ATCC.
将22RV1细胞1×106细胞/只/200μL(含50％matrigel)接种于NOG小鼠右肋部皮下，次日将复苏后状态良好的人PBMC以4.5×106细胞/100μL注射到NOG小鼠腹腔，当荷瘤小鼠肿瘤体积达到110mm3左右时，去除体积过大过小、体重过小的进行分组给药。将分组当天定义为该实验Day0(第零天)，Day0开始腹腔注射各抗体，每周2次，共给药4次。每周2次监测肿瘤体积、动物重量并记录数据。当实验动物肿瘤体积超过2000mm3或多数肿瘤出现破溃或体重下降20％时，将荷瘤动物进行安乐死作为实验终点。22RV1 cells 1×10 6 cells/mouse/200 μL (containing 50% matrigel) were inoculated subcutaneously into the right flank of NOG mice. The next day, human PBMC in good condition after recovery were injected into NOG mice at 4.5×10 6 cells/100 μL. In the abdominal cavity of mice, when the tumor volume of tumor-bearing mice reaches about 110 mm 3 , the tumors that are too large, too small, or those with too small body weight will be removed and administered in groups. The day of grouping was defined as Day 0 (day zero) of the experiment. On Day 0, each antibody was injected intraperitoneally, twice a week, for a total of 4 times. Tumor volume and animal weight were monitored twice a week and data were recorded. When the tumor volume of the experimental animals exceeds 2000 mm 3 or most tumors appear to be ruptured or the body weight decreases by 20%, the tumor-bearing animals will be euthanized as the end point of the experiment.
肿瘤体积(V)计算公式为：V＝1/2×a×b2，其中a、b分别表示长、宽。The calculation formula of tumor volume (V) is: V=1/2×a×b 2 , where a and b represent length and width respectively.
相对肿瘤增殖率T/C(％)＝(T-T0)/(C-C0)×100％，其中T、C为实验结束时治疗组和对照组的肿瘤体积；T0、C0为实验开始时的肿瘤体积。Relative tumor proliferation rate T/C (%) = (T-T0)/(C-C0)×100%, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are at the beginning of the experiment tumor volume.
抑瘤率TGI(％)＝1-T/C(％)。Tumor inhibition rate TGI(%)=1-T/C(%).
表48.PSMA/CD28与PSMA/CD3双特异性抗体15-F5-2联用的抗肿瘤活性

注：“/”表示无抑制肿瘤的作用。Table 48. Anti-tumor activity of PSMA/CD28 combined with PSMA/CD3 bispecific antibody 15-F5-2

Note: “/” means no tumor inhibitory effect.
结果显示，本披露PSMA/CD28双抗能有效提高低剂量PSMA/CD3双抗的药效。PSMA/CD28双抗本身对该模型无明显肿瘤抑制作用。实验期间未观测到药物相关动物死亡及其他明显药物相关毒副反应。The results show that the disclosed PSMA/CD28 dual antibodies can effectively improve the efficacy of low-dose PSMA/CD3 dual antibodies. The PSMA/CD28 double antibody itself has no obvious tumor inhibitory effect on this model. During the experiment, no drug-related animal deaths or other obvious drug-related toxic and side effects were observed.
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本披露的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。 Although the above invention has been described in detail by means of the drawings and examples for the purpose of clear understanding, the description and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety.

Claims (22)

  1. 一种抗原结合分子，其特异性结合CD28和PSMA，所述抗原结合分子包含至少一个特异性结合CD28的抗原结合模块和至少一个特异性结合PSMA的抗原结合模块，所述特异性结合CD28的抗原结合模块包含重链可变区CD28-VH和轻链可变区CD28-VL，所述特异性结合PSMA的抗原结合模块包含重链可变区PSMA-VH和轻链可变区PSMA-VL；其中：An antigen-binding molecule that specifically binds CD28 and PSMA, the antigen-binding molecule comprising at least one antigen-binding module that specifically binds CD28 and at least one antigen-binding module that specifically binds PSMA, and the antigen that specifically binds CD28 The binding module includes the heavy chain variable region CD28-VH and the light chain variable region CD28-VL, and the antigen-binding module that specifically binds PSMA includes the heavy chain variable region PSMA-VH and the light chain variable region PSMA-VL; in:
    i)所述CD28-VH具有：包含SEQ ID NO：19的氨基酸序列的CD28-HCDR1，包含SEQ ID NO：20的氨基酸序列的CD28-HCDR2和包含SEQ ID NO：21的氨基酸序列的CD28-HCDR3；和所述CD28-VL具有：包含SEQ ID NO：22的氨基酸序列的CD28-LCDR1，包含SEQ ID NO：23、81、82、83、84、85、86、87、88、89、90、91、92或93的氨基酸序列的CD28-LCDR2，和包含SEQ ID NO：24的氨基酸序列的CD28-LCDR3；或i) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 and CD28-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21 ; and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, including SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, CD28-LCDR2 with the amino acid sequence 91, 92 or 93, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24; or
    ii)所述CD28-VH具有：包含SEQ ID NO：13的氨基酸序列的CD28-HCDR1，包含SEQ ID NO：14的氨基酸序列的CD28-HCDR2，和包含SEQ ID NO：15的氨基酸序列的CD28-HCDR3；和所述CD28-VL具有：包含SEQ ID NO：16的氨基酸序列的CD28-LCDR1，包含SEQ ID NO：62、17、54、55、56、57、58、59、60或61的氨基酸序列的CD28-LCDR2，和包含SEQ ID NO：18的氨基酸序列的CD28-LCDR3；或ii) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and CD28- comprising the amino acid sequence of SEQ ID NO: 15 HCDR3; and the CD28-VL having: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, comprising the amino acid sequence of SEQ ID NO: 62, 17, 54, 55, 56, 57, 58, 59, 60 or 61 CD28-LCDR2 of the sequence, and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18; or
    iii)所述CD28-VH具有：包含SEQ ID NO：25或117的氨基酸序列的CD28-HCDR1，包含SEQ ID NO：26或116的氨基酸序列的CD28-HCDR2，和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3；和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1，包含SEQ ID NO：29、118、119、120、121或122的氨基酸序列的CD28-LCDR2，和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或iii) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or 117, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 116, and comprising the amino acid sequence of SEQ ID NO: 27 a CD28-HCDR3 of the sequence; and the CD28-VL has: a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 LCDR2, and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30; or
    iv)所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1，包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2，和包含SEQ ID NO：9、39、40、41或42的氨基酸序列的CD28-HCDR3；和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1，包含SEQ ID NO：43或11的氨基酸序列的CD28-LCDR2，和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3；iv) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, 39, 40, 41 or a CD28-HCDR3 with an amino acid sequence of 42; and the CD28-VL has: a CD28-LCDR1 comprising an amino acid sequence of SEQ ID NO: 10, a CD28-LCDR2 comprising an amino acid sequence of SEQ ID NO: 43 or 11, and comprising SEQ ID NO: 12 amino acid sequence of CD28-LCDR3;
    优选地，Preferably,
    i)所述CD28-VH具有：包含SEQ ID NO：19的氨基酸序列的CD28-HCDR1，包含SEQ ID NO：20的氨基酸序列的CD28-HCDR2，和包含SEQ ID NO：21的氨基酸序列的CD28-HCDR3；和所述CD28-VL具有：包含SEQ ID NO：22的氨基酸序列的CD28-LCDR1，包含SEQ ID NO：23的氨基酸序列的CD28-LCDR2，和包含SEQ ID NO：24的氨基酸序列的CD28-LCDR3；或 i) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, and CD28- comprising the amino acid sequence of SEQ ID NO: 21 HCDR3; and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and CD28 comprising the amino acid sequence of SEQ ID NO: 24 -LCDR3; or
    ii)所述CD28-VH具有：包含SEQ ID NO：13的氨基酸序列的CD28-HCDR1，包含SEQ ID NO：14的氨基酸序列的CD28-HCDR2，和包含SEQ ID NO：15的氨基酸序列的CD28-HCDR3；和所述CD28-VL具有：包含SEQ ID NO：16的氨基酸序列的CD28-LCDR1，包含SEQ ID NO：62的氨基酸序列的CD28-LCDR2，和包含SEQ ID NO：18的氨基酸序列的CD28-LCDR3；或ii) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and CD28- comprising the amino acid sequence of SEQ ID NO: 15 HCDR3; and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 62, and CD28 comprising the amino acid sequence of SEQ ID NO: 18 -LCDR3; or
    iii)所述CD28-VH具有：包含SEQ ID NO：25的氨基酸序列的CD28-HCDR1，包含SEQ ID NO：26的氨基酸序列的CD28-HCDR2，和包含SEQ ID NO：27的氨基酸序列的CD28-HCDR3；和所述CD28-VL具有：包含SEQ ID NO：28的氨基酸序列的CD28-LCDR1，包含SEQ ID NO：29的氨基酸序列的CD28-LCDR2，和包含SEQ ID NO：30的氨基酸序列的CD28-LCDR3；或iii) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and CD28- comprising the amino acid sequence of SEQ ID NO: 27 HCDR3; and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, and CD28 comprising the amino acid sequence of SEQ ID NO: 30 -LCDR3; or
    iv)所述CD28-VH具有：包含SEQ ID NO：7的氨基酸序列的CD28-HCDR1，包含SEQ ID NO：8的氨基酸序列的CD28-HCDR2，和包含SEQ ID NO：9的氨基酸序列的CD28-HCDR3；和所述CD28-VL具有：包含SEQ ID NO：10的氨基酸序列的CD28-LCDR1，包含SEQ ID NO：43的氨基酸序列的CD28-LCDR2，和包含SEQ ID NO：12的氨基酸序列的CD28-LCDR3；iv) The CD28-VH has: CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, and CD28- comprising the amino acid sequence of SEQ ID NO: 9 HCDR3; and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43, and CD28 comprising the amino acid sequence of SEQ ID NO: 12 -LCDR3;
    更优选地，More preferably,
    所述抗原结合分子在25℃条件下以小于2×10-8M的KD结合人CD28，所述KD是通过表面等离子体共振法测量的。The antigen-binding molecule binds to human CD28 with a KD of less than 2×10 -8 M at 25°C, as measured by surface plasmon resonance.
