WO2023198042A1 - Antigen-binding molecule specifically binding to egfr and cd28, and medical use thereof - Google Patents

Antigen-binding molecule specifically binding to egfr and cd28, and medical use thereof Download PDF

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Publication number
WO2023198042A1
WO2023198042A1 PCT/CN2023/087574 CN2023087574W WO2023198042A1 WO 2023198042 A1 WO2023198042 A1 WO 2023198042A1 CN 2023087574 W CN2023087574 W CN 2023087574W WO 2023198042 A1 WO2023198042 A1 WO 2023198042A1
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Prior art keywords
seq
amino acid
acid sequence
antigen
egfr
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PCT/CN2023/087574
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French (fr)
Chinese (zh)
Inventor
石金平
王倩
黎婷婷
金薪盛
应华
陶维康
Original Assignee
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 WO2023198042A1 publication Critical patent/WO2023198042A1/en

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    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

  • the present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to EGFR/CD28 antigen-binding molecules and their applications.
  • EGFR is an antigen widely expressed on a variety of tumors. After it binds to ligands such as EGF, it phosphorylates the intracellular region, thereby initiating downstream signaling pathways, affecting cell proliferation, apoptosis and migration, and can affect angiogenesis. . When EGFR is mutated or overexpressed, downstream phosphorylation is not inhibited, thereby promoting the malignant proliferation of tumor cells.
  • EGFR monoclonal antibodies that have been approved or are in the clinical stage, such as cetuximab, panitumumab, nimotuzumab, nituzumab, matuzumab, etc., which can be used to treat colorectal cancer, head and neck cancer, etc. cancer, non-small cell lung cancer, glioma, gastric cancer and other cancers.
  • monoclonal antibodies only bind to the ECD region of EGFR, mutations in the intracellular region and activation of the alternative pathway make patients prone to drug resistance.
  • T cells relies on the first signal provided by TCR activated by MHC-peptide complexes on the surface of APC cells or other cells.
  • costimulatory factors are required to provide the second signal to completely activate T cells.
  • CD28 as one of the major costimulatory molecules on the surface of T cells, 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.
  • Agonistic anti-CD28 mAbs can be used for sustained ex vivo expansion of cultured T cells; however, a series of acute and serious adverse events occurred in phase I clinical trials of superagonist anti-CD28 mAbs, and the use of anti-CD28 antibodies has been discouraged. (Hünig, Nature Reviews Immunology. 2012; 12:317-318).
  • anti-PD-1 and anti-PD-L1 antibody treatment can directly alleviate tumor-mediated T cell exhaustion and effectively modulate anti-tumor immune responses in vivo.
  • anti-PD-1/PD-L1 therapy there are still many unresolved problems in the clinical application of anti-PD-1/PD-L1 therapy. For example, more than 50% of tumors that strongly express PD-L1 do not respond to PD-1/PD-L1 inhibitors.
  • EGFR-targeted therapy can be combined with PD1 antibody immunotherapy, but the results of clinical trials are not ideal, which should be related to the immunosuppressive state of the tumor.
  • the present disclosure provides an antigen-binding molecule that specifically binds CD28 and EGFR.
  • the present disclosure provides an antigen-binding molecule comprising at least one antigen-binding moiety that specifically binds CD28 and at least one antigen-binding moiety that specifically binds EGFR, the antigen-binding moiety that specifically binds CD28 comprises a heavy The chain variable region CD28-VH and the light chain variable region CD28-VL, the antigen-binding module that specifically binds to EGFR includes the heavy chain variable region EGFR-VH and the light chain variable region EGFR-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 groups of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33 acid sequence
  • 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: 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: 46
  • 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: 52;
  • 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: 32.
  • 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: an amino group containing SEQ ID NO: 16 CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 62, 17, 54, 55, 56, 57, 58, 59, 60 or 61 and CD28 comprising the amino acid sequence of SEQ ID NO: 18 -LCDR3; 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: 39, 9, 40, 41 or CD28-HCDR3 with an amino acid sequence of 42, 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 or 11, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 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: an amino group containing SEQ ID NO: 28 CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 30.
  • 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 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: 84, and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; 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 containing the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 containing the amino acid sequence of SEQ ID NO: 8, and an amino group containing SEQ ID NO: 39 CD28-HCDR3 with acid sequence
  • 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-HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and
  • the CD28-VL has: CD28-LCDR1 including the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 including the amino acid sequence of SEQ ID NO: 23, and CD28-LCDR3 including the amino acid sequence of SEQ ID NO: 24.
  • 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: 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 CD28-VL has: CD28-LCDR1 including the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 including the amino acid sequence of SEQ ID NO: 62, and CD28-LCDR3 including the amino acid sequence of SEQ ID NO: 18.
  • 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 including the amino acid sequence of SEQ ID NO:28, CD28-LCDR2 including the amino acid sequence of SEQ ID NO:29, and CD28-LCDR3 including the amino acid sequence of 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: 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, CD28-LCDR2 as shown in SEQ ID NO: 62, 17, 54, 55, 56, 57, 58, 59, 60 or 61 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- as shown in SEQ ID NO: 27 HCDR3, and the CD28-VL comprises 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 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: 39, 9, 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: 43 or 11, 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 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 .
  • 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, such as SEQ ID NO: CD28-HCDR2 shown in 20 and CD28-HCDR3 shown in SEQ ID NO: 21, and the CD28-VL includes CD28-LCDR1 shown in SEQ ID NO: 22, CD28-LCDR1 shown in SEQ ID NO: 23 CD28-LCDR2 and CD28-LCDR3 as set forth in SEQ ID NO: 24; or
  • 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: 84, 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:39, 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 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 is CD28-LCDR1 as shown in SEQ ID NO:28, CD28-LCDR2 as shown in SEQ ID NO:29, and CD28-LCDR3 as shown 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, as shown in SEQ ID NO: CD28-HCDR2 shown in 8 and CD28-HCDR3 shown in SEQ ID NO: 39, and the CD28-VL is CD28-LCDR1 shown in SEQ ID NO: 10, CD28 shown in SEQ ID NO: 43 -LCDR2 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 is CD28-LCDR1 as shown in SEQ ID NO:22, CD28-LCDR2 as shown in SEQ ID NO:84, and CD28-LCDR3 as shown in SEQ ID NO:24.
  • 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 as described in any one of the preceding items wherein the CD28-VH and CD28-VL are humanized and comprise the FR region of a human antibody.
  • 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 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.
  • CD28-LCDR3 of the amino acid sequence, and the FR region of the 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 CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 27
  • the CD28-HCDR3 of the sequence, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 30S, 71V and 78A; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 having the amino acid sequence of NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and CD28-LCDR2 comprising 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 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-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 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: 19, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 20, and CD28-HCDR3 including the amino acid sequence of SEQ ID NO: 21, 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 NO: 22 CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 84, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 84, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, and the FR region of the CD28-VL contains a protein selected from the group consisting of 41D, One or more amino acid substitutions from the group consisting of 42G, 43T, 44I and 71Y; 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 FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 66K, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 containing the amino acid sequence of NO: 16, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 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
  • 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.
  • the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 30S, 71V and 78A; and the CD28-VL has: comprising SEQ ID NO: 28
  • the CD28-LCDR1 of the amino acid sequence, the CD28-LCDR2 of the amino acid sequence of SEQ ID NO: 29 and the CD28-LCDR3 of the amino acid sequence of SEQ ID NO: 30, and the FR region of the CD28-VL is selected from the group consisting of 41D and 42G.
  • 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: 39 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 containing the amino acid sequence of SEQ ID NO: 43, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 12; and the FR region of the CD28-VL contains a protein selected from the group consisting of 43S 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 has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 20, and CD28-HCDR3 including the amino acid sequence of SEQ ID NO: 21, 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 NO: 22 CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 23, 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 the CD28-VL contains a protein selected from the group consisting of 41D, One or more amino acid substitutions from the group consisting of 42G, 43T, 44I and 71Y.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • 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-HCDR3 including 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, 66K, 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: 62, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 18, 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.
  • 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 FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 30S, 71V and 78A; and the CD28-VL has: comprising the amino acid sequence of 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 from the group consisting of 44V and 73L.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the EGFR-VH includes EGFR-HCDR1 as shown in SEQ ID NO: 160, EGFR-HCDR2 as shown in SEQ ID NO: 161, and EGFR-HCDR3 as shown in SEQ ID NO: 162; and the EGFR- VL contains EGFR-LCDR1 as shown in SEQ ID NO: 163, EGFR-LCDR2 as shown in SEQ ID NO: 164, and EGFR-LCDR3 as shown in SEQ ID NO: 165.
  • 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: 46, 44, 31, 45, 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 group of SEQ ID NO: 68, 63, 64, 65, 66, 67 or 69 or
  • 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:
  • said CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98
  • said CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102
  • 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: 46
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52 or 50, 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: 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
  • the CD28-VL comprises 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
  • the amino acid sequence of CD28-VH is shown in SEQ ID NO: 68, and the amino acid sequence of CD28-VL is The amino acid sequence 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
  • 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: 68, and the CD28-VL contains Containing 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: 46
  • 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:
  • 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
  • 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 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 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: 106 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: 46, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 52 shown.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 158
  • the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • amino acid sequence of EGFR-VH is shown in SEQ ID NO: 158
  • EGFR-VL The amino acid sequence is shown in SEQ ID NO: 159.
  • 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 EGFR includes a Titin chain and an Obscurin chain, wherein the Titin chain and Obscurin chains are able to form dimers.
  • 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 EGFR 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 Titin chain includes an amino acid sequence selected from the group consisting of SEQ ID NO: 218 to SEQ ID NO: 236, and the Obscurin chain includes an amino acid sequence selected from the group consisting of The amino acid sequence of the group consisting of SEQ ID NO: 237 to SEQ ID NO: 277.
  • 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: 272.
  • the antigen-binding molecule of any one of the preceding items wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 234, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 272.
  • the antigen-binding molecule of any one of the preceding items wherein the antigen-binding molecule comprises a human IgG Fc region.
  • the antigen-binding molecule of any one of the preceding items wherein the antigen-binding molecule comprises a human 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 acid residues at positions 234 and 235 are A, respectively,
  • 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 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 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 hole structure according to the pestle and mortar technology; and vice versa.
  • 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: 167; and the Fc2 includes the amino acid sequence of SEQ ID NO: 168.
  • 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 EGFR.
  • 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 EGFR, wherein:
  • the antigen-binding module that specifically binds to EGFR 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.
  • 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 with the structure shown in formula (c) and a fourth chain with the structure shown in formula (d) ( Figure 1A),
  • the Fc1 and the Fc2 are interchangeable;
  • 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) , one with A third chain having the structure shown in formula (c) and a fourth chain having the structure shown in formula (d) ( Figure 1A),
  • the Fc1 and the Fc2 are interchangeable;
  • 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 has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 20, and CD28-HCDR3 including the amino acid sequence of SEQ ID NO: 21; and the CD28-VL having: 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-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24 ;and
  • the EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
  • 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 having: 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-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30 ;and
  • the EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
  • 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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159; 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
  • the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 158
  • the amino acid sequence of EGFR-VL includes the amino acid sequence of SEQ ID NO: 159.
  • 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 EGFR-VH is shown in SEQ ID NO: 158 is shown, and the amino acid sequence of the EGFR-VL is shown in SEQ ID NO: 159.
  • 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: 126, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 134; and the amino acid sequence of EGFR-VH is shown in SEQ ID NO: 158 is shown, and the amino acid sequence of the EGFR-VL is shown in SEQ ID NO: 159.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain and Obscurin chain are any Titin chains in Table 3-1 and Table 3-2 of the present disclosure that can form dimers 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: 234.
  • 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: 272.
  • 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 the linker 1 and linker 2 are both GGGGS (SEQ ID NO: 169).
  • 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: 166.
  • 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 a kappa chain or a lamada chain. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 145.
  • 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 EGFR, and the antigen-binding module that specifically binds EGFR
  • the antigen-binding module is a replaced Fab, which contains a Titin chain and an Obscurin chain capable of forming dimers; the antigen-binding module that specifically binds to CD28 is Fab.