  2. 根据权利要求1所述的抗原结合分子，其中：The antigen-binding molecule according to claim 1, wherein:
    i)所述PSMA-VH具有：包含SEQ ID NO：170的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：171的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：172的氨基酸序列的PSMA-HCDR3；和所述PSMA-VL具有：包含SEQ ID NO：173的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：174的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：175的氨基酸序列的PSMA-LCDR3；或i) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 172 HCDR3; and the PSMA-VL having: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 174, and PSMA comprising the amino acid sequence of SEQ ID NO: 175 -LCDR3; or
    ii)所述PSMA-VH具有：包含SEQ ID NO：164的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：165的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：166的氨基酸序列的PSMA-HCDR3；和所述PSMA-VL具有：包含SEQ ID NO：167的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：168的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：169的氨基酸序列的PSMA-LCDR3；或ii) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 166 HCDR3; and the PSMA-VL having: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 168, and PSMA comprising the amino acid sequence of SEQ ID NO: 169 -LCDR3; or
    iii)所述PSMA-VH具有：包含SEQ ID NO：158的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：159的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：160的氨基酸序列的PSMA-HCDR3；和所述PSMA-VL具有：包含SEQ ID NO：161的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：162的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：163的氨基酸序列的PSMA-LCDR3；或 iii) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 158, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 159, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 161, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 162, and PSMA comprising the amino acid sequence of SEQ ID NO: 163 -LCDR3; or
    iv)所述PSMA-VH具有：包含SEQ ID NO：176的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：177的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：178的氨基酸序列的PSMA-HCDR3；和所述PSMA-VL具有：包含SEQ ID NO：179的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：180的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：181的氨基酸序列的PSMA-LCDR3；或iv) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 176, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 177, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 178 HCDR3; and the PSMA-VL having: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 179, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 180, and PSMA comprising the amino acid sequence of SEQ ID NO: 181 -LCDR3; or
    v)所述PSMA-VH具有：包含SEQ ID NO：213的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：214的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：215的氨基酸序列的PSMA-HCDR3；和所述PSMA-VL具有：包含SEQ ID NO：216的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：217的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：218的氨基酸序列的PSMA-LCDR3。v) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 213, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 214, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 215 HCDR3; and the PSMA-VL having: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 216, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 217, and PSMA comprising the amino acid sequence of SEQ ID NO: 218 -LCDR3.
  3. 根据权利要求1或2所述的抗原结合分子，其中：The antigen-binding molecule according to claim 1 or 2, wherein:
    i)所述CD28-VH包含SEQ ID NO：95、35、94、96、97或98的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101、36、99、100、102、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列；或i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 35, 94, 96, 97 or 98, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 36, 99, 100, 102, 103, The amino acid sequence of 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 or 115; or
    ii)所述CD28-VH包含SEQ ID NO：68、33、63、64、65、66、67或69的氨基酸序列，和所述CD28-VL包含SEQ ID NO：80、34、70、71、72、73、74、75、76、77、78或79的氨基酸序列；或ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69, and the CD28-VL comprises SEQ ID NO: 80, 34, 70, 71, The amino acid sequence of 72, 73, 74, 75, 76, 77, 78 or 79; or
    iii)所述CD28-VH包含SEQ ID NO：126、37、123、124、125、127、128、129、130、131、132或133的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、38、135、136、137、138、139、140、141、142或143的氨基酸序列；或iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, 37, 123, 124, 125, 127, 128, 129, 130, 131, 132 or 133, and the CD28-VL comprises SEQ ID NO: The amino acid sequence of 134, 38, 135, 136, 137, 138, 139, 140, 141, 142 or 143; or
    iv)所述CD28-VH包含SEQ ID NO：44、31、45、46、47、48、49或53的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52、32、50或51的氨基酸序列；iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, 31, 45, 46, 47, 48, 49 or 53, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52, 32, 50 or 51 amino acid sequence;
    优选地，Preferably,
    i)所述CD28-VH包含SEQ ID NO：95、94、96、97或98的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101、99、100或102的氨基酸序列，或i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102, or
    所述CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101、99、100、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列，或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes SEQ ID NO: 101, 99, 100, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
    所述CD28-VH包含SEQ ID NO：94的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99、100、101、103、104、105、106、107、108、109、110、111、112、113、114或115的氨基酸序列，或The CD28-VH includes the amino acid sequence of SEQ ID NO: 94, and the CD28-VL includes SEQ ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, the amino acid sequence of 112, 113, 114 or 115, or
    所述CD28-VH包含SEQ ID NO：96的氨基酸序列，和所述CD28-VL包含SEQ ID NO：99或100的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 96, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 99 or 100, or
    所述CD28-VH包含SEQ ID NO：97的氨基酸序列，和所述CD28-VL包含SEQ  ID NO：99、100或101的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 97, and the CD28-VL comprises SEQ ID NO: 99, 100 or 101 amino acid sequence, or
    ii)所述CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述CD28-VL包含SEQ ID NO：80、72、73、74、75、76、77、78或79的氨基酸序列，或ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80, 72, 73, 74, 75, 76, 77, 78 or 79, or
    所述CD28-VH包含SEQ ID NO：63的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70或71的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 63, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70 or 71, or
    所述CD28-VH包含SEQ ID NO：64的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70、71或72的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 64, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
    所述CD28-VH包含SEQ ID NO：65的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70、71或72的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 65, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71 or 72, or
    所述CD28-VH包含SEQ ID NO：66的氨基酸序列，和所述CD28-VL包含SEQ ID NO：70、71、72、74、75、76或79氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 66, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 70, 71, 72, 74, 75, 76 or 79, or
    所述CD28-VH包含SEQ ID NO：67的氨基酸序列，和所述CD28-VL包含SEQ ID NO：72、74、75、76或79的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 67, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 72, 74, 75, 76 or 79, or
    iii)所述CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、135、136、137、138或139的氨基酸序列，或iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135, 136, 137, 138 or 139, or
    所述CD28-VH包含SEQ ID NO：123的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、135或136的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 123, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 135 or 136, or
    所述CD28-VH包含SEQ ID NO：124的氨基酸序列，和所述CD28-VL包含SEQ ID NO：135的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 124, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 135; or
    所述CD28-VH包含SEQ ID NO：125的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134或135的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 125, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134 or 135, or
    所述CD28-VH包含SEQ ID NO：127的氨基酸序列，和所述CD28-VL包含SEQ ID NO：137、138或139的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 127, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 137, 138 or 139; or
    所述CD28-VH包含SEQ ID NO：128的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、137、138、139或142的氨基酸序列；或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 128, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 137, 138, 139 or 142; or
    所述CD28-VH包含SEQ ID NO：129的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134、138、139、140、141、142或143的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 129, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134, 138, 139, 140, 141, 142 or 143, or
    所述CD28-VH包含SEQ ID NO：130的氨基酸序列，和所述CD28-VL包含SEQ ID NO：138、140、141、142或143的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 130, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
    所述CD28-VH包含SEQ ID NO：131的氨基酸序列，和所述CD28-VL包含SEQ ID NO：138、140、141、142或143的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 131, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 138, 140, 141, 142 or 143, or
    iv)所述CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52、50或51的氨基酸序列，或iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52, 50 or 51, or
    所述CD28-VH包含SEQ ID NO：45的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 45, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
    所述CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述CD28-VL包含SEQ  ID NO：50或52的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 46, and the CD28-VL comprises SEQ ID NO: 50 or 52 amino acid sequence, or
    所述CD28-VH包含SEQ ID NO：47的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列，或The CD28-VH comprises the amino acid sequence of SEQ ID NO: 47, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51, or
    所述CD28-VH包含SEQ ID NO：48的氨基酸序列，和所述CD28-VL包含SEQ ID NO：50或51的氨基酸序列；The CD28-VH comprises the amino acid sequence of SEQ ID NO: 48, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 50 or 51;
    更优选地：More preferably:
    i)所述CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述CD28-VL包含SEQ ID NO：101的氨基酸序列；或i) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 101; or
    ii)所述CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述CD28-VL包含SEQ ID NO：80的氨基酸序列；或ii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 68, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 80; or
    iii)所述CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述CD28-VL包含SEQ ID NO：134的氨基酸序列；或iii) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 126, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 134; or
    iv)所述CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52的氨基酸序列。iv) the CD28-VH comprises the amino acid sequence of SEQ ID NO: 44, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52.
  4. 根据权利要求1至3中任一项所述的抗原结合分子，其中：The antigen-binding molecule according to any one of claims 1 to 3, wherein:
    i)所述PSMA-VH包含SEQ ID NO：204、184或203的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：206、185或205的氨基酸序列；或i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, 184 or 203, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206, 185 or 205; or
    ii)所述PSMA-VH包含SEQ ID NO：197、332、194、195、196、198或199的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：201、333、200或202的氨基酸序列；或ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, 332, 194, 195, 196, 198 or 199, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201, 333, 200 or 202 ;or
    iii)所述PSMA-VH包含SEQ ID NO：188、182、189或190的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：191、183、192或193的氨基酸序列；或iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, 182, 189 or 190, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191, 183, 192 or 193; or
    iv)所述PSMA-VH包含SEQ ID NO：207或186的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：208、187、209或210的氨基酸序列；或iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207 or 186, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208, 187, 209 or 210; or
    v)所述PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：212的氨基酸序列；v) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212;
    优选地，Preferably,
    i)所述PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：206的氨基酸序列；或i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
    ii)所述PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：201的氨基酸序列；或ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201; or
    iii)所述PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：191的氨基酸序列；或iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191; or
    iv)所述PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：208的氨基酸序列。 iv) The PSMA-VH comprises the amino acid sequence of SEQ ID NO:207, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO:208.
  5. 根据权利要求1至4中任一项所述的抗原结合分子，其中所述特异性结合CD28的抗原结合模块或所述特异性结合PSMA的抗原结合模块包含能够形成二聚体的Titin链和Obscurin链；The antigen-binding molecule according to any one of claims 1 to 4, wherein the antigen-binding module that specifically binds CD28 or the antigen-binding module that specifically binds PSMA comprises a Titin chain capable of forming a dimer and Obscurin chain;
    优选地，Preferably,
    所述Titin链包含选自由SEQ ID NO：334至SEQ ID NO：352组成的组的氨基酸序列，所述Obscurin链包含选自由SEQ ID NO：353至SEQ ID NO：393组成的组的氨基酸序列；The Titin chain includes an amino acid sequence selected from the group consisting of SEQ ID NO: 334 to SEQ ID NO: 352, and the Obscurin chain includes an amino acid sequence selected from the group consisting of SEQ ID NO: 353 to SEQ ID NO: 393;
    更优选地，More preferably,
    所述Titin链包含SEQ ID NO：350的氨基酸序列，所述Obscurin链包含SEQ ID NO：388的氨基酸序列。The Titin chain includes the amino acid sequence of SEQ ID NO: 350, and the Obscurin chain includes the amino acid sequence of SEQ ID NO: 388.
  6. 根据权利要求1至5中任一项所述的抗原结合分子，其中所述抗原结合分子进一步包含Fc区，所述Fc区优选为IgG Fc区，进一步优选为IgG1 Fc区；The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule further comprises an Fc region, and the Fc region is preferably an IgG Fc region, and further preferably an IgG1 Fc region;
    更优选地，所述Fc区包含一个或多个能够减少Fc区与Fcγ受体结合的氨基酸取代；More preferably, the Fc region contains one or more amino acid substitutions capable of reducing the binding of the Fc region to Fcγ receptors;
    最优选地，所述Fc区是人IgG1 Fc区，并且在234和235位置的氨基酸为A，编号依据为EU索引。Most preferably, the Fc region is a human IgG1 Fc region, and the amino acids at positions 234 and 235 are A, and the numbering basis is the EU index.
  7. 根据权利要求6所述的抗原结合分子，其中所述Fc区包含能够相互缔合的第一亚基Fc1和第二亚基Fc2，所述Fc1和Fc2各自独立地具有一个或多个减少Fc区同源二聚化的氨基酸取代；The antigen-binding molecule according to claim 6, wherein the Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, and each of the Fc1 and Fc2 independently has one or more reducing Fc regions. Amino acid substitutions for homodimerization;
    优选地，所述Fc1具有根据杵臼技术的凸起结构，和所述Fc2具有根据杵臼技术的孔结构；Preferably, the Fc1 has a convex structure according to the pestle and mortar technology, and the Fc2 has a hole structure according to the pestle and mortar technology;
    更优选地，所述Fc1在366位置的氨基酸为W；并且所述Fc2在366位置的氨基酸为S，在368位置的氨基酸为A，和在407位置的氨基酸为V，编号依据为EU索引；More preferably, the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, and the numbering basis is the EU index;
    最优选地，所述Fc1包含SEQ ID NO：220的氨基酸序列；所述Fc2包含SEQ ID NO：221或222的氨基酸序列。Most preferably, the Fc1 includes the amino acid sequence of SEQ ID NO: 220; the Fc2 includes the amino acid sequence of SEQ ID NO: 221 or 222.