  • 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) ( Figure 1B),
  • Fc1 and Fc2 are interchangeable
  • 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, a third chain having the structure shown in formula (g) and a fourth chain having the structure shown in formula (h) ( Figure 1B),
  • Fc1 and Fc2 are interchangeable
  • 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 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-HCDR3 including the amino acid sequence of SEQ ID NO: 15; and the CD28-VL having: 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-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18 ;and
  • the EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: containing SEQ ID NO: 163 EGFR-LCDR1 having the amino acid sequence of SEQ ID NO: 164, EGFR-LCDR2 having the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 having the amino acid sequence of SEQ ID NO: 165.
  • the antigen-binding molecule of any one of the preceding items wherein:
  • the CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 20, and CD28-HCDR3 including the amino acid sequence of SEQ ID NO: 21; and the CD28-VL having: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84, and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24 ;and
  • the EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
  • 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-HCDR3 comprising the amino acid sequence of SEQ ID NO: 39; and the CD28-VL having: 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-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12 ;and
  • the EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
  • 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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159; 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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159; 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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL includes the amino acid sequence of SEQ ID NO: 159.
  • 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 package Contains the amino acid sequence of SEQ ID NO: 80; and the EGFR-VH includes the amino acid sequence of SEQ ID NO: 158, and the EGFR-VL includes the amino acid sequence of SEQ ID NO: 159.
  • 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 EGFR-VH is SEQ ID NO: shown in 158, and the amino acid sequence of the EGFR-VL is shown in SEQ ID NO: 159.
  • the antigen-binding molecule as described in any one of the preceding items, wherein the Titin chain and Obscurin chain are any Titin chains in Table 3-1 and Table 3-2 of the present disclosure that can form dimers 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: 234.
  • 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: 272.
  • 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: 169).
  • 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: 166.
  • 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.
  • 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, and the CD28- CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 points in VL specifically comprising 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: 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 contain the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 32 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-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- 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 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 CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 27
  • the sequence of CD28-HCDR3, and the CD28-VL has: contains SEQ. CD28-LCDR1 having the amino acid sequence of ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 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, 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 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: an amino group containing SEQ ID NO: 22 or
  • 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: 84, and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; 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; 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: 39, and
  • the CD28-VL has: CD28-LCDR1 including the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 including the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR3 including the amino acid sequence of 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, 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, CD28-LCDR2 set forth in 55, 56, 57, 58, 59, 60, 61 or 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 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 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: 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: 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: 84, 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 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; 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:39, and the CD28- VL contains 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 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 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 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 is CD28-LCDR1 as shown in SEQ ID NO:28, CD28-LCDR2 as shown in SEQ ID NO:29, and CD28-LCDR3 as shown in SEQ ID NO:30.
  • 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 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 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 FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 66K, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 of the amino acid sequence of NO: 16, CD28-LCDR2 of the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and SEQ ID NO: 18
  • the CD28-LCDR3 of the amino acid sequence, and the FR region of the 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 CD28-HCDR3 of the sequence, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 30S, 71V and 78A
  • the CD28-VL has: comprising SEQ ID 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
  • the FR region of CD28-VL comprises 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-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 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-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 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: 19, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 20, and CD28-HCDR3 including the amino acid sequence of SEQ ID NO: 21, 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 NO: 22 CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 84, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 84, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, and the FR region of the CD28-VL contains a protein selected from the group consisting of 41D, One or more amino acid substitutions from the group consisting of 42G, 43T, 44I and 71Y; 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 FR region of CD28-VH includes 1E, 28S, 66K, One or more amino acid substitutions in the group consisting of 69L, 71V, 73K and 94S; and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, and comprising the amino acid sequence of SEQ ID NO: 62 A CD28-LCDR2 and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18, and the FR region of the CD28-VL comprises one or more amino acid substitutions selected from the group consisting of 43S and 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.
  • the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 30S, 71V and 78A; and the CD28-VL has: comprising SEQ ID NO: 28
  • the CD28-LCDR1 of the amino acid sequence, the CD28-LCDR2 of the amino acid sequence of SEQ ID NO: 29 and the CD28-LCDR3 of the amino acid sequence of SEQ ID NO: 30, and the FR region of the CD28-VL is selected from the group consisting of 41D and 42G.
  • 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 containing the amino acid sequence of SEQ ID NO: 43, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 12; and the FR region of the CD28-VL contains a protein selected from the group consisting of 43S One or more amino acid substitutions in the group consisting of 73F; 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: 39, 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 the amino acid of SEQ ID NO: 10
  • the CD28-LCDR1 of the sequence, the CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43 and the CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and the FR region of the CD28-VL is composed of 43S and 73F.
  • One or more amino acids in the group are substituted.
  • 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 comprises the amino acid sequence of SEQ ID NO: 98 or a variant thereof, wherein the variant contains the FR region of SEQ ID NO: 98 selected from the group consisting of 26A, 29L, 69L, 71V, 78A and 93S consisting of one or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 102 or a variant thereof, the variant being the FR region of SEQ ID NO: 102 comprising a member selected from 41D 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 includes a FR region of SEQ ID NO: 69 selected from the group consisting of 28S, 66K, 69L, 71V, 73K and 94S consisting of 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 comprising 43S selected from the group consisting of and one or more amino acid substitutions in the group consisting of 70K; or
  • the CD28-VH comprises the amino acid sequence of SEQ ID NO: 133 or a variant thereof, wherein the FR region of SEQ ID NO: 133 includes a group selected from the group consisting of 25P, 30S, 71V and 78A.
  • One or more amino acid substitutions, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 137 or a variant thereof, wherein 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: 46 or a variant thereof, the variant comprising an amino acid substitution selected from 69L in the FR region of SEQ ID NO: 46, and the CD28-VL comprises SEQ ID The amino acid sequence of NO: 52 or a variant thereof, which variant is one or more amino acid substitutions selected from the group consisting of 43S and 73F in the FR region of SEQ ID NO: 52
  • 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, 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 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: 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:
  • said CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98
  • said CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102
  • 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: 46
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52 or 50, 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: 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: 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: 94
  • the CD28-VL comprises 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, 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: 95
  • the CD28-VL comprises the amino acid sequence of SEQ ID NO: 84; 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: 46
  • 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: 95, and the amino acid sequence of the CD28-VL is as shown in SEQ ID NO: 106 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 CD28-VH
  • the amino acid sequence is shown in SEQ ID NO:44
  • the amino acid sequence of CD28-VL is shown in SEQ ID NO:52.
  • 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 comprises the amino acid sequence of SEQ ID NO: 146, and the light chain comprises 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 EGFR.
  • the present disclosure provides a pharmaceutical composition, which contains: a therapeutically effective amount of the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing, and one or more A pharmaceutically acceptable carrier, diluent, buffer or excipient.
  • the pharmaceutical composition further includes at least one second therapeutic agent.
  • the second therapeutic agent includes an antineoplastic agent, radiation therapy, an antibody drug conjugate, a bispecific antibody, a bispecific antibody conjugated to an antineoplastic agent, an immune checkpoint inhibitor, or the like. combination.
  • the second therapeutic agent is any agent that is advantageously combined with an EGFR/CD28 bispecific antigen-binding molecule.
  • the second therapeutic agent is an antibody capable of specifically binding CD3.
  • the second therapeutic agent is a bispecific antibody capable of specifically binding CD3.
  • the second therapeutic agent is a bispecific antibody capable of specifically binding CD3 and a tumor cell surface antigen (TAA).
  • TAA tumor cell surface antigen
  • the second therapeutic agent is a bispecific antibody capable of specifically binding to CD3 and MUC16.
  • the second therapeutic agent is an anti-PD-L1 antibody.
  • the anti-PD-1 antibody includes any known anti-PD-1 antibody with therapeutic activity, including but not limited to Camrelizumab, Keytruda , nivolumab (Opdivo) and cemiplimab (cemiplimab).
  • the anti-PD-1 antibody comprises 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 comprising the amino acid sequence of SEQ ID NO: 206, PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO: 207, and comprising the amino acid sequence of SEQ ID NO: 208 the sequence of PD-1-HCDR3; and the PD-1-VL having: PD-1-LCDR1 comprising the amino acid sequence of SEQ ID NO: 209, PD-1-LCDR2 comprising the amino acid sequence of SEQ ID NO: 210, and PD-1-LCDR3 comprising the amino acid sequence of SEQ ID NO: 211.
  • the anti-PD-1 antibody comprises a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, wherein:
  • the PD-1-VH includes PD-1-HCDR1 as shown in SEQ ID NO: 206, includes PD-1-HCDR2 as shown in SEQ ID NO: 207, and as shown in SEQ ID NO: 208 PD-1-HCDR3; and said PD-1-VL comprising PD-1-LCDR1 as set forth in SEQ ID NO:209, comprising PD-1-LCDR2 as set forth in SEQ ID NO:210, and said PD-1-VL as set forth in SEQ ID NO:210 PD-1-LCDR3 shown in NO:211.
  • the anti-PD-1 antibody comprises 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:212
  • the PD-1-VL includes the amino acid sequence of SEQ ID NO:213.
  • the anti-PD-1 antibody comprises a heavy chain and a light chain, wherein:
  • the anti-PD-1 antibody includes a heavy chain of the amino acid sequence of SEQ ID NO: 215, and a light chain of the amino acid sequence of SEQ ID NO: 216.
  • the second therapeutic agent is a bispecific antibody that specifically binds MUC16 and CD3.
  • the second therapeutic agent is a bispecific antibody that specifically binds PSMA and CD3.
  • the second therapeutic agent is a bispecific antibody that specifically binds STEAP2 and CD3.
  • the second therapeutic agent is a bispecific antibody that specifically binds MUC16 and CD3, comprising at least one antigen-binding moiety that specifically binds MUC16 and at least one antigen-binding moiety that specifically binds CD3.
  • the second therapeutic agent is a bispecific antibody that specifically binds MUC16 and CD3, comprising at least one antigen-binding moiety that specifically binds MUC16 and at least one antigen-binding moiety that specifically binds CD3,
  • the antigen-binding module that specifically binds to MUC16 includes a heavy chain variable region MUC16-VH and a light chain variable region MUC16-VL
  • the antigen-binding module that specifically binds to CD3 includes a heavy chain variable region CD3-VH and a light chain variable region. Chain variable region CD3-VL.
  • the MUC16-VH has: MUC16-HCDR1 comprising the amino acid sequence of SEQ ID NO: 187, MUC16-HCDR2 comprising the amino acid sequence of SEQ ID NO: 188, and MUC16-HCDR3 comprising the amino acid sequence of SEQ ID NO: 189; and the MUC16-VL has: MUC16-LCDR1 comprising the amino acid sequence of SEQ ID NO: 190, MUC16-LCDR2 comprising the amino acid sequence of SEQ ID NO: 191 , and MUC16-LCDR3 comprising the amino acid sequence of SEQ ID NO:192.
  • the MUC16-VH includes MUC16-HCDR1 as set forth in SEQ ID NO: 187, MUC16-HCDR2 as set forth in SEQ ID NO: 188, and MUC16-HCDR1 as set forth in SEQ ID NO: 189 HCDR3; and the MUC16-VL includes MUC16-LCDR1 as shown in SEQ ID NO: 190, MUC16-LCDR2 as shown in SEQ ID NO: 191, and MUC16-LCDR3 as shown in SEQ ID NO: 192.
  • said MUC16-VH comprises the amino acid sequence of SEQ ID NO: 199
  • said MUC16-VL comprises the amino acid sequence of SEQ ID NO: 200.
  • 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:
  • the CD3-VH has: CD3-HCDR1 including the amino acid sequence of SEQ ID NO: 193, CD3-HCDR2 including the amino acid sequence of SEQ ID NO: 194, and CD3-HCDR3 including the amino acid sequence of SEQ ID NO: 195; And the CD3-VL has: CD3-LCDR1 including the amino acid sequence of SEQ ID NO: 196, CD3-LCDR2 including the amino acid sequence of SEQ ID NO: 197, and CD3-LCDR3 including the amino acid sequence of SEQ ID NO: 198 .
  • the CD3-VH comprises CD3-HCDR1 as set forth in SEQ ID NO: 193, CD3-HCDR2 as set forth in SEQ ID NO: 194, and CD3-HCDR1 as set forth in SEQ ID NO: 195 HCDR3; and the CD3-VL includes CD3-LCDR1 as shown in SEQ ID NO: 196, CD3-LCDR2 as shown in SEQ ID NO: 197, and CD3-LCDR3 as shown in SEQ ID NO: 198.