  8. 根据权利要求7所述的抗原结合分子，其中所述抗原结合分子包含一个特异性结合CD28的抗原结合模块和一个特异性结合PSMA的抗原结合模块，所述特异性结合CD28的抗原结合模块是Fab；所述特异性结合PSMA的抗原结合模块是经替换的Fab，其包含能够形成二聚体的Titin链和Obscurin链；The antigen-binding molecule according to claim 7, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds to CD28 and an antigen-binding module that specifically binds to PSMA, and the antigen-binding module that specifically binds to CD28 is Fab. ; The antigen-binding module that specifically binds PSMA is a replaced Fab, which contains a Titin chain and an Obscurin chain capable of forming dimers;
    优选地，Preferably,
    所述抗原结合分子包含一条具有式(a)所示结构的第一链、一条具有式(b)所示 结构的第二链、一条具有式(c)所示结构的第三链和一条具有式(d)所示结构的第四链，The antigen-binding molecule includes a first chain having a structure represented by formula (a), a first chain having a structure represented by formula (b) The second chain of the structure, a third chain having the structure shown in formula (c) and a fourth chain having the structure shown in formula (d),
    (a)[CD28-VH]-[CH1]-[Fc1]，(a)[CD28-VH]-[CH1]-[Fc1],
    (b)[CD28-VL]-[CL]，(b)[CD28-VL]-[CL],
    (c)[PSMA-VH]-[连接子1]-[Titin链]-[Fc2]，(c)[PSMA-VH]-[linker 1]-[Titin chain]-[Fc2],
    (d)[PSMA-VL]-[连接子2]-[Obscurin链]，(d)[PSMA-VL]-[Linker 2]-[Obscurin chain],
    其中：所述连接子1和所述连接子2是相同或不同的肽连接子；或连接子1和/或连接子2不存在；Wherein: the linker 1 and the linker 2 are the same or different peptide linkers; or the linker 1 and/or the linker 2 do not exist;
    式(a)、(b)、(c)和(d)所示的结构是从N端至C端排列的；The structures shown in formulas (a), (b), (c) and (d) are arranged from the N end to the C end;
    更优选地，其中：More preferably, wherein:
    i)所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：80的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或i) The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204 sequence, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 206; or
    所述的CD28-VH包含SEQ ID NO：33的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：34的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 33, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 34; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：65的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：71的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 65, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 71; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：78的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 78; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：80的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：80的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
    所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：80的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：191的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
    所述的CD28-VH包含SEQ ID NO：68的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：80的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：208的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 80; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
    ii)所述的CD28-VH包含SEQ ID NO：31的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：32的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211 的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或ii) The CD28-VH includes the amino acid sequence of SEQ ID NO: 31, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 32; and the PSMA-VH includes SEQ ID NO: 211 The amino acid sequence of SEQ ID NO: 212, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：50的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 50; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
    所述的CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 206; or
    所述的CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
    所述的CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 206; or
    所述的CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：191的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
    所述的CD28-VH包含SEQ ID NO：46的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：208的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 46, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
    iii)所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或iii) The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 204 sequence, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 206; or
    所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
    所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211 的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 The amino acid sequence of SEQ ID NO: 212, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：191的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
    所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：208的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
    iv)所述的CD28-VH包含SEQ ID NO：37的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：38的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或iv) the CD28-VH includes the amino acid sequence of SEQ ID NO: 37, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 38; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212;
    最优选地，所述抗原结合分子具有：Most preferably, the antigen binding molecule has:
    一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：239的氨基酸序列的第二链、一条包含SEQ ID NO：249的氨基酸序列的第三链和一条包含SEQ ID NO：250的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 239, a third strand comprising the amino acid sequence of SEQ ID NO: 249 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of the amino acid sequence of 250; or
    一条包含SEQ ID NO：226的氨基酸序列的第一链、一条包含SEQ ID NO：227的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 226, a second strand comprising the amino acid sequence of SEQ ID NO: 227, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 226 : The fourth strand of the amino acid sequence 229; or
    一条包含SEQ ID NO：230的氨基酸序列的第一链、一条包含SEQ ID NO：231的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 230, a second strand comprising the amino acid sequence of SEQ ID NO: 231, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 230 : The fourth strand of the amino acid sequence 229; or
    一条包含SEQ ID NO：230的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 230, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 232 : The fourth strand of the amino acid sequence 229; or
    一条包含SEQ ID NO：233的氨基酸序列的第一链、一条包含SEQ ID NO：234的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 233, a second strand comprising the amino acid sequence of SEQ ID NO: 234, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 233 : The fourth strand of the amino acid sequence 229; or
    一条包含SEQ ID NO：235的氨基酸序列的第一链、一条包含SEQ ID NO：236的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 235, a second strand comprising the amino acid sequence of SEQ ID NO: 236, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 235 : The fourth strand of the amino acid sequence 229; or
    一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：238的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 238, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of the amino acid sequence 229; or
    一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO： 239的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或One contains the first strand of the amino acid sequence of SEQ ID NO: 237, one contains the first strand of the amino acid sequence of SEQ ID NO: or
    一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO：241的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 240, a second strand comprising the amino acid sequence of SEQ ID NO: 241, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 240 : The fourth strand of the amino acid sequence 229; or
    一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO：242的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 240, a second strand comprising the amino acid sequence of SEQ ID NO: 242, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 240 : The fourth strand of the amino acid sequence 229; or
    一条包含SEQ ID NO：243的氨基酸序列的第一链、一条包含SEQ ID NO：244的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 243, a second strand comprising the amino acid sequence of SEQ ID NO: 244, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 243 : The fourth strand of the amino acid sequence 229; or
    一条包含SEQ ID NO：245的氨基酸序列的第一链、一条包含SEQ ID NO：246的氨基酸序列的第二链、一条包含SEQ ID NO：228的氨基酸序列的第三链和一条包含SEQ ID NO：229的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 245, a second strand comprising the amino acid sequence of SEQ ID NO: 246, a third strand comprising the amino acid sequence of SEQ ID NO: 228 and a third strand comprising the amino acid sequence of SEQ ID NO: 245 : The fourth strand of the amino acid sequence 229; or