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO:201
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO:202.
  • the bispecific antibody that specifically binds MUC16 and CD3 comprises:
  • the MUC16-VH includes MUC16-HCDR1 as shown in SEQ ID NO: 187, MUC16-HCDR2 as shown in SEQ ID NO: 188, and MUC16-HCDR3 as shown in SEQ ID NO: 189; and the MUC16 -VL contains MUC16-LCDR1 as shown in SEQ ID NO:190, MUC16-LCDR2 as shown in SEQ ID NO:191, and MUC16-LCDR3 as shown in SEQ ID NO:192; and
  • the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 193, CD3-HCDR2 as shown in SEQ ID NO: 194, and CD3-HCDR3 as shown in SEQ ID NO: 195; and the CD3 -VL contains CD3-LCDR1 as set forth in SEQ ID NO: 196, CD3-LCDR2 as set forth in SEQ ID NO: 197, and CD3-LCDR3 as set forth in SEQ ID NO: 198.
  • the bispecific antibody that specifically binds MUC16 and CD3 comprises:
  • the MUC16-VH comprises the amino acid sequence of SEQ ID NO: 199, and the MUC16-VL Comprising the amino acid sequence of SEQ ID NO: 200;
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO:201
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO:202.
  • the bispecific antibody that specifically binds MUC16 and CD3 includes: a first chain comprising the amino acid sequence of SEQ ID NO: 203, and two first chains comprising the amino acid sequence of SEQ ID NO: 204. The second strand and a third strand containing the amino acid sequence of SEQ ID NO: 205.
  • 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 or preventing a hyperproliferative disease, the method comprising administering to a subject a therapeutically effective amount of the antigen-binding molecule of any one of the foregoing or any one of the foregoing.
  • the present disclosure also provides the use of the antigen-binding molecule described in any one of the foregoing, the anti-CD28 antibody described in any of the foregoing, or the pharmaceutical composition described in any of the foregoing in the preparation of the treatment or prevention of hyperproliferative diseases. uses in medicines.
  • the present disclosure provides the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing or the pharmaceutical composition of any of the foregoing, which is used to treat or prevent hyperproliferative disease.
  • the present disclosure provides the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing or the pharmaceutical composition of any of the foregoing, which is used to treat or prevent hyperproliferative disease.
  • the antigen-binding molecule and the second therapeutic agent are administered simultaneously, sequentially, or separately.
  • the antigen binding molecule and the second therapeutic agent are in the same or different containers.
  • the present disclosure also provides the antigen-binding molecule according to any one of the foregoing items or the anti-CD28 antibody according to any one of the foregoing items or the pharmaceutical composition according to any one of the foregoing items for use as a medicament.
  • the drugs are used to treat hyperproliferative diseases.
  • the hyperproliferative disease of any one of the preceding items is cancer.
  • the cancer of any one of the preceding items is selected from the group consisting of lung cancer, lung adenocarcinoma, non-small cell lung cancer, lung squamous cell carcinoma, esophageal cancer, cervical squamous cell carcinoma, endometrial cancer, endometrium Adenocarcinoma, bladder cancer, bladder urothelial cancer, colorectal cancer (including colon and rectal cancer), head and neck cancer, head and neck squamous cell carcinoma, glioma, brain cancer, glioblastoma multiforme tumors, gastric cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, prostate cancer, ovarian cancer, kidney cancer, liver cancer, pancreatic cancer, melanoma, osteosarcoma and skin cancer.
  • the cancer as described above is an EGFR-expressing cancer.
  • the cancer as described above is a solid tumor.
  • the cancer as described above is lung cancer.
  • the cancer as described above is non-small cell lung cancer.
  • the method of treating a hyperproliferative disease is as described above, further comprising using a second therapeutic agent.
  • the second therapeutic agent includes an antineoplastic agent, radiation therapy, an antibody drug conjugate, a bispecific antibody, a bispecific antibody conjugated to an antineoplastic agent, an immune checkpoint inhibitor, or the like. combination.
  • the second therapeutic agent is selected from the group consisting of a PD-1 inhibitor, an anti-PD-1 antibody, a PD-L1 inhibitor, an anti-PD-L1 antibody, Bispecific antibodies that bind to tumor target antigens and CD3, platinum anticancer chemotherapeutic agents, paclitaxel, docetaxel, vincristine, cisplatin, carboplatin, oroxin oxaliplatin, anti-cancer antibodies, trastuzumab, cetuximab, bevacizumab and cemiplimab.
  • the second therapeutic agent and the antigen-binding molecule of any one of the present disclosure are administered simultaneously, sequentially, or separately.
  • the method of treating a hyperproliferative disease is as described above, wherein the second therapeutic agent is an immune checkpoint inhibitor.
  • the method of treating a hyperproliferative disease is as described above, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
  • the method of treating a hyperproliferative disease as described above, wherein the anti-PD-1 antibody includes any known anti-PD-1 antibody with therapeutic activity including but not limited to camrelizumab Camrelizumab, Keytruda, Opdivo and cemiplimab.
  • the method of treating a hyperproliferative disease as described above, wherein the anti-PD-1 antibody comprises a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL in:
  • the PD-1-VH has: PD-1-HCDR1 comprising the amino acid sequence of SEQ ID NO: 206, PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO: 207, and comprising the amino acid sequence of SEQ ID NO: 208 the sequence of PD-1-HCDR3; and the PD-1-VL having: PD-1-LCDR1 comprising the amino acid sequence of SEQ ID NO: 209, PD-1-LCDR2 comprising the amino acid sequence of SEQ ID NO: 210, and PD-1-LCDR3 comprising the amino acid sequence of SEQ ID NO: 211.
  • the method of treating a hyperproliferative disease as described above, wherein the anti-PD-1 antibody comprises a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL in:
  • the PD-1-VH includes PD-1-HCDR1 as shown in SEQ ID NO: 206, includes PD-1-HCDR2 as shown in SEQ ID NO: 207, and as shown in SEQ ID NO: 208 PD-1-HCDR3; and said PD-1-VL comprising PD-1-LCDR1 as set forth in SEQ ID NO:209, comprising PD-1-LCDR2 as set forth in SEQ ID NO:210, and said PD-1-VL as set forth in SEQ ID NO:210 PD-1-LCDR3 shown in NO:211.
  • the method of treating a hyperproliferative disease as described above, wherein the anti-PD-1 antibody comprises a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL in:
  • the PD-1-VH includes the amino acid sequence of SEQ ID NO:212
  • the PD-1-VL includes the amino acid sequence of SEQ ID NO:213.
  • the anti-PD-1 antibody includes a heavy chain of the amino acid sequence of SEQ ID NO: 215, and a light chain of the amino acid sequence of SEQ ID NO: 216.
  • the second therapeutic agent is a bispecific antibody that specifically binds to MUC16 and CD3.
  • the second therapeutic agent is a bispecific antibody that specifically binds PSMA and CD3.
  • the second therapeutic agent is a bispecific antibody that specifically binds STEAP2 and CD3.
  • the second therapeutic agent is a bispecific antibody that specifically binds MUC16 and CD3, which includes at least one antigen-binding module that specifically binds MUC16. and at least one antigen-binding module that specifically binds CD3.
  • the second therapeutic agent is a bispecific antibody that specifically binds MUC16 and CD3, which includes at least one antigen-binding module that specifically binds MUC16. and at least one antigen-binding module that specifically binds to CD3, the antigen-binding module that specifically binds to MUC16 includes the heavy chain variable region MUC16-VH and the light chain variable region MUC16-VL, and the antigen-binding module that specifically binds to CD3 The module contains the heavy chain variable domain CD3-VH and the light chain variable domain CD3-VL.
  • a method of treating a hyperproliferative disease as described above wherein:
  • the MUC16-VH has: MUC16-HCDR1 including the amino acid sequence of SEQ ID NO: 187, MUC16-HCDR2 including the amino acid sequence of SEQ ID NO: 188, and MUC16-HCDR3 including the amino acid sequence of SEQ ID NO: 189; And the MUC16-VL has: MUC16-LCDR1 including the amino acid sequence of SEQ ID NO: 190, MUC16-LCDR2 including the amino acid sequence of SEQ ID NO: 191, and MUC16-LCDR3 including the amino acid sequence of SEQ ID NO: 192 .
  • a method of treating a hyperproliferative disease as described above wherein:
  • the MUC16-VH includes MUC16-HCDR1 as shown in SEQ ID NO: 187, MUC16-HCDR2 as shown in SEQ ID NO: 188, and MUC16-HCDR3 as shown in SEQ ID NO: 189; and the MUC16 -VL contains MUC16-LCDR1 as shown in SEQ ID NO:190, MUC16-LCDR2 as shown in SEQ ID NO:191, and MUC16-LCDR3 as shown in SEQ ID NO:192.
  • the method of treating a hyperproliferative disease as described above wherein the MUC16-VH comprises the amino acid sequence of SEQ ID NO: 199, and the MUC16-VL comprises the amino acid sequence of SEQ ID NO: 200.
  • a method of treating a hyperproliferative disease as described above, wherein the specific The antigen-binding module that binds CD3 contains the 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: 193, CD3-HCDR2 including the amino acid sequence of SEQ ID NO: 194, and CD3-HCDR3 including the amino acid sequence of SEQ ID NO: 195; And the CD3-VL has: CD3-LCDR1 including the amino acid sequence of SEQ ID NO: 196, CD3-LCDR2 including the amino acid sequence of SEQ ID NO: 197, and CD3-LCDR3 including the amino acid sequence of SEQ ID NO: 198 .
  • a method of treating a hyperproliferative disease as described above wherein:
  • the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 193, CD3-HCDR2 as shown in SEQ ID NO: 194, and CD3-HCDR3 as shown in SEQ ID NO: 195; and the CD3 -VL contains CD3-LCDR1 as set forth in SEQ ID NO: 196, CD3-LCDR2 as set forth in SEQ ID NO: 197, and CD3-LCDR3 as set forth in SEQ ID NO: 198.
  • the method of treating a hyperproliferative disease as described above comprises the amino acid sequence of SEQ ID NO: 201, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 202.
  • the method of treating a hyperproliferative disease as described above, wherein the bispecific antibody that specifically binds MUC16 and CD3 comprises:
  • the MUC16-VH includes MUC16-HCDR1 as shown in SEQ ID NO: 187, MUC16-HCDR2 as shown in SEQ ID NO: 188, and MUC16-HCDR3 as shown in SEQ ID NO: 189; and the MUC16 -VL contains MUC16-LCDR1 as shown in SEQ ID NO:190, MUC16-LCDR2 as shown in SEQ ID NO:191, and MUC16-LCDR3 as shown in SEQ ID NO:192; and
  • the CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 193, CD3-HCDR2 as shown in SEQ ID NO: 194, and CD3-HCDR3 as shown in SEQ ID NO: 195; and the CD3 -VL contains CD3-LCDR1 as set forth in SEQ ID NO: 196, CD3-LCDR2 as set forth in SEQ ID NO: 197, and CD3-LCDR3 as set forth in SEQ ID NO: 198.
  • the method of treating a hyperproliferative disease as described above, wherein the bispecific antibody that specifically binds MUC16 and CD3 comprises:
  • the MUC16-VH comprises the amino acid sequence of SEQ ID NO: 199
  • the MUC16-VL comprises the amino acid sequence of SEQ ID NO: 200
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO:201
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO:202.
  • the method for treating hyperproliferative diseases as described above, wherein the bispecific antibody that specifically binds MUC16 and CD3 comprises: a first chain comprising the amino acid sequence of SEQ ID NO: 203, Two second strands comprising the amino acid sequence of SEQ ID NO: 204 and one third strand comprising the amino acid sequence of SEQ ID NO: 205.
  • the antigen-binding molecules provided by this disclosure have therapeutic activity, safety, and pharmacokinetic properties. and good drugability (such as stability).
  • Figure 1A shows a schematic structural diagram of Format1 of EGFR/CD28 bispecific antibody
  • Figure 1B shows a schematic structural diagram of Format2 of EGFR/CD28 bispecific antibody
  • Ob represents Obscurin.