    一条包含SEQ ID NO：230的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：247的氨基酸序列的第三链和一条包含SEQ ID NO：248的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 230, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 247 and a third strand comprising the amino acid sequence of SEQ ID NO: 232 : The fourth strand of an amino acid sequence of 248; or
    一条包含SEQ ID NO：230的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：249的氨基酸序列的第三链和一条包含SEQ ID NO：250的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 230, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 249 and a third strand comprising the amino acid sequence of SEQ ID NO: 232 : The fourth strand of the amino acid sequence of 250; or
    一条包含SEQ ID NO：251的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：247的氨基酸序列的第三链和一条包含SEQ ID NO：248的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 251, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 247 and a third strand comprising the amino acid sequence of SEQ ID NO: 247 : The fourth strand of an amino acid sequence of 248; or
    一条包含SEQ ID NO：251的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：249的氨基酸序列的第三链和一条包含SEQ ID NO：250的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 251, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 249 and a third strand comprising the amino acid sequence of SEQ ID NO: 249 : The fourth strand of the amino acid sequence of 250; or
    一条包含SEQ ID NO：251的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：252的氨基酸序列的第三链和一条包含SEQ ID NO：253的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 251, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 252 and a third strand comprising the amino acid sequence of SEQ ID NO: 251 : The fourth strand of the amino acid sequence 253; or
    一条包含SEQ ID NO：251的氨基酸序列的第一链、一条包含SEQ ID NO：232的氨基酸序列的第二链、一条包含SEQ ID NO：254的氨基酸序列的第三链和一条包含SEQ ID NO：255的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 251, a second strand comprising the amino acid sequence of SEQ ID NO: 232, a third strand comprising the amino acid sequence of SEQ ID NO: 254 and a third strand comprising the amino acid sequence of SEQ ID NO: 251 : The fourth strand of an amino acid sequence of 255; or
    一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：239的氨基酸序列的第二链、一条包含SEQ ID NO：247的氨基酸序列的第三链和一条包含SEQ ID NO：248的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 239, a third strand comprising the amino acid sequence of SEQ ID NO: 247 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of an amino acid sequence of 248; or
    一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO： 239的氨基酸序列的第二链、一条包含SEQ ID NO：252的氨基酸序列的第三链和一条包含SEQ ID NO：253的氨基酸序列的第四链；或One contains the first strand of the amino acid sequence of SEQ ID NO: 237, one contains the first strand of the amino acid sequence of SEQ ID NO: or
    一条包含SEQ ID NO：237的氨基酸序列的第一链、一条包含SEQ ID NO：239的氨基酸序列的第二链、一条包含SEQ ID NO：254的氨基酸序列的第三链和一条包含SEQ ID NO：255的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 237, a second strand comprising the amino acid sequence of SEQ ID NO: 239, a third strand comprising the amino acid sequence of SEQ ID NO: 254 and a third strand comprising the amino acid sequence of SEQ ID NO: 237 : The fourth strand of an amino acid sequence of 255; or
    一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO：242的氨基酸序列的第二链、一条包含SEQ ID NO：247的氨基酸序列的第三链和一条包含SEQ ID NO：248的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 240, a second strand comprising the amino acid sequence of SEQ ID NO: 242, a third strand comprising the amino acid sequence of SEQ ID NO: 247 and a third strand comprising the amino acid sequence of SEQ ID NO: 240 : The fourth strand of an amino acid sequence of 248; or
    一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO：242的氨基酸序列的第二链、一条包含SEQ ID NO：249的氨基酸序列的第三链和一条包含SEQ ID NO：250的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 240, a second strand comprising the amino acid sequence of SEQ ID NO: 242, a third strand comprising the amino acid sequence of SEQ ID NO: 249 and a third strand comprising the amino acid sequence of SEQ ID NO: 242 : The fourth strand of the amino acid sequence of 250; or
    一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO：242的氨基酸序列的第二链、一条包含SEQ ID NO：252的氨基酸序列的第三链和一条包含SEQ ID NO：253的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 240, a second strand comprising the amino acid sequence of SEQ ID NO: 242, a third strand comprising the amino acid sequence of SEQ ID NO: 252 and a third strand comprising the amino acid sequence of SEQ ID NO: 240 : The fourth strand of the amino acid sequence 253; or
    一条包含SEQ ID NO：240的氨基酸序列的第一链、一条包含SEQ ID NO：242的氨基酸序列的第二链、一条包含SEQ ID NO：254的氨基酸序列的第三链和一条包含SEQ ID NO：255的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 240, a second strand comprising the amino acid sequence of SEQ ID NO: 242, a third strand comprising the amino acid sequence of SEQ ID NO: 254 and a third strand comprising the amino acid sequence of SEQ ID NO: 240 : The fourth strand of the amino acid sequence of 255.
  9. 根据权利要求7所述的抗原结合分子，其中所述抗原结合分子包含一个特异性结合CD28的抗原结合模块和一个特异性结合PSMA的抗原结合模块，所述特异性结合PSMA的抗原结合模块是Fab；所述特异性结合CD28的抗原结合模块是经替换的Fab，其包含能够形成二聚体的Titin链和Obscurin链；The antigen-binding molecule according to claim 7, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds to CD28 and an antigen-binding module that specifically binds to PSMA, and the antigen-binding module that specifically binds to PSMA is Fab. ; The antigen-binding module that specifically binds to CD28 is a replaced Fab, which contains a Titin chain and an Obscurin chain capable of forming dimers;
    优选地，Preferably,
    所述抗原结合分子包含一条具有式(e)所示结构的第一链、一条具有式(f)所示结构的第二链、一条具有式(g)所示结构的第三链和一条具有式(h)所示结构的第四链，The antigen-binding molecule includes a first chain having a structure represented by formula (e), a second chain having a structure represented by formula (f), a third chain having a structure represented by formula (g), and a third chain having a structure represented by formula (g). The fourth chain of the structure shown in formula (h),
    (e)[CD28-VH]-[连接子3]-[Titin链]-[Fc1]，(e)[CD28-VH]-[Linker 3]-[Titin chain]-[Fc1],
    (f)[CD28-VL]-[连接子4]-[Obscurin链]，(f)[CD28-VL]-[linker 4]-[Obscurin chain],
    (g)[PSMA-VH]-[CH1]-[Fc2]，(g)[PSMA-VH]-[CH1]-[Fc2],
    (h)[PSMA-VL]-[CL]，(h)[PSMA-VL]-[CL],
    其中：所述连接子3和连接子4是相同或不同的肽连接子；或连接子3和/或连接子4不存在；Wherein: the linker 3 and the linker 4 are the same or different peptide linkers; or the linker 3 and/or the linker 4 do not exist;
    式(e)、(f)、(g)和(h)所示的结构是从N端至C端排列的；The structures shown in formulas (e), (f), (g) and (h) are arranged from the N end to the C end;
    更优选地，其中：More preferably, wherein:
    i)所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO： 204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或i) The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO: 204, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 206; or
    所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
    所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：106的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 106; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：94的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 94, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：191的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
    所述的CD28-VH包含SEQ ID NO：95的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：101的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：208的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 95, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 101; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208; or
    ii)所述的CD28-VH包含SEQ ID NO：44的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：52的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或ii) The CD28-VH includes the amino acid sequence of SEQ ID NO: 44, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 52; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