  • Figure 2A shows the release of IFN- ⁇ from PBMC cells after fixing the concentration of MUC16/CD3 double antibody and combined with EGFR/CD28 double antibody
  • Figure 2B shows the concentration of fixed MUC16/CD3 double antibody and combined with EGFR/CD28 double antibody after PBMC The release of TNF- ⁇ from cells
  • Figure 2C shows the release of IFN- ⁇ from PBMC cells after the concentration of fixed EGFR/CD28 double antibodies, combined with MUC16/CD3 double antibodies
  • Figure 2D shows the concentration of fixed EGFR/CD28 double antibodies, combined with MUC16/CD3 double antibodies Release of TNF- ⁇ from PBMC cells after using MUC16/CD3 double antibody.
  • CD28 cluster of differentiation 28, Tp44
  • CD28 is the receptor for CD80 (B7.1) protein and CD86 (B7.2) protein.
  • B7.1 The exemplary amino acid sequence of human CD28 is shown in UniProt accession number P10747.
  • EGFR EGFR fragment
  • EGFR EGFR fragment
  • 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 refer to compounds that have the same basic chemical structure (i.e., alpha carbon bonded to hydrogen, carboxyl, amino, and R groups) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide , methionine methyl sulfonium.
  • 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. It should be understood that whether defined as T366W or 366W, the original residue at this position does not limit the scope of the claims.
  • 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 light and two 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).
  • 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 can be divided into symmetric structure bispecific antibodies and asymmetric structure bispecific antibodies according to whether the structure is symmetrical or not.
  • fragment-type bispecific antibodies such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining two or more Fab fragments in one molecule, have lower immunogenicity and small molecular weight. , with high tumor tissue penetration.
  • Typical antibody structures of this type include F(ab)2, scFv-Fab, (scFv)2-Fab.
  • IgG-like bispecific antibodies for example, with Fc fragment
  • the Fc fragment helps to purify the antibody and improve its solubility and stability.
  • the Fc part may also bind to the receptor FcRn.
  • variable region refers to the domain of an antigen-binding molecule that binds the antigen/epitope.
  • the heavy chain variable region in the antigen-binding module that specifically binds to EGFR is labeled EGFR-VH
  • the light chain variable region is labeled EGFR-VL
  • the heavy-chain variable region in the antigen-binding module that specifically binds to CD28 is labeled EGFR-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).
  • 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 EGFR-VH are labeled EGFR-HCDR1, EGFR-HCDR2 and EGFR-HCDR3 respectively;
  • the three CDR regions in EGFR-VL are labeled EGFR-LCDR1, EGFR-LCDR2 and EGFR-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, for example: the "Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", pp. 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia” numbering scheme, “ABM” numbering scheme, "contact” numbering scheme (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [ J].2001) and ImMunoGenTics (IMGT) numbering rules (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; various numbering systems The corresponding relationship is well known to those skilled in the art.
  • 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 of 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 Obscurin to form a dimerization complex.
  • 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 is capable of binding to Titin 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 part of the heavy and/or light chain is derived from a specific source. source or species, whereas the remainder 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.
  • humanization can be achieved by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions and 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 interaction between the members of a binding pair (eg, antibody and antigen).
  • KD equilibrium dissociation constant
  • 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 bound by a selective binding agent of an antigen-binding molecule, such as 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)).
  • KD equilibrium dissociation constant
  • 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 an antibody binds an antigen to its epitope with an equilibrium dissociation constant (KD) of approximately 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.
  • an 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 the antigen-binding molecule refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), included in a single vector or separate vectors. such one or more nucleic acid molecules in the body, and such one or more nucleic acid molecules present at one or more locations in the 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 (together with the host cell) operably linked thereto.
  • 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 do not Restricted to mammalian cells, insect cell lines, 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, 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 genus, Saccharomycescerevisiae, Saccharomyces cerevisiae , Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysospor
  • 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, diluents, buffers or excipients 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.
  • administer when applied to animals, humans, experimental subjects, cells, tissues, organs or organisms
  • fluid means the contact of an exogenous drug, therapeutic, diagnostic, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid.
  • 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, abdominal cavity and other body cavities, fluid collected from bronchial lavage, synovial fluid, fluid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ conditions culture medium), 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, mucosal secretions of secretor
  • 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.
  • 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 may 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, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5) and/or inhibitors of 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 the present disclosure may include, but are 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 .
  • CD28 should be understood broadly and is intended to cover various forms of CD28 molecules at various stages in the mammalian body, such as but not limited to the amplification, replication, transcription, splicing, processing, translation, and modification of the CD28 gene.
  • Produced molecules eg, precursor CD28, mature CD28, membrane-expressed CD28, CD28 splice variants, modified CD28, or fragments thereof; this term also encompasses artificially prepared or in vitro-expressed CD28.
  • EGFR should be understood broadly and is intended to cover various forms of EGFR molecules at various stages in the mammalian body, such as but not limited to the amplification, replication, transcription, splicing, processing, translation, and modification processes of the EGFR gene.
  • Produced molecules eg, precursor EGFR, mature EGFR, membrane-expressed EGFR, EGFR splice variants, modified EGFR, or fragments thereof; the term also encompasses artificially prepared or in vitro-expressed EGFR.
  • 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 (such as bispecific antibodies) that specifically bind to CD28 and EGFR and anti-CD28 antibodies.
  • the antigen-binding molecules of the present disclosure have any one of the following properties or a combination thereof:
  • the anti-CD28 antibody binds human CD28 with an EC50 of less than 1 nM, as measured by ELISA.
  • the antigen-binding molecule is present at no more than 24, 23, 22, 21, 20, 18, 15, 13, 10, 9, 8, 7, 6, 5, 4, 3 nM
  • the EC50 of the antigen-binding molecule binds to human CD28, and the antigen-binding molecule binds to human CD28 with an EC50 of no more than 35, 34, 30, 28, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 10, 9,
  • the EC50 of 8, 7, 6, and 5 nM binds to cynomolgus monkey CD28, and the antigen-binding molecule binds to cynomolgus monkey CD28 at an EC50 of no more than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, and 0.8 nM.
  • EC50 binds human EGFR.
  • the antigen The binding molecule activates Jurkat expressing IL-2 with an EC50 of less than 0.15 ⁇ g/mL (e.g., 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05 ⁇ g/mL) in the presence of the first activation signal cell.
  • combination with MUC16/CD3 bispecific antibody or anti-PD-1 antibody has a synergistic effect and shows better in vivo therapeutic activity.
  • the killing effect of EGFR/CD3 bispecific antibody on tumor cells can be significantly activated.
  • the present disclosure provides an antigen-binding molecule comprising at least one antigen-binding module that specifically binds CD28 and at least one antigen-binding module that specifically binds EGFR, wherein the antigen-binding module that specifically binds CD28 includes CD28-VH and CD28 -VL, the antigen-binding module that specifically binds to EGFR includes EGFR-VH and EGFR-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 based on clones 81, 94, 97, and 129. The antibodies or antigen-binding molecules herein are described below by taking antibody clones 97 and 94 as examples.
  • An antigen-binding molecule or anti-CD28 antibody that specifically binds CD28 and EGFR 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, CD28-LCDR2 as shown in SEQ ID NO: 62, 17, 54, 55, 56, 57, 58, 59, 60 or 61 and CD28-LCDR3 shown in SEQ ID NO:18.
  • An antigen-binding molecule or anti-CD28 antibody that specifically binds CD28 and EGFR 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, FR2, FR3 derived from IGHV1-3*01 and FR4 derived from IGHJ1*01, and is unsubstituted or has FR4 derived from IGHJ1*01 , one or more amino acid substitutions in the group consisting of 28S, 66K, 69L, 71V, 73K and 94S; and/or the humanized CD28-VL has FR1, FR2, FR3 derived from IGKV1-12*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 43S and 70K.
  • 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 in the FR region of SEQ ID NO: 69 selected from the group consisting of 28S, 66K, 69L, 71V, 73K, and 94S.
  • 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 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:106 shown.
  • the antigen-binding molecule or anti-CD28 antibody of any one of the above wherein the amino acid sequence of CD28-VH is consistent with SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69 Having 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 amino acid sequence of CD28-VH is set forth in SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69
  • the amino acid sequence of CD28-VL is 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 CD28-VH is shown in SEQ ID NO: 63, and the amino acid sequence of CD28-VL As shown in SEQ ID NO: 70 or 71, 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 EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
  • the antigen-binding molecule of any one of the preceding items, the EGFR-VH and/or the EGFR-VL is murine or humanized. In some embodiments, the EGFR-VH and/or the EGFR-VL are humanized.
  • the antigen-binding molecule as described in any one of the preceding items wherein the amino acid sequence of the EGFR-VH is shown in SEQ ID NO: 158, and the amino acid sequence of the EGFR-VL is shown in SEQ ID NO: 159 Show.
  • variable regions and CDRs described above are defined according to the Kabat numbering rule.
  • the antigen-binding molecule as described in any one of the preceding items comprising:
  • the antigen-binding molecule as described in any one of the preceding items comprising:
  • antigen-binding molecules comprising antibody series 81 and 129 disclosed according to the embodiments herein have a similar scope of technical solutions as the above-described antibody series 97 and 94.
  • 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 4
  • 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: 284), GGGS (SEQ ID NO: 285), SGGGGS (SEQ ID NO: 286), GGGTKLTVLGGG (SEQ ID NO: 287), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is bond, G, GG, GGG (SEQ ID NO:288) or GGGG (SEQ ID NO:289), 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: 169.
  • the antigen-binding molecule of the present disclosure has a first chain having a structure represented by formula (a-1), a second chain having a structure represented by formula (b-1), and a second chain having a structure represented by formula (c). a third chain having the structure shown and a fourth chain having the structure shown in formula (d),
  • antigen binding molecules include:
  • the antigen-binding molecule includes a first chain having a structure shown in formula (e), a first chain having There is a second chain having the structure shown in formula (f), a third chain having the structure shown in formula (g-1) and a fourth chain having the structure shown in formula (h-1),
  • 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. Such changes can be made to CDR "hotspots," residues encoded by codons that undergo mutations at high frequency during the somatic maturation process, and/or residues that contact the antigen, while simultaneously modifying the resulting variant VH or VL tests binding 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 Another way to introduce diversity involves methods of CDR orientation, where several CDR residues (e.g. 4-6 residues at a time) are randomized change.
  • 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 EGFR 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
  • substitution of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 can reduce effector function.
  • the Fc region is a human IgGl 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 readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes capable of binding specifically to genes encoding 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 used for vectors encoding antibodies.
  • Suitable cloning or expression hosts include fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of 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, US6040498, US6420548, US7125978 and US6417429; Vertebrate animal cells can also be used as hosts, for example mammalian cell lines adapted for 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 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 African green monkey kidney cells
  • HELA human cervical cancer cells
  • MDCK canine kidney cells
  • BRL3A buffalo rat liver cells
  • W138 human liver cells
  • Hep G2 human liver cells
  • MMT 060562 mouse breast tumors
  • TRI cells MRC 5 cells; and
  • 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 also provides immunoconjugates comprising an antigen-binding molecule conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (such as protein toxins, enzymatically active toxins, or fragments thereof of bacterial, fungal, plant or animal origin), or radioactive isotopes.
  • cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (such as protein toxins, enzymatically active toxins, or fragments thereof of bacterial, fungal, plant or animal origin), or radioactive isotopes.
  • the present disclosure provides antigen-binding molecules that can be used to detect the presence of EGFR and/or CD28 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 EGFR and/or CD28 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, EGFR and/or CD28 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.
  • Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent, chromogenic, electron dense, chemiluminescent, and radioactive labels), and indirectly detected moieties (e.g., indirectly detected via enzymatic reactions or molecular interactions).
  • module 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 MUCl6 and CD3.
  • the antigen-binding molecule and the additional therapeutic agent can be administered simultaneously, sequentially, or separately.
  • the time interval of administration can be determined by those skilled in the art, as long as the two can produce a synergistic effect.
  • 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.
  • Table 9 Correspondence table of Obscurin chain/Titin chain and linker in PL bispecific antibodies Note: The numbers of Titin chain and Obscurin chain in the table are shown in Table 3-1 and Table 3-2.
  • 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-APC-hEGFR CHO-K1-hCD28, HEK293-hCD28, CHO-K1-cyno CD28, CHO-APC-hEGFR-PDL1 and Jurkat-PD1 cells
  • relevant protein sequences used are as follows:
  • HEK293T cells co-transfect HEK293T cells (ATCC, CRL-11268) with three plasmids: pVSV-G, pCMV-dR8.91 and pCDH-h EGFR.