    iii)所述的CD28-VH包含SEQ ID NO：37的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：38的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或iii) The CD28-VH includes the amino acid sequence of SEQ ID NO: 37, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 38; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211 sequence, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：129的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：138的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：211的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：212的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 129, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 138; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 211, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 212; or
    所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：201的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 201; or
    所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO： 204的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：206的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO: 204, and the PSMA-VL includes the amino acid sequence of SEQ ID NO: 206; or
    所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：191的氨基酸序列；或The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 191; or
    所述的CD28-VH包含SEQ ID NO：126的氨基酸序列，和所述的CD28-VL包含SEQ ID NO：134的氨基酸序列；并且所述的PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述的PSMA-VL包含SEQ ID NO：208的氨基酸序列；The CD28-VH includes the amino acid sequence of SEQ ID NO: 126, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 134; and the PSMA-VH includes the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprising the amino acid sequence of SEQ ID NO: 208;
    最优选地，所述抗原结合分子具有：Most preferably, the antigen binding molecule has:
    一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：274的氨基酸序列的第三链和一条包含SEQ ID NO：275的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 274 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence 275; or
    一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：272的氨基酸序列的第三链和一条包含SEQ ID NO：273的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 272 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence 273; or
    一条包含SEQ ID NO：257的氨基酸序列的第一链、一条包含SEQ ID NO：258的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 257, a second strand comprising the amino acid sequence of SEQ ID NO: 258, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 257 : The fourth strand of the amino acid sequence of 260; or
    一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence of 260; or
    一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：263的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 263, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence of 260; or
    一条包含SEQ ID NO：264的氨基酸序列的第一链、一条包含SEQ ID NO：265的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 264, a second strand comprising the amino acid sequence of SEQ ID NO: 265, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 264 : The fourth strand of the amino acid sequence of 260; or
    一条包含SEQ ID NO：266的氨基酸序列的第一链、一条包含SEQ ID NO：267的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 266, a second strand comprising the amino acid sequence of SEQ ID NO: 267, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 266 : The fourth strand of the amino acid sequence of 260; or
    一条包含SEQ ID NO：268的氨基酸序列的第一链、一条包含SEQ ID NO：269的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 268, a second strand comprising the amino acid sequence of SEQ ID NO: 269, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 268 : The fourth strand of the amino acid sequence of 260; or
    一条包含SEQ ID NO：270的氨基酸序列的第一链、一条包含SEQ ID NO：271的氨基酸序列的第二链、一条包含SEQ ID NO：259的氨基酸序列的第三链和一条包含SEQ ID NO：260的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 270, a second strand comprising the amino acid sequence of SEQ ID NO: 271, a third strand comprising the amino acid sequence of SEQ ID NO: 259 and a third strand comprising the amino acid sequence of SEQ ID NO: 271 : The fourth strand of the amino acid sequence of 260; or
    一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO： 262的氨基酸序列的第二链、一条包含SEQ ID NO：276的氨基酸序列的第三链和一条包含SEQ ID NO：277的氨基酸序列的第四链；或One contains the first strand of the amino acid sequence of SEQ ID NO: 261, one contains the first strand of the amino acid sequence of SEQ ID NO: or
    一条包含SEQ ID NO：261的氨基酸序列的第一链、一条包含SEQ ID NO：262的氨基酸序列的第二链、一条包含SEQ ID NO：278的氨基酸序列的第三链和一条包含SEQ ID NO：279的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 261, a second strand comprising the amino acid sequence of SEQ ID NO: 262, a third strand comprising the amino acid sequence of SEQ ID NO: 278 and a third strand comprising the amino acid sequence of SEQ ID NO: 261 : The fourth strand of the amino acid sequence 279; or
    一条包含SEQ ID NO：268的氨基酸序列的第一链、一条包含SEQ ID NO：269的氨基酸序列的第二链、一条包含SEQ ID NO：272的氨基酸序列的第三链和一条包含SEQ ID NO：273的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 268, a second strand comprising the amino acid sequence of SEQ ID NO: 269, a third strand comprising the amino acid sequence of SEQ ID NO: 272 and a third strand comprising the amino acid sequence of SEQ ID NO: 268 : The fourth strand of the amino acid sequence 273; or
    一条包含SEQ ID NO：268的氨基酸序列的第一链、一条包含SEQ ID NO：269的氨基酸序列的第二链、一条包含SEQ ID NO：274的氨基酸序列的第三链和一条包含SEQ ID NO：275的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 268, a second strand comprising the amino acid sequence of SEQ ID NO: 269, a third strand comprising the amino acid sequence of SEQ ID NO: 274 and a third strand comprising the amino acid sequence of SEQ ID NO: 268 : The fourth strand of the amino acid sequence 275; or
    一条包含SEQ ID NO：268的氨基酸序列的第一链、一条包含SEQ ID NO：269的氨基酸序列的第二链、一条包含SEQ ID NO：276的氨基酸序列的第三链和一条包含SEQ ID NO：277的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 268, a second strand comprising the amino acid sequence of SEQ ID NO: 269, a third strand comprising the amino acid sequence of SEQ ID NO: 276 and a third strand comprising the amino acid sequence of SEQ ID NO: 268 : The fourth strand of the amino acid sequence 277; or
    一条包含SEQ ID NO：268的氨基酸序列的第一链、一条包含SEQ ID NO：269的氨基酸序列的第二链、一条包含SEQ ID NO：278的氨基酸序列的第三链和一条包含SEQ ID NO：279的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 268, a second strand comprising the amino acid sequence of SEQ ID NO: 269, a third strand comprising the amino acid sequence of SEQ ID NO: 278 and a third strand comprising the amino acid sequence of SEQ ID NO: 268 : The fourth strand of the amino acid sequence of 279.
  10. 一种药物组合物，其含有：A pharmaceutical composition containing:
    权利要求1至9中任一项所述的抗原结合分子，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂；The antigen-binding molecule of any one of claims 1 to 9, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients;
    优选地，所述的药物组合物中还包含第二治疗剂；Preferably, the pharmaceutical composition also contains a second therapeutic agent;
    更优选地，所述第二治疗剂是能够特异性结合CD3的抗体。More preferably, the second therapeutic agent is an antibody capable of specifically binding CD3.
  11. 分离的核酸，其编码权利要求1至9中任一项所述的抗原结合分子。An isolated nucleic acid encoding the antigen-binding molecule of any one of claims 1 to 9.
  12. 一种宿主细胞，其包含如权利要求11所述的分离的核酸。A host cell comprising the isolated nucleic acid of claim 11.
  13. 一种治疗疾病的方法，所述方法包括向受试者施用治疗有效量的权利要求1至9中任一项所述的抗原结合分子，或权利要求10所述的药物组合物；A method of treating a disease, the method comprising administering to a subject a therapeutically effective amount of the antigen-binding molecule of any one of claims 1 to 9, or the pharmaceutical composition of claim 10;
    优选地，所述疾病是肿瘤；更优选地，所述的肿瘤为前列腺癌、肾癌、尿路上皮癌、乳腺癌、膀胱癌、原发性肝癌、胰腺癌、黑色素瘤、胶质母细胞瘤、骨肉瘤、卵巢癌、食管癌、宫颈鳞状细胞癌、肺癌、结直肠癌、子宫内膜癌、皮肤癌、头颈部鳞状细胞癌、脑癌、胃食管癌、肝脏转移性癌、多发性骨髓瘤、淋巴瘤、急性髓样白血病(AML)、急性淋巴母细胞性白血病(ALL)、慢性淋巴细胞白血病(CLL)或B细胞淋巴瘤；最优选地，其中所述的肿瘤的细胞或靠近该肿瘤的血管 内皮细胞表达PSMA。Preferably, the disease is a tumor; more preferably, the tumor is prostate cancer, renal cancer, urothelial cancer, breast cancer, bladder cancer, primary liver cancer, pancreatic cancer, melanoma, glioblastoma tumor, osteosarcoma, ovarian cancer, esophageal cancer, cervical squamous cell carcinoma, lung cancer, colorectal cancer, endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, gastroesophageal cancer, liver metastatic cancer , multiple myeloma, lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) or B-cell lymphoma; most preferably, wherein the tumor cells or blood vessels close to the tumor Endothelial cells express PSMA.
  14. 根据权利要求13所述的方法，所述方法进一步包括使用第二治疗剂；优选地，所述所述第二治疗剂包括抗肿瘤药剂、放射疗法、抗体药物缀合物、双特异性抗体、与抗肿瘤药剂缀合的双特异性抗体、免疫检查点抑制剂或其组合。The method of claim 13, further comprising using a second therapeutic agent; preferably, the second therapeutic agent includes an anti-tumor agent, radiotherapy, antibody drug conjugates, bispecific antibodies, Bispecific antibodies, immune checkpoint inhibitors, or combinations thereof conjugated to anti-tumor agents.