  • Packaging viruses 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.
  • CHO-APC-hEGFR, CHO-K1-hCD28, HEK293-hCD28 and CHO-K1-cyno CD28 cell lines with high expression of EGFR 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-hEGFR ( CRL-11268) packaged virus, 48 hours after transfection, collect the virus to infect CHO-APC-hEGFR 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-hEGFR-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 high affinity, cross-bind to cynomolgus CD28, and bind to human CD28 and cynomolgus CD28 on the cell surface. Has better 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 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, TB326Rev.B 0503). The amino acid sequences of the CDR and variable regions of the mouse antibody are as follows:
  • the underlined part is the CDR area obtained according to Kabat numbering rules.
  • variable region sequence of the mouse anti-CD28 antibody was combined with the constant region 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 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:
  • the single underlined part is the CDR
  • the double underlined part is the amino acid substitution site
  • the remaining parts are FR zone.
  • the humanized antibody of m94 antibody 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 IGHJ1*01 FR4 serves as the 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:
  • the single underlined part is the CDR
  • the double underlined part is the amino acid substitution site
  • the remaining parts are FR zone.
  • 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:
  • the single underlined part is the CDR
  • the double underlined part is the amino acid substitution site
  • the remaining parts are FR zone.
  • 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, 53, 54, 55 and/or 73 of the light chain variable region of the humanized antibody are substituted; and/or the humanized antibody The amino acid residues at positions 1, 25, 30, 34, 52, 71 and/or 78 of the heavy chain variable region of the antibody are substituted.
  • the CDRs of the humanized antibody to m129 are as follows:
  • variable region sequence of the humanized antibody of m129 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 EGFR arm of the EGFR/CD28 bispecific antibodies of the present disclosure can be derived from any suitable anti-EGFR antibody.
  • the CDR and variable region sequences of the anti-EGFR arm in the bispecific antibody of the present disclosure are prepared with reference to zalutumumab 2F8 in the WO2002100348 patent, and its variable region and CDR sequences are as follows:
  • the structure of the disclosed EGFR-CD28 bispecific antibody molecule is as follows:
  • Format1 is an asymmetric structure molecule, containing four chains, all of which are different. The details are as follows:
  • Chain 1 CD28-VH-linker 1a-Titin-IgG1Fc (Knob);
  • Chain 2 CD28-VL-linker 1a-Obscurin
  • Chain 3 EGFR-VH-IgG 1 (CH1)-IgG1Fc(Hole);
  • Chain 4 EGFR-VL-CL;
  • Chain 1 CD28-VH-IgG1(CH1)-IgG1Fc(Knob);
  • Chain 2 CD28-VL-CL;
  • Chain 3 EGFR-VH-linker 1a-Obscurin-IgG1Fc (Hole);
  • Chain 4 EGFR-VL-linker 1a-Titin;
  • sequences of exemplary bispecific antibodies of the present disclosure are as follows:

Abstract

The present disclosure relates to an antigen-binding molecule specifically binding to EGFR and CD28, and a medical use thereof. The antigen-binding molecule can be used in the treatment of tumors.

Description

特异性结合EGFR和CD28的抗原结合分子及其医药用途Antigen-binding molecules that specifically bind to EGFR 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202210371538.8的优先权。This disclosure claims priority from Chinese patent application 202210371538.8 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EGFR/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 EGFR/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.
EGFR是一个在多种肿瘤上广泛表达的抗原，它与EGF等配体结合后，胞内区发生磷酸化，进而启动下游信号通路，影响细胞的增殖、凋亡及迁移，并可影响血管生成。当EGFR发生突变或者过表达后，下游磷酸化不受抑制，从而促进肿瘤细胞的恶性增殖。目前，已有获批或处于临床阶段的EGFR单克隆抗体，如西妥昔单抗、帕尼单抗、尼妥珠单抗、尼尼单抗、Matuzumab等，可用于治疗结直肠癌、头颈癌、非小细胞肺癌、神经胶质瘤、胃癌等癌症。但由于单克隆抗体只结合EGFR的ECD区，胞内区的突变以及旁路途径的激活导致病人容易发生耐药。EGFR is an antigen widely expressed on a variety of tumors. After it binds to ligands such as EGF, it phosphorylates the intracellular region, thereby initiating downstream signaling pathways, affecting cell proliferation, apoptosis and migration, and can affect angiogenesis. . When EGFR is mutated or overexpressed, downstream phosphorylation is not inhibited, thereby promoting the malignant proliferation of tumor cells. Currently, there are EGFR monoclonal antibodies that have been approved or are in the clinical stage, such as cetuximab, panitumumab, nimotuzumab, nituzumab, matuzumab, etc., which can be used to treat colorectal cancer, head and neck cancer, etc. cancer, non-small cell lung cancer, glioma, gastric cancer and other cancers. However, because monoclonal antibodies only bind to the ECD region of EGFR, mutations in the intracellular region and activation of the alternative pathway make patients prone to drug resistance.
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分子之一，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。激动性抗CD28mAb可以应用于经培养的T细胞的持续的离体扩增；然而，超级激动剂抗CD28mAb的I期临床试验出现了一系列急性且严重不良事件，已经不鼓励使用抗CD28的抗体(Hünig，Nature Reviews Immunology.2012；12:317-318)。The activation of T cells relies on the first signal provided by TCR activated by MHC-peptide complexes on the surface of APC cells or other cells. At the same time, costimulatory factors are required to provide the second signal to completely activate T cells. CD28, as one of the major costimulatory molecules on the surface of T cells, 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. Agonistic anti-CD28 mAbs can be used for sustained ex vivo expansion of cultured T cells; however, a series of acute and serious adverse events occurred in phase I clinical trials of superagonist anti-CD28 mAbs, and the use of anti-CD28 antibodies has been discouraged. (Hünig, Nature Reviews Immunology. 2012; 12:317-318).
与化疗或放疗等传统治疗方法相比，抗PD-1和抗PD-L1抗体治疗可以直接减轻肿瘤介导的T细胞衰竭，并在体内有效调节抗肿瘤免疫反应。然而，抗PD-1/PD-L1治疗在临床应用中仍存在许多未解决的问题，比如，超过50％的强表达PD-L1的肿瘤对PD-1/PD-L1抑制剂没有反应。有文献证明可以将EGFR的靶向治疗与PD1抗体免疫治疗联合，但临床实验的效果并不理想，这应该与肿瘤的免疫抑制状态有关。Compared with traditional treatments such as chemotherapy or radiotherapy, anti-PD-1 and anti-PD-L1 antibody treatment can directly alleviate tumor-mediated T cell exhaustion and effectively modulate anti-tumor immune responses in vivo. However, there are still many unresolved problems in the clinical application of anti-PD-1/PD-L1 therapy. For example, more than 50% of tumors that strongly express PD-L1 do not respond to PD-1/PD-L1 inhibitors. There is literature proving that EGFR-targeted therapy can be combined with PD1 antibody immunotherapy, but the results of clinical trials are not ideal, which should be related to the immunosuppressive state of the tumor.
发明内容 Contents of the invention
本披露提供了一种特异性结合CD28和EGFR的抗原结合分子。The present disclosure provides an antigen-binding molecule that specifically binds CD28 and EGFR.
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EGFR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EGFR的抗原结合模块包含重链可变区EGFR-VH和轻链可变区EGFR-VL。In one aspect, the present disclosure provides an antigen-binding molecule comprising at least one antigen-binding moiety that specifically binds CD28 and at least one antigen-binding moiety that specifically binds EGFR, the antigen-binding moiety that specifically binds CD28 comprises a heavy The chain variable region CD28-VH and the light chain variable region CD28-VL, the antigen-binding module that specifically binds to EGFR includes the heavy chain variable region EGFR-VH and the light chain variable region EGFR-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 groups of CD28-HCDR1, CD28-HCDR2 and CD28-HCDR3 in SEQ ID NO: 33 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: 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：46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；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: 46, 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: 52; 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：32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: 32.
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：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 has: an amino group containing SEQ ID NO: 16 CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 62, 17, 54, 55, 56, 57, 58, 59, 60 or 61 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：39、9、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: 39, 9, 40, 41 or CD28-HCDR3 with an amino acid sequence of 42, 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 or 11, and CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 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: an amino group containing SEQ ID NO: 28 CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and CD28-LCDR3 containing 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
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：84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: 84, and CD28-LCDR3 comprising 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：39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 containing the amino acid sequence of SEQ ID NO: 7, CD28-HCDR2 containing the amino acid sequence of SEQ ID NO: 8, and an amino group containing SEQ ID NO: 39 CD28-HCDR3 with acid sequence, 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:
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。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 including the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 including the amino acid sequence of SEQ ID NO: 23, and CD28-LCDR3 including the amino acid sequence of 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。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 CD28-VL has: CD28-LCDR1 including the amino acid sequence of SEQ ID NO: 16, CD28-LCDR2 including the amino acid sequence of SEQ ID NO: 62, and CD28-LCDR3 including the amino acid sequence of 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，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: 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 including the amino acid sequence of SEQ ID NO:28, CD28-LCDR2 including the amino acid sequence of SEQ ID NO:29, and CD28-LCDR3 including 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, 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：62、17、54、55、56、57、58、59、60或61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: 62, 17, 54, 55, 56, 57, 58, 59, 60 or 61 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- as shown in SEQ ID NO: 27 HCDR3, and the CD28-VL comprises 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 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：39、9、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或11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: 39, 9, 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: 43 or 11, 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 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 .
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, such as SEQ ID NO: CD28-HCDR2 shown in 20 and CD28-HCDR3 shown in SEQ ID NO: 21, and the CD28-VL includes CD28-LCDR1 shown in SEQ ID NO: 22, CD28-LCDR1 shown in SEQ ID NO: 23 CD28-LCDR2 and CD28-LCDR3 as set forth in SEQ ID NO: 24; or
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：84所示的CD28-LCDR2和如SEQ ID NO：24所示的CD28-LCDR3；或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: 84, 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：39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:39, 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，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。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 is CD28-LCDR1 as shown in SEQ ID NO:28, CD28-LCDR2 as shown in SEQ ID NO:29, and CD28-LCDR3 as shown 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：39所示的CD28-HCDR3，和所述CD28-VL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, as shown in SEQ ID NO: CD28-HCDR2 shown in 8 and CD28-HCDR3 shown in SEQ ID NO: 39, and the CD28-VL is CD28-LCDR1 shown in SEQ ID NO: 10, CD28 shown in SEQ ID NO: 43 -LCDR2 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如SEQ ID NO：22所示的CD28-LCDR1、如SEQ ID NO：84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 is CD28-LCDR1 as shown in SEQ ID NO:22, CD28-LCDR2 as shown in SEQ ID NO:84, 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，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，其包含人抗体的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 and 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(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、66K、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 CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 66K, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 of the amino acid sequence of NO: 16, CD28-LCDR2 of the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and SEQ ID NO: 18 The CD28-LCDR3 of the amino acid sequence, and the FR region of the 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、30S、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 CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 27 The CD28-HCDR3 of the sequence, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 30S, 71V and 78A; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 having the amino acid sequence of NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 and CD28-LCDR2 comprising 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，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-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 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-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
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：84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The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 20, and CD28-HCDR3 including the amino acid sequence of SEQ ID NO: 21, 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 NO: 22 CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 84, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 84, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, and the FR region of the CD28-VL contains a protein selected from the group consisting of 41D, One or more amino acid substitutions from the group consisting of 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、66K、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 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 FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 66K, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 containing the amino acid sequence of NO: 16, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 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、30S、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 the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 30S, 71V and 78A; and the CD28-VL has: comprising SEQ ID NO: 28 The CD28-LCDR1 of the amino acid sequence, the CD28-LCDR2 of the amino acid sequence of SEQ ID NO: 29 and the CD28-LCDR3 of the amino acid sequence of SEQ ID NO: 30, and the FR region of the CD28-VL is selected from the group consisting of 41D and 42G. One or more amino acid substitutions from the group consisting of , 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：39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: 39 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 containing the amino acid sequence of SEQ ID NO: 43, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 12; and the FR region of the CD28-VL contains a protein selected from the group consisting of 43S 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:
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。The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 20, and CD28-HCDR3 including the amino acid sequence of SEQ ID NO: 21, 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 NO: 22 CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 23, 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 the CD28-VL contains a protein selected from the group consisting of 41D, One or more amino acid substitutions from the group consisting of 42G, 43T, 44I and 71Y.