  15. 根据权利要求14所述的方法，所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体，其包含至少一个特异性结合PSMA的抗原结合模块和至少一个特异性结合CD3的抗原结合模块，所述特异性结合PSMA的抗原结合模块包含重链可变区PSMA-VH和轻链可变区PSMA-VL，所述特异性结合CD3的抗原结合模块包含重链可变区CD3-VH和轻链可变区CD3-VL；The method of claim 14, wherein the second therapeutic agent is a bispecific antibody that specifically binds PSMA and CD3, comprising at least one antigen-binding module that specifically binds PSMA and at least one antigen that specifically binds CD3. Binding module, the antigen-binding module that specifically binds to PSMA includes the heavy chain variable region PSMA-VH and the light chain variable region PSMA-VL, and the antigen-binding module that specifically binds to CD3 includes the heavy chain variable region CD3- VH and light chain variable region CD3-VL;
    优选地，Preferably,
    i)所述PSMA-VH具有：包含SEQ ID NO：164的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：165的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：166的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：167的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：168的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：169的氨基酸序列的PSMA-LCDR3；或i) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 164, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 165, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 166 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 167, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 168, and PSMA comprising the amino acid sequence of SEQ ID NO: 169 -LCDR3; or
    ii)所述PSMA-VH具有：包含SEQ ID NO：170的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：171的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：172的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：173的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：174的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：175的氨基酸序列的PSMA-LCDR3；或ii) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 170, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 171, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 172 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 173, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 174, and PSMA comprising the amino acid sequence of SEQ ID NO: 175 -LCDR3; or
    iii)所述PSMA-VH具有：包含SEQ ID NO：158的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：159的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：160的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：161的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：162的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：163的氨基酸序列的PSMA-LCDR3；或iii) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 158, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 159, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 161, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 162, and PSMA comprising the amino acid sequence of SEQ ID NO: 163 -LCDR3; or
    iv)所述PSMA-VH具有：包含SEQ ID NO：176的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：177的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：178的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：179的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：180的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：181的氨基酸序列的PSMA-LCDR3；或iv) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 176, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 177, and PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 178 HCDR3; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 179, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 180, and PSMA comprising the amino acid sequence of SEQ ID NO: 181 -LCDR3; or
    v)所述PSMA-VH具有：包含SEQ ID NO：301的氨基酸序列的PSMA-HCDR1，包含SEQ ID NO：302的氨基酸序列的PSMA-HCDR2，和包含SEQ ID NO：303 的氨基酸序列的PSMA-HCDR3；并且所述PSMA-VL具有：包含SEQ ID NO：304的氨基酸序列的PSMA-LCDR1，包含SEQ ID NO：305的氨基酸序列的PSMA-LCDR2，和包含SEQ ID NO：306的氨基酸序列的PSMA-LCDR3；v) The PSMA-VH has: PSMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 301, PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 302, and PSMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 303 PSMA-HCDR3 of the amino acid sequence; and the PSMA-VL has: PSMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 304, PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 305, and PSMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: PSMA-LCDR3 with an amino acid sequence of 306;
    更优选地，More preferably,
    i)所述PSMA-VH包含SEQ ID NO：197、199、332、194、195、196或198的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：201、202、333或200的氨基酸序列；或i) The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, 199, 332, 194, 195, 196 or 198, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201, 202, 333 or 200 ;or
    ii)所述PSMA-VH包含SEQ ID NO：204、184或203的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：206、185或205的氨基酸序列；或ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, 184 or 203, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206, 185 or 205; or
    iii)所述PSMA-VH包含SEQ ID NO：188、182、189或190的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：191、183、192或193的氨基酸序列；或iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, 182, 189 or 190, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191, 183, 192 or 193; or
    iv)所述PSMA-VH包含SEQ ID NO：207或186的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：208、187、209或210的氨基酸序列；或iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207 or 186, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208, 187, 209 or 210; or
    v)所述PSMA-VH包含SEQ ID NO：313的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：314的氨基酸序列；v) said PSMA-VH comprises the amino acid sequence of SEQ ID NO: 313, and said PSMA-VL comprises the amino acid sequence of SEQ ID NO: 314;
    最优选地，Most preferably,
    i)所述PSMA-VH包含SEQ ID NO：197的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：201的氨基酸序列；或i) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 197, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 201; or
    所述PSMA-VH包含SEQ ID NO：199的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：202的氨基酸序列；或The PSMA-VH comprises the amino acid sequence of SEQ ID NO: 199, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 202; or
    ii)所述PSMA-VH包含SEQ ID NO：204的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：206的氨基酸序列；或ii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 204, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 206; or
    iii)所述PSMA-VH包含SEQ ID NO：188的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：191的氨基酸序列；或iii) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 188, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 191; or
    iv)所述PSMA-VH包含SEQ ID NO：207的氨基酸序列，和所述PSMA-VL包含SEQ ID NO：208的氨基酸序列。iv) the PSMA-VH comprises the amino acid sequence of SEQ ID NO: 207, and the PSMA-VL comprises the amino acid sequence of SEQ ID NO: 208.
  16. 根据权利要求15所述的方法，其中所述特异性结合CD3的抗原结合模块包含重链可变区CD3-VH和轻链可变区CD3-VL，其中：The method of claim 15, wherein the antigen-binding module that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein:
    i)所述CD3-VH具有：包含SEQ ID NO：283的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：284的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：285的氨基酸序列的CD3-HCDR3；并且所述CD3-VL具有：包含SEQ ID NO：286的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：287的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：288的氨基酸序列的CD3-LCDR3；或i) The CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 283, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 284, and CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 285 HCDR3; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 286, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 287, and CD3 comprising the amino acid sequence of SEQ ID NO: 288 -LCDR3; or
    ii)所述CD3-VH具有：包含SEQ ID NO：307的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：308的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：309的 氨基酸序列的CD3-HCDR3；并且所述CD3-VL具有：包含SEQ ID NO：310的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：311的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：312的氨基酸序列的CD3-LCDR3；ii) The CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 307, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 308, and CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 309 CD3-HCDR3 of the amino acid sequence; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO: 310, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 311, and CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO: 312 The amino acid sequence of CD3-LCDR3;
    更优选地，More preferably,
    所述CD3-VH包含SEQ ID NO：289的氨基酸序列，和所述CD3-VL包含SEQ ID NO：290的氨基酸序列；或The CD3-VH comprises the amino acid sequence of SEQ ID NO: 289, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 290; or
    所述CD3-VH包含SEQ ID NO：315的氨基酸序列，和所述CD3-VL包含SEQ ID NO：316的氨基酸序列。The CD3-VH comprises the amino acid sequence of SEQ ID NO: 315, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 316.
  17. 根据权利要求15或16所述的方法，其中所述的特异性结合PSMA和CD3的双特异性抗体包含Fc区；The method according to claim 15 or 16, wherein the bispecific antibody that specifically binds PSMA and CD3 comprises an Fc region;
    优选地，所述Fc区包含能够相互缔合的第一亚基Fc1和第二亚基Fc2，所述Fc1具有根据杵臼技术的凸起结构，和所述Fc2具有根据杵臼技术的孔结构；Preferably, the Fc region includes a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 has a convex structure according to the pestle and mortar technology, and the Fc2 has a pore structure according to the pestle and mortar technology;
    更优选地，所述Fc1在366位置的氨基酸为W；并且所述Fc2在366位置的氨基酸为S，在368位置的氨基酸为A，和在407位置的氨基酸为V，编号依据为EU索引；More preferably, the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, and the numbering basis is the EU index;
    最优选地，所述Fc1包含SEQ ID NO：220的氨基酸序列；所述Fc2包含SEQ ID NO：221或222的氨基酸序列。Most preferably, the Fc1 includes the amino acid sequence of SEQ ID NO: 220; the Fc2 includes the amino acid sequence of SEQ ID NO: 221 or 222.
  18. 根据权利要求15至17中任一项所述的方法，其中所述的特异性结合PSMA和CD3的双特异性抗体包含一个特异性结合PSMA的抗原结合模块和一个特异性结合CD3的抗原结合模块，所述的特异性结合PSMA的抗原结合模块和特异性结合CD3的抗原结合模块均是scFv；The method according to any one of claims 15 to 17, wherein the bispecific antibody that specifically binds PSMA and CD3 comprises an antigen-binding module that specifically binds PSMA and an antigen-binding module that specifically binds CD3. , the antigen-binding module that specifically binds to PSMA and the antigen-binding module that specifically binds to CD3 are both scFv;
    优选地，Preferably,
    所述特异性结合PSMA和CD3的双特异性抗体包含一条具有式(a)所示结构的第一链和一条具有式(b)所示结构的第二链，The bispecific antibody that specifically binds PSMA and CD3 includes a first chain having a structure shown in formula (a) and a second chain having a structure shown in formula (b),
    (a)[PSMA-VH]-[连接子6]-[PSMA-VL]-[连接子7]-[CD3-VH]-[连接子8]-[CD3-VL]-[连接子9]-[Fc2]，(a) [PSMA-VH]-[Linker 6]-[PSMA-VL]-[Linker 7]-[CD3-VH]-[Linker 8]-[CD3-VL]-[Linker 9] -[Fc2],
    (b)[Fc1]，(b)[Fc1],
    其中，式(a)和(b)所示的结构是从N端至C端排列的，所述连接子6、连接子7、连接子8和连接子9是相同或不同的肽连接子；Wherein, the structures shown in formulas (a) and (b) are arranged from the N end to the C end, and the linker 6, linker 7, linker 8 and linker 9 are the same or different peptide linkers;
    最优选地，Most preferably,
    所述特异性结合PSMA和CD3的双特异性抗体具有：一条包含SEQ ID NO：296的氨基酸序列的第一链和一条包含SEQ ID NO：220的氨基酸序列的第二链；或The bispecific antibody that specifically binds PSMA and CD3 has: a first chain including the amino acid sequence of SEQ ID NO: 296 and a second chain including the amino acid sequence of SEQ ID NO: 220; or
    所述特异性结合PSMA和CD3的双特异性抗体具有：一条包含SEQ ID NO： 297的氨基酸序列的第一链和一条包含SEQ ID NO：220的氨基酸序列的第二链。The bispecific antibody that specifically binds PSMA and CD3 has: one block containing SEQ ID NO: A first strand containing the amino acid sequence of SEQ ID NO: 297 and a second strand containing the amino acid sequence of SEQ ID NO: 220.
  19. 根据权利要求15至17中任一项所述的方法，其中所述的特异性结合PSMA和CD3的双特异性抗体包含一个特异性结合PSMA的抗原结合模块和一个特异性结合CD3的抗原结合模块，所述特异性结合PSMA的抗原结合模块是Fab；所述特异性结合CD3的抗原结合模块是经替换的Fab，其包含能够形成二聚体的Titin链和Obscurin链；The method according to any one of claims 15 to 17, wherein the bispecific antibody that specifically binds PSMA and CD3 comprises an antigen-binding module that specifically binds PSMA and an antigen-binding module that specifically binds CD3. , the antigen-binding module that specifically binds to PSMA is Fab; the antigen-binding module that specifically binds to CD3 is a replaced Fab, which includes a Titin chain and an Obscurin chain capable of forming dimers;
    优选地，Preferably,
    所述特异性结合PSMA和CD3的双特异性抗体包含一条具有式(c)所示结构的第一链、一条具有式(d)所示结构的第二链、一条具有式(e)所示结构的第三链和一条具有式(f)所示结构的第四链，The bispecific antibody that specifically binds to PSMA and CD3 includes a first chain having a structure shown in formula (c), a second chain having a structure shown in formula (d), and a second chain having a structure shown in formula (e) The third chain of the structure and a fourth chain having the structure shown in formula (f),
    (c)[PSMA-VH]-[CH1]-[Fc1]，(c)[PSMA-VH]-[CH1]-[Fc1],
    (d)[PSMA-VL]-[CL]，(d)[PSMA-VL]-[CL],
    (e)[CD3-VH]-[连接子10]-[Obscurin链]-[Fc2]，(e)[CD3-VH]-[linker 10]-[Obscurin chain]-[Fc2],
    (f)[CD3-VL]-[连接子11]-[Titin链]，(f)[CD3-VL]-[linker 11]-[Titin chain],
    其中：式(c)、(d)、(e)和(f)所示的结构是从N端至C端排列的，所述连接子10和连接子11是相同或不同的肽连接子；Wherein: the structures shown in formulas (c), (d), (e) and (f) are arranged from the N end to the C end, and the linker 10 and the linker 11 are the same or different peptide linkers;
    更优选地，More preferably,
    所述特异性结合PSMA和CD3的双特异性抗体具有：一条包含SEQ ID NO：298的氨基酸序列的第一链、一条包含SEQ ID NO：275的氨基酸序列的第二链、一条包含SEQ ID NO：291的氨基酸序列的第三链和一条包含SEQ ID NO：292的氨基酸序列的第四链。The bispecific antibody that specifically binds to PSMA and CD3 has: a first chain including the amino acid sequence of SEQ ID NO: 298, a second chain including the amino acid sequence of SEQ ID NO: 275, and a second chain including the amino acid sequence of SEQ ID NO: 275. : the third strand of the amino acid sequence of SEQ ID NO: 291 and a fourth strand containing the amino acid sequence of SEQ ID NO: 292.
  20. 根据权利要求15至17中任一项所述的方法，其中所述的特异性结合PSMA和CD3的双特异性抗体包含两条式(a)所示结构的第一链和两条式(b)所示结构的第二链：The method according to any one of claims 15 to 17, wherein the bispecific antibody that specifically binds PSMA and CD3 comprises two first chains of the structure represented by formula (a) and two formulas (b ) of the second strand of the structure shown:
    (a)[PSMA-VH]-[IgG恒定区]-[连接子12]-[CD3-VL]-[连接子13]-[CD3-VH]；(a) [PSMA-VH]-[IgG constant region]-[linker 12]-[CD3-VL]-[linker 13]-[CD3-VH];
    (b)[PSMA-VL]-CL；(b)[PSMA-VL]-CL;
    式(a)和(b)所示的结构是从N端至C端排列的，所述连接子12和连接子13是相同或不同的肽连接子；优选地，The structures shown in formulas (a) and (b) are arranged from the N end to the C end, and the linker 12 and the linker 13 are the same or different peptide linkers; preferably,
    其中所述的特异性结合PSMA和CD3的双特异性抗体具有两条包含SEQ ID NO：299的氨基酸序列的第一链和两条包含SEQ ID NO：300的氨基酸序列的第二链。The bispecific antibody that specifically binds PSMA and CD3 has two first chains including the amino acid sequence of SEQ ID NO: 299 and two second chains including the amino acid sequence of SEQ ID NO: 300.
  21. 根据权利要求14所述的方法，其中所述的第二治疗剂是免疫检查点抑制剂，其中所述的免疫检查点抑制剂靶向PD-1或CTLA4；优选地，其中所述的免 疫检查点抑制剂是抗PD-1抗体。The method of claim 14, wherein the second therapeutic agent is an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor targets PD-1 or CTLA4; preferably, the immune checkpoint inhibitor targets PD-1 or CTLA4; preferably, the immune checkpoint inhibitor targets PD-1 or CTLA4; Checkpoint inhibitors are anti-PD-1 antibodies.
  22. 根据权利要求21所述的方法，其中所述的抗PD-1抗体包含重链可变区PD-1-VH和轻链可变区PD-1-VL，其中：The method of claim 21, wherein the anti-PD-1 antibody comprises a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, wherein:
    所述PD-1-VH具有：包含SEQ ID NO：319的氨基酸序列的PD-1-HCDR1，包含SEQ ID NO：320的氨基酸序列的PD-1-HCDR2，和包含SEQ ID NO：321的氨基酸序列的PD-1-HCDR3；和所述PD-1-VL具有：包含SEQ ID NO：322的氨基酸序列的PD-1-LCDR1，包含SEQ ID NO：323的氨基酸序列的PD-1-LCDR2，和包含SEQ ID NO：324的氨基酸序列的PD-1-LCDR3；The PD-1-VH has: PD-1-HCDR1 comprising the amino acid sequence of SEQ ID NO: 319, PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO: 320, and the amino acid sequence comprising SEQ ID NO: 321 the sequence of PD-1-HCDR3; and the PD-1-VL having: PD-1-LCDR1 comprising the amino acid sequence of SEQ ID NO: 322, PD-1-LCDR2 comprising the amino acid sequence of SEQ ID NO: 323, and PD-1-LCDR3 comprising the amino acid sequence of SEQ ID NO: 324;
    优选地，Preferably,
    所述PD-1-VH包含SEQ ID NO：325的氨基酸序列，和所述PD-1-VL包含SEQ ID NO：326的氨基酸序列；The PD-1-VH includes the amino acid sequence of SEQ ID NO: 325, and the PD-1-VL includes the amino acid sequence of SEQ ID NO: 326;
    更优选地，More preferably,
    所述抗PD-1抗体包含SEQ ID NO：328的氨基酸序列的重链，和SEQ ID NO：329的氨基酸序列的轻链。 The anti-PD-1 antibody comprises a heavy chain of the amino acid sequence of SEQ ID NO:328, and a light chain of the amino acid sequence of SEQ ID NO:329.
PCT/CN2023/087463 2022-04-11 2023-04-11 Antigen-binding molecule specifically binding to psma and cd28 and pharmaceutical use thereof WO2023198015A1 (en)

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