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：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、66K、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。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-HCDR3 including 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, 66K, 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: 62, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 18, 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.
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，且所述CD28-VH的FR区包含选自1E、25P、30S、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。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 FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 30S, 71V and 78A; and the CD28-VL has: comprising the amino acid sequence of 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 from the group consisting of 44V and 73L.
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EGFR-VH具有：包含SEQ ID NO：160的氨基酸序列的EGFR-HCDR1，包含SEQ ID NO：161的氨基酸序列的EGFR-HCDR2，和包含SEQ ID NO：162的氨基酸序列的EGFR-HCDR3；和所述EGFR-VL具有：包含SEQ ID NO：163的氨基酸序列的EGFR-LCDR1，包含SEQ ID NO：164的氨基酸序列的EGFR-LCDR2，和包含SEQ ID NO：165的氨基酸序列的EGFR-LCDR3。The EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
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EGFR-VH包含如SEQ ID NO：160所示的EGFR-HCDR1，如SEQ ID NO：161所示EGFR-HCDR2，和如SEQ ID NO：162所示的EGFR-HCDR3；并且所述EGFR-VL包含如SEQ ID NO：163所示的EGFR-LCDR1，如SEQ ID NO：164所示的EGFR-LCDR2，和如SEQ ID NO：165所示的EGFR-LCDR3。The EGFR-VH includes EGFR-HCDR1 as shown in SEQ ID NO: 160, EGFR-HCDR2 as shown in SEQ ID NO: 161, and EGFR-HCDR3 as shown in SEQ ID NO: 162; and the EGFR- VL contains EGFR-LCDR1 as shown in SEQ ID NO: 163, EGFR-LCDR2 as shown in SEQ ID NO: 164, and EGFR-LCDR3 as shown in SEQ ID NO: 165.
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：46、44、31、45、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: 46, 44, 31, 45, 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 group of SEQ ID NO: 68, 63, 64, 65, 66, 67 or 69 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，i) said CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98, and said CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102,
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：46的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52或50的氨基酸序列，或iv) 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 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、50或51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, 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：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 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 amino acid sequence of CD28-VL 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 The amino acid sequence 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 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
(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 contains Containing 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：46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: 46, 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
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
(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：46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: 46, 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 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: 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：106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: 106 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：46所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：52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: 46, 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原结合分子，其中：In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein:
所述EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述EGFR-VL包含SEQ ID NO：159的氨基酸序列。The EGFR-VH comprises the amino acid sequence of SEQ ID NO: 158, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159.
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EGFR-VH的氨基酸序列如SEQ ID NO：158所示，和所述EGFR-VL的 氨基酸序列如SEQ ID NO：159所示。The amino acid sequence of EGFR-VH is shown in SEQ ID NO: 158, and the EGFR-VL The amino acid sequence is shown in SEQ ID NO: 159.
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EGFR的抗原结合模块包含Titin链和Obscurin链，其中Titin链和Obscurin链能够形成二聚体。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 EGFR includes a Titin chain and an Obscurin chain, wherein the Titin chain and Obscurin chains are able to form 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EGFR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 EGFR 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Titin链包含选自由SEQ ID NO：218至SEQ ID NO：236组成的组的氨基酸序列，所述Obscurin链包含选自由SEQ ID NO：237至SEQ ID NO：277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 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 218 to SEQ ID NO: 236, and the Obscurin chain includes an amino acid sequence selected from the group consisting of The amino acid sequence of the group consisting of SEQ ID NO: 237 to SEQ ID NO: 277.
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：234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: 234.
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：272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: 272.
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：234的氨基酸序列，和所述Obscurin链包含SEQ ID NO：272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: 234, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 272.
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人IgG Fc区。In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein the antigen-binding molecule comprises a human 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人IgG1Fc区。In some embodiments, the antigen-binding molecule of any one of the preceding items, wherein the antigen-binding molecule comprises a human 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位置的氨基酸残基分别为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 acid residues at positions 234 and 235 are A, respectively, 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；反之 亦然。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 hole structure according to the pestle and mortar technology; and vice versa. Likewise.
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：167的氨基酸序列；和所述Fc2包含SEQ ID NO：168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: 167; and the Fc2 includes the amino acid sequence of SEQ ID NO: 168.
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EGFR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 EGFR.
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EGFR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 EGFR, wherein:
所述特异性结合EGFR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 module that specifically binds to EGFR 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.
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(图1A)，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 with the structure shown in formula (c) and a fourth chain with the structure shown in formula (d) (Figure 1A),
式(a)[CD28-VH]-[连接子1]-[Titin链]-[Fc1]，Formula (a) [CD28-VH]-[linker 1]-[Titin chain]-[Fc1],
式(b)[CD28-VL]-[连接子2]-[Obscurin链]，Formula (b) [CD28-VL]-[linker 2]-[Obscurin chain],
式(c)[EGFR-VH]-[CH1]-[Fc2]，Formula (c) [EGFR-VH]-[CH1]-[Fc2],
式(d)[EGFR-VL]-[CL]，Formula (d)[EGFR-VL]-[CL],
所述Fc1和所述Fc2可互换；The Fc1 and the Fc2 are interchangeable;
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(图1A)，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) , one with A third chain having the structure shown in formula (c) and a fourth chain having the structure shown in formula (d) (Figure 1A),
式(a)[CD28-VH]-[连接子1]-[Titin链]-[Fc1]，Formula (a) [CD28-VH]-[linker 1]-[Titin chain]-[Fc1],
式(b)[CD28-VL]-[连接子2]-[Obscurin链]，Formula (b) [CD28-VL]-[linker 2]-[Obscurin chain],
式(c)[EGFR-VH]-[CH1]-[Fc2]，Formula (c) [EGFR-VH]-[CH1]-[Fc2],
式(d)[EGFR-VL]-[CL]，Formula (d)[EGFR-VL]-[CL],
所述Fc1和所述Fc2可互换；The Fc1 and the Fc2 are interchangeable;
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:
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；和The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 20, and CD28-HCDR3 including the amino acid sequence of SEQ ID NO: 21; and the CD28-VL having: 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-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24 ;and
所述EGFR-VH具有：包含SEQ ID NO：160的氨基酸序列的EGFR-HCDR1，包含SEQ ID NO：161的氨基酸序列的EGFR-HCDR2，和包含SEQ ID NO：162的氨基酸序列的EGFR-HCDR3；和所述EGFR-VL具有：包含SEQ ID NO：163的氨基酸序列的EGFR-LCDR1，包含SEQ ID NO：164的氨基酸序列的EGFR-LCDR2，和包含SEQ ID NO：165的氨基酸序列的EGFR-LCDR3。The EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
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；和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 having: 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-LCDR3 comprising the amino acid sequence of SEQ ID NO: 30 ;and
所述EGFR-VH具有：包含SEQ ID NO：160的氨基酸序列的EGFR-HCDR1，包含SEQ ID NO：161的氨基酸序列的EGFR-HCDR2，和包含SEQ ID NO：162的氨基酸序列的EGFR-HCDR3；和所述EGFR-VL具有：包含SEQ ID NO：163的氨基酸序列的EGFR-LCDR1，包含SEQ ID NO：164的氨基酸序列的EGFR-LCDR2，和包含SEQ ID NO：165的氨基酸序列的EGFR-LCDR3。The EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
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EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述的EGFR-VL包含SEQ ID NO：159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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159; or
ii)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EGFR-VH包含SEQ ID NO：158 的氨基酸序列，和所述的EGFR-VL包含SEQ ID NO：159的氨基酸序列。ii) 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; and the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 158 The amino acid sequence of EGFR-VL includes the amino acid sequence of SEQ ID NO: 159.
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EGFR-VH的氨基酸序列如SEQ ID NO：158所示，和所述的EGFR-VL的氨基酸序列如SEQ ID NO：159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 EGFR-VH is shown in SEQ ID NO: 158 is shown, and the amino acid sequence of the EGFR-VL is shown in SEQ ID NO: 159.
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：126所示，和所述的CD28-VL的氨基酸序列如SEQ ID NO：134所示；并且所述的EGFR-VH的氨基酸序列如SEQ ID NO：158所示，和所述的EGFR-VL的氨基酸序列如SEQ ID NO：159所示。The amino acid sequence of CD28-VH is shown in SEQ ID NO: 126, and the amino acid sequence of CD28-VL is shown in SEQ ID NO: 134; and the amino acid sequence of EGFR-VH is shown in SEQ ID NO: 158 is shown, and the amino acid sequence of the EGFR-VL is shown in SEQ ID NO: 159.
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：234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：272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 are any Titin chains in Table 3-1 and Table 3-2 of the present disclosure that can form dimers 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: 234. 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: 272.
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的序列均为GGGGS(SEQ ID NO：169)。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 the linker 1 and linker 2 are both GGGGS (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：166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: 166.
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链或lamada链的轻链恒定区。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 a kappa chain or a lamada chain. 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：174的氨基酸序列的第一链、一条包含SEQ ID NO：176的氨基酸序列的第二链、一条包含SEQ ID NO：180的氨基酸序列的第三链和一条包含SEQ ID NO：182的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 174, a second strand comprising the amino acid sequence of SEQ ID NO: 176, a third strand comprising the amino acid sequence of SEQ ID NO: 180 and a third strand comprising the amino acid sequence of SEQ ID NO: 174 : The fourth strand of the amino acid sequence of 182.
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：178的氨基酸序列的第一链、一条包含SEQ ID NO：179的氨基酸序列的第二链、一条包含SEQ ID NO：180的氨基酸序列的第三链和一条包含SEQ ID NO：182的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 178, a second strand comprising the amino acid sequence of SEQ ID NO: 179, a third strand comprising the amino acid sequence of SEQ ID NO: 180 and a third strand comprising the amino acid sequence of SEQ ID NO: 179 : The fourth strand of the amino acid sequence of 182.
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EGFR的抗原结合模块，所述特异性结合EGFR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；所述特异性结合CD28的抗原结合模块是Fab。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 EGFR, and the antigen-binding module that specifically binds EGFR The antigen-binding module is a replaced Fab, which contains a Titin chain and an Obscurin chain capable of forming dimers; the antigen-binding module that specifically binds to CD28 is Fab.
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(图1B)，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) (Figure 1B),
式(e)[CD28-VH]-[CH1]-[Fc1]，Formula (e)[CD28-VH]-[CH1]-[Fc1],
式(f)[CD28-VL]-[CL]，Formula (f)[CD28-VL]-[CL],
式(g)[EGFR-VH]-[连接子3]-[Obscurin链]-[Fc2]，Formula (g) [EGFR-VH]-[linker 3]-[Obscurin chain]-[Fc2],
式(h)[EGFR-VL]-[连接子4]-[Titin链]，Formula (h) [EGFR-VL]-[linker 4]-[Titin chain],
Fc1和Fc2可互换；Fc1 and Fc2 are interchangeable;
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(图1B)，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) (Figure 1B),
式(e)[CD28-VH]-[CH1]-[Fc1]，Formula (e)[CD28-VH]-[CH1]-[Fc1],
式(f)[CD28-VL]-[CL]，Formula (f)[CD28-VL]-[CL],
式(g)[EGFR-VH]-[连接子3]-[Obscurin链]-[Fc2]，Formula (g) [EGFR-VH]-[linker 3]-[Obscurin chain]-[Fc2],
式(h)[EGFR-VL]-[连接子4]-[Titin链]，Formula (h) [EGFR-VL]-[linker 4]-[Titin chain],
Fc1和Fc2可互换；Fc1 and Fc2 are interchangeable;
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:
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；和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-HCDR3 including the amino acid sequence of SEQ ID NO: 15; and the CD28-VL having: 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-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18 ;and
所述EGFR-VH具有：包含SEQ ID NO：160的氨基酸序列的EGFR-HCDR1，包含SEQ ID NO：161的氨基酸序列的EGFR-HCDR2，和包含SEQ ID NO：162的氨基酸序列的EGFR-HCDR3；和所述EGFR-VL具有：包含SEQ ID NO：163 的氨基酸序列的EGFR-LCDR1，包含SEQ ID NO：164的氨基酸序列的EGFR-LCDR2，和包含SEQ ID NO：165的氨基酸序列的EGFR-LCDR3。The EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: containing SEQ ID NO: 163 EGFR-LCDR1 having the amino acid sequence of SEQ ID NO: 164, EGFR-LCDR2 having the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 having the amino acid sequence of SEQ ID NO: 165.
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：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：84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；和The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 20, and CD28-HCDR3 including the amino acid sequence of SEQ ID NO: 21; and the CD28-VL having: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84, and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24 ;and
所述EGFR-VH具有：包含SEQ ID NO：160的氨基酸序列的EGFR-HCDR1，包含SEQ ID NO：161的氨基酸序列的EGFR-HCDR2，和包含SEQ ID NO：162的氨基酸序列的EGFR-HCDR3；和所述EGFR-VL具有：包含SEQ ID NO：163的氨基酸序列的EGFR-LCDR1，包含SEQ ID NO：164的氨基酸序列的EGFR-LCDR2，和包含SEQ ID NO：165的氨基酸序列的EGFR-LCDR3。The EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
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：39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；和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: 39; and the CD28-VL having: 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-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12 ;and
所述EGFR-VH具有：包含SEQ ID NO：160的氨基酸序列的EGFR-HCDR1，包含SEQ ID NO：161的氨基酸序列的EGFR-HCDR2，和包含SEQ ID NO：162的氨基酸序列的EGFR-HCDR3；和所述EGFR-VL具有：包含SEQ ID NO：163的氨基酸序列的EGFR-LCDR1，包含SEQ ID NO：164的氨基酸序列的EGFR-LCDR2，和包含SEQ ID NO：165的氨基酸序列的EGFR-LCDR3。The EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
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EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述的EGFR-VL包含SEQ ID NO：159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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159; or
ii)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EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述的EGFR-VL包含SEQ ID NO：159的氨基酸序列；或ii) 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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159; 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EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述的EGFR-VL包含SEQ ID NO：159的氨基酸序列。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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL includes the amino acid sequence of SEQ ID NO: 159.
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EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述的EGFR-VL包含SEQ ID NO：159的氨基酸序列。The CD28-VH includes the amino acid sequence of SEQ ID NO: 68, and the CD28-VL package Contains the amino acid sequence of SEQ ID NO: 80; and the EGFR-VH includes the amino acid sequence of SEQ ID NO: 158, and the EGFR-VL includes the amino acid sequence of SEQ ID NO: 159.
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SEQ ID NO：80所示；并且所述的EGFR-VH的氨基酸序列SEQ ID NO：158所示，和所述的EGFR-VL的氨基酸序列SEQ ID NO：159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 EGFR-VH is SEQ ID NO: shown in 158, and the amino acid sequence of the EGFR-VL is shown in SEQ ID NO: 159.
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：234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：272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 are any Titin chains in Table 3-1 and Table 3-2 of the present disclosure that can form dimers 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: 234. 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: 272.
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：169)。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: 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：166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: 166.
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：172的氨基酸序列的第一链、一条包含SEQ ID NO：173的氨基酸序列的第二链、一条包含SEQ ID NO：181的氨基酸序列的第三链和一条包含SEQ ID NO：183的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 172, a second strand comprising the amino acid sequence of SEQ ID NO: 173, a third strand comprising the amino acid sequence of SEQ ID NO: 181 and a third strand comprising the amino acid sequence of SEQ ID NO: 172 : The fourth strand of the amino acid sequence of 183.
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：170的氨基酸序列的第一链、一条包含SEQ ID NO：171的氨基酸序列的第二链、一条包含SEQ ID NO：181的氨基酸序列的第三链和一条包含SEQ ID NO：183的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 170, a second strand comprising the amino acid sequence of SEQ ID NO: 171, a third strand comprising the amino acid sequence of SEQ ID NO: 181 and a third strand comprising the amino acid sequence of SEQ ID NO: 171 : The fourth strand of the amino acid sequence of 183.
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：175的氨基酸序列的第一链、一条包含SEQ ID NO：177的氨基酸序列的第二链、一条包含SEQ ID NO：181的氨基酸序列的第三链和一条包含SEQ ID NO：183的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 175, a second strand comprising the amino acid sequence of SEQ ID NO: 177, a third strand comprising the amino acid sequence of SEQ ID NO: 181 and a third strand comprising the amino acid sequence of SEQ ID NO: 175 : The fourth strand of the amino acid sequence of 183.
抗CD28抗体anti-CD28 antibody
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 points in VL specifically comprising the amino acid sequences of CD28-LCDR1, CD28-LCDR2 and CD28-LCDR3 in SEQ ID NO: 80, 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 contain 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- 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 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 CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 27 The sequence of CD28-HCDR3, and the CD28-VL has: contains SEQ. CD28-LCDR1 having the amino acid sequence of ID NO: 28, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 29, 118, 119, 120, 121 or 122 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, 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: an amino group containing SEQ ID NO: 22 or
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：84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: 84, and CD28-LCDR3 comprising 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；或(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; 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：39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-HCDR3 comprising the amino acid sequence of SEQ ID NO: 39, and The CD28-VL has: CD28-LCDR1 including the amino acid sequence of SEQ ID NO: 10, CD28-LCDR2 including the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR3 including the amino acid sequence of 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:
(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；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, such as SEQ ID NO: 17, 54, CD28-LCDR2 set forth in 55, 56, 57, 58, 59, 60, 61 or 62 and CD28-LCDR3 set forth 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 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
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：84所示的CD28-LCDR2和如SEQ ID NO：24所示的CD28-LCDR3；或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: 84, 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；或(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 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; 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：39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-HCDR3 as shown in SEQ ID NO:39, and the CD28- VL contains 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 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：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。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 is CD28-LCDR1 as shown in SEQ ID NO:28, CD28-LCDR2 as shown in SEQ ID NO:29, and CD28-LCDR3 as shown in SEQ ID NO:30.
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，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 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、66K、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 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 FR region of CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 28S, 66K, 69L, 71V, 73K and 94S; and the CD28-VL has: comprising SEQ ID CD28-LCDR1 of the amino acid sequence of NO: 16, CD28-LCDR2 of the amino acid sequence of SEQ ID NO: 17, 54, 55, 56, 57, 58, 59, 60, 61 or 62 and SEQ ID NO: 18 The CD28-LCDR3 of the amino acid sequence, and the FR region of the 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、30S、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 CD28-HCDR3 of the sequence, and the FR region of the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 30S, 71V and 78A; and the CD28-VL has: comprising SEQ ID 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 The FR region of CD28-VL comprises 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-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 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-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
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：84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The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 20, and CD28-HCDR3 including the amino acid sequence of SEQ ID NO: 21, 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 NO: 22 CD28-LCDR1 containing the amino acid sequence of SEQ ID NO: 84, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 84, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 24, and the FR region of the CD28-VL contains a protein selected from the group consisting of 41D, One or more amino acid substitutions from the group consisting of 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、66K、 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 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 FR region of CD28-VH includes 1E, 28S, 66K, One or more amino acid substitutions in the group consisting of 69L, 71V, 73K and 94S; and the CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, and comprising the amino acid sequence of SEQ ID NO: 62 A CD28-LCDR2 and a CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 18, and the FR region of the CD28-VL comprises 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、30S、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 the CD28-VH comprises one or more amino acid substitutions selected from the group consisting of 1E, 25P, 30S, 71V and 78A; and the CD28-VL has: comprising SEQ ID NO: 28 The CD28-LCDR1 of the amino acid sequence, the CD28-LCDR2 of the amino acid sequence of SEQ ID NO: 29 and the CD28-LCDR3 of the amino acid sequence of SEQ ID NO: 30, and the FR region of the CD28-VL is selected from the group consisting of 41D and 42G. One or more amino acid substitutions from the group consisting of , 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；或(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 containing the amino acid sequence of SEQ ID NO: 43, CD28-LCDR2 containing the amino acid sequence of SEQ ID NO: 43, and CD28-LCDR3 containing the amino acid sequence of SEQ ID NO: 12; and the FR region of the CD28-VL contains a protein selected from the group consisting of 43S One or more amino acid substitutions in the group consisting of 73F; 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：39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。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: 39, 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 the amino acid of SEQ ID NO: 10 The CD28-LCDR1 of the sequence, the CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 43 and the CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and the FR region of the CD28-VL is composed of 43S and 73F. 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 contains the FR region of SEQ ID NO: 98 selected from the group consisting of 26A, 29L, 69L, 71V, 78A and 93S consisting of one or more amino acid substitutions in the group, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 102 or a variant thereof, the variant being the FR region of SEQ ID NO: 102 comprising a member selected from 41D 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、66K、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 includes a FR region of SEQ ID NO: 69 selected from the group consisting of 28S, 66K, 69L, 71V, 73K and 94S consisting of 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 comprising 43S selected from the group consisting of and one or more amino acid substitutions in the group consisting of 70K; 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、30S、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 FR region of SEQ ID NO: 133 includes a group selected from the group consisting of 25P, 30S, 71V and 78A. One or more amino acid substitutions, and the CD28-VL includes the amino acid sequence of SEQ ID NO: 137 or a variant thereof, wherein the variant is in the FR region of SEQ ID NO: 137. One or more amino acid substitutions in the group consisting of , 44V and 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：46的氨基酸序列或其变体，所述变体为在SEQ ID NO：46的FR区包含选自69L的氨基酸取代，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: 46 or a variant thereof, the variant comprising an amino acid substitution selected from 69L in the FR region of SEQ ID NO: 46, and the CD28-VL comprises SEQ ID The amino acid sequence of NO: 52 or a variant thereof, which variant is one or more amino acid substitutions selected from the group consisting of 43S and 73F in the FR region 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：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, 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 group of SEQ ID NO: 68, 63, 64, 65, 66, 67 or 69 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：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，i) said CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98, and said CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102,
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：46的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52或50的氨基酸序列，或iv) 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 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、50或51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, 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：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：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：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(i) 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, 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: 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、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, 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
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：84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: 84; 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：46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: 46, 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：95所示，和所述CD28-VL的氨基酸序列如SEQ ID NO：106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: 106 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 CD28-VH The amino acid sequence 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 comprises the amino acid sequence of SEQ ID NO: 146, and the light chain comprises 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EGFR。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 EGFR.
在另一个方面，本披露提供了一种药物组合物，其含有：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第二治疗剂。在一些实施方案中，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。In another aspect, the present disclosure provides a pharmaceutical composition, which contains: a therapeutically effective amount of the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing, and one or more A pharmaceutically acceptable carrier, diluent, buffer or excipient. In some embodiments, the pharmaceutical composition further includes at least one second therapeutic agent. In some embodiments, the second therapeutic agent includes an antineoplastic agent, radiation therapy, an antibody drug conjugate, a bispecific antibody, a bispecific antibody conjugated to an antineoplastic agent, an immune checkpoint inhibitor, or the like. combination.
在一些实施方案中，所述第二治疗剂是有利地与EGFR/CD28双特异性抗原结合分子组合的任何药剂。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。在一些实施方案中，所述第二治疗剂是能够特异性结合CD3的双特异性抗体。在一些实施方案中，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。在一些实施方案中，所述第二治疗剂是能够特异性结合CD3和MUC16的双特异性的抗体。在一些实施方案中，所述第二治疗剂是抗PD-L1抗体。在一些实施方案中，所述的抗PD-1抗体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 is any agent that is advantageously combined with an EGFR/CD28 bispecific antigen-binding molecule. In some embodiments, the second therapeutic agent is an antibody capable of specifically binding CD3. In some embodiments, the second therapeutic agent is a bispecific antibody capable of specifically binding CD3. In some embodiments, the second therapeutic agent is a bispecific antibody capable of specifically binding CD3 and a tumor cell surface antigen (TAA). In some embodiments, the second therapeutic agent is a bispecific antibody capable of specifically binding to CD3 and MUC16. In some embodiments, the second therapeutic agent is an anti-PD-L1 antibody. In some embodiments, the anti-PD-1 antibody includes any known anti-PD-1 antibody with therapeutic activity, including but not limited to Camrelizumab, Keytruda , nivolumab (Opdivo) and cemiplimab (cemiplimab).
在一些实施方案中，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：In some embodiments, 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：206的氨基酸序列的PD-1-HCDR1，包含SEQ ID NO：207的氨基酸序列的PD-1-HCDR2，和包含SEQ ID NO：208的氨基酸序列的PD-1-HCDR3；和所述PD-1-VL具有：包含SEQ ID NO：209的氨基酸序列的PD-1-LCDR1，包含SEQ ID NO：210的氨基酸序列的PD-1-LCDR2，和包含SEQ ID NO：211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: 206, PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO: 207, and comprising the amino acid sequence of SEQ ID NO: 208 the sequence of PD-1-HCDR3; and the PD-1-VL having: PD-1-LCDR1 comprising the amino acid sequence of SEQ ID NO: 209, PD-1-LCDR2 comprising the amino acid sequence of SEQ ID NO: 210, and PD-1-LCDR3 comprising the amino acid sequence of SEQ ID NO: 211.
在一些实施方案中，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：In some embodiments, 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包含如SEQ ID NO：206所示的的PD-1-HCDR1，包含如SEQ ID NO：207所示的PD-1-HCDR2，和如SEQ ID NO：208所示的PD-1-HCDR3；和所述PD-1-VL包含如SEQ ID NO：209所示的PD-1-LCDR1，包含如SEQ ID NO：210所示的PD-1-LCDR2，和如SEQ ID NO：211所示的PD-1-LCDR3。The PD-1-VH includes PD-1-HCDR1 as shown in SEQ ID NO: 206, includes PD-1-HCDR2 as shown in SEQ ID NO: 207, and as shown in SEQ ID NO: 208 PD-1-HCDR3; and said PD-1-VL comprising PD-1-LCDR1 as set forth in SEQ ID NO:209, comprising PD-1-LCDR2 as set forth in SEQ ID NO:210, and said PD-1-VL as set forth in SEQ ID NO:210 PD-1-LCDR3 shown in NO:211.
在一些实施方案中，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：In some embodiments, 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包含SEQ ID NO：212的氨基酸序列，和所述PD-1-VL包含SEQ ID NO：213的氨基酸序列。The PD-1-VH includes the amino acid sequence of SEQ ID NO:212, and the PD-1-VL includes the amino acid sequence of SEQ ID NO:213.
在一些实施方案中，所述的抗PD-1抗体包含重链和轻链，其中：In some embodiments, the anti-PD-1 antibody comprises a heavy chain and a light chain, wherein:
所述抗PD-1抗体包含SEQ ID NO：215的氨基酸序列的重链，和SEQ ID NO：216的氨基酸序列的轻链。The anti-PD-1 antibody includes a heavy chain of the amino acid sequence of SEQ ID NO: 215, and a light chain of the amino acid sequence of SEQ ID NO: 216.
在一些实施方案中，所述的第二治疗剂是特异性结合MUC16和CD3的双特异性抗体。In some embodiments, the second therapeutic agent is a bispecific antibody that specifically binds MUC16 and CD3.
在一些实施方案中，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 is a bispecific antibody that specifically binds PSMA and CD3.
在一些实施方案中，所述的第二治疗剂是特异性结合STEAP2和CD3的双特异性抗体。In some embodiments, the second therapeutic agent is a bispecific antibody that specifically binds STEAP2 and CD3.
在一些实施方案中，所述的第二治疗剂是特异性结合MUC16和CD3的双特异性抗体，其包含至少一个特异性结合MUC16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 is a bispecific antibody that specifically binds MUC16 and CD3, comprising at least one antigen-binding moiety that specifically binds MUC16 and at least one antigen-binding moiety that specifically binds CD3.
在一些实施方案中，所述的第二治疗剂是特异性结合MUC16和CD3的双特异性抗体，其包含至少一个特异性结合MUC16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MUC16的抗原结合模块包含重链可变区MUC16-VH和轻链可变区MUC16-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 is a bispecific antibody that specifically binds MUC16 and CD3, comprising at least one antigen-binding moiety that specifically binds MUC16 and at least one antigen-binding moiety that specifically binds CD3, The antigen-binding module that specifically binds to MUC16 includes a heavy chain variable region MUC16-VH and a light chain variable region MUC16-VL, and the antigen-binding module that specifically binds to CD3 includes a heavy chain variable region CD3-VH and a light chain variable region. Chain variable region CD3-VL.
在一些实施方案中，所述MUC16-VH具有：包含SEQ ID NO：187的氨基酸序列的MUC16-HCDR1，包含SEQ ID NO：188的氨基酸序列的MUC16-HCDR2， 和包含SEQ ID NO：189的氨基酸序列的MUC16-HCDR3；并且所述MUC16-VL具有：包含SEQ ID NO：190的氨基酸序列的MUC16-LCDR1，包含SEQ ID NO：191的氨基酸序列的MUC16-LCDR2，和包含SEQ ID NO：192的氨基酸序列的MUC16-LCDR3。In some embodiments, the MUC16-VH has: MUC16-HCDR1 comprising the amino acid sequence of SEQ ID NO: 187, MUC16-HCDR2 comprising the amino acid sequence of SEQ ID NO: 188, and MUC16-HCDR3 comprising the amino acid sequence of SEQ ID NO: 189; and the MUC16-VL has: MUC16-LCDR1 comprising the amino acid sequence of SEQ ID NO: 190, MUC16-LCDR2 comprising the amino acid sequence of SEQ ID NO: 191 , and MUC16-LCDR3 comprising the amino acid sequence of SEQ ID NO:192.
在一些实施方案中，所述MUC16-VH包含如SEQ ID NO：187所示的MUC16-HCDR1，如SEQ ID NO：188所示的MUC16-HCDR2，和如SEQ ID NO：189所示的MUC16-HCDR3；并且所述MUC16-VL包含如SEQ ID NO：190所示的MUC16-LCDR1，如SEQ ID NO：191所示的MUC16-LCDR2，和如SEQ ID NO：192所示的MUC16-LCDR3。In some embodiments, the MUC16-VH includes MUC16-HCDR1 as set forth in SEQ ID NO: 187, MUC16-HCDR2 as set forth in SEQ ID NO: 188, and MUC16-HCDR1 as set forth in SEQ ID NO: 189 HCDR3; and the MUC16-VL includes MUC16-LCDR1 as shown in SEQ ID NO: 190, MUC16-LCDR2 as shown in SEQ ID NO: 191, and MUC16-LCDR3 as shown in SEQ ID NO: 192.
在一些实施方案中，其中所述MUC16-VH包含SEQ ID NO：199的氨基酸序列，和所述MUC16-VL包含SEQ ID NO：200的氨基酸序列。In some embodiments, wherein said MUC16-VH comprises the amino acid sequence of SEQ ID NO: 199, and said MUC16-VL comprises the amino acid sequence of SEQ ID NO: 200.
在一些实施方案中，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 antigen-binding module that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein:
所述CD3-VH具有：包含SEQ ID NO：193的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：194的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：195的氨基酸序列的CD3-HCDR3；并且所述CD3-VL具有：包含SEQ ID NO：196的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：197的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：198的氨基酸序列的CD3-LCDR3。The CD3-VH has: CD3-HCDR1 including the amino acid sequence of SEQ ID NO: 193, CD3-HCDR2 including the amino acid sequence of SEQ ID NO: 194, and CD3-HCDR3 including the amino acid sequence of SEQ ID NO: 195; And the CD3-VL has: CD3-LCDR1 including the amino acid sequence of SEQ ID NO: 196, CD3-LCDR2 including the amino acid sequence of SEQ ID NO: 197, and CD3-LCDR3 including the amino acid sequence of SEQ ID NO: 198 .
在一些实施方案中，所述CD3-VH包含如SEQ ID NO：193所示的CD3-HCDR1，如SEQ ID NO：194所示的CD3-HCDR2，和如SEQ ID NO：195所示的CD3-HCDR3；并且所述CD3-VL包含如SEQ ID NO：196所示的CD3-LCDR1，如SEQ ID NO：197所示的CD3-LCDR2，和如SEQ ID NO：198所示的CD3-LCDR3。In some embodiments, the CD3-VH comprises CD3-HCDR1 as set forth in SEQ ID NO: 193, CD3-HCDR2 as set forth in SEQ ID NO: 194, and CD3-HCDR1 as set forth in SEQ ID NO: 195 HCDR3; and the CD3-VL includes CD3-LCDR1 as shown in SEQ ID NO: 196, CD3-LCDR2 as shown in SEQ ID NO: 197, and CD3-LCDR3 as shown in SEQ ID NO: 198.
在一些实施方案中，所述CD3-VH包含SEQ ID NO：201的氨基酸序列，和所述CD3-VL包含SEQ ID NO：202的氨基酸序列。In some embodiments, the CD3-VH comprises the amino acid sequence of SEQ ID NO:201, and the CD3-VL comprises the amino acid sequence of SEQ ID NO:202.
在一些实施方案中，所述的特异性结合MUC16和CD3的双特异性抗体包含：In some embodiments, the bispecific antibody that specifically binds MUC16 and CD3 comprises:
所述MUC16-VH包含如SEQ ID NO：187所示的MUC16-HCDR1，如SEQ ID NO：188所示的MUC16-HCDR2，和如SEQ ID NO：189所示的MUC16-HCDR3；并且所述MUC16-VL包含如SEQ ID NO：190所示的MUC16-LCDR1，如SEQ ID NO：191所示的MUC16-LCDR2，和如SEQ ID NO：192所示的MUC16-LCDR3；且The MUC16-VH includes MUC16-HCDR1 as shown in SEQ ID NO: 187, MUC16-HCDR2 as shown in SEQ ID NO: 188, and MUC16-HCDR3 as shown in SEQ ID NO: 189; and the MUC16 -VL contains MUC16-LCDR1 as shown in SEQ ID NO:190, MUC16-LCDR2 as shown in SEQ ID NO:191, and MUC16-LCDR3 as shown in SEQ ID NO:192; and
所述CD3-VH包含如SEQ ID NO：193所示的CD3-HCDR1，如SEQ ID NO：194所示的CD3-HCDR2，和如SEQ ID NO：195所示的CD3-HCDR3；并且所述CD3-VL包含如SEQ ID NO：196所示的CD3-LCDR1，如SEQ ID NO：197所示的CD3-LCDR2，和如SEQ ID NO：198所示的CD3-LCDR3。The CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 193, CD3-HCDR2 as shown in SEQ ID NO: 194, and CD3-HCDR3 as shown in SEQ ID NO: 195; and the CD3 -VL contains CD3-LCDR1 as set forth in SEQ ID NO: 196, CD3-LCDR2 as set forth in SEQ ID NO: 197, and CD3-LCDR3 as set forth in SEQ ID NO: 198.
在一些实施方案中，所述的特异性结合MUC16和CD3的双特异性抗体包含：In some embodiments, the bispecific antibody that specifically binds MUC16 and CD3 comprises:
所述MUC16-VH包含SEQ ID NO：199的氨基酸序列，和所述MUC16-VL 包含SEQ ID NO：200的氨基酸序列；且The MUC16-VH comprises the amino acid sequence of SEQ ID NO: 199, and the MUC16-VL Comprising the amino acid sequence of SEQ ID NO: 200; and
所述CD3-VH包含SEQ ID NO：201的氨基酸序列，和所述CD3-VL包含SEQ ID NO：202的氨基酸序列。The CD3-VH comprises the amino acid sequence of SEQ ID NO:201, and the CD3-VL comprises the amino acid sequence of SEQ ID NO:202.
在一些实施方案中，所述的特异性结合MUC16和CD3的双特异性抗体包含：一条包含SEQ ID NO：203的氨基酸序列的第一链、两条包含SEQ ID NO：204的氨基酸序列的第二链和一条包含SEQ ID NO：205的氨基酸序列的第三链。In some embodiments, the bispecific antibody that specifically binds MUC16 and CD3 includes: a first chain comprising the amino acid sequence of SEQ ID NO: 203, and two first chains comprising the amino acid sequence of SEQ ID NO: 204. The second strand and a third strand containing the amino acid sequence of SEQ ID NO: 205.
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或预防过度增殖性疾病的方法，所述方法包括向受试者施用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药物组合物。In another aspect, the present disclosure also provides a method of treating or preventing a hyperproliferative disease, the method comprising administering to a subject a therapeutically effective amount of the antigen-binding molecule of any one of the foregoing or any one of the foregoing. The anti-CD28 antibody described above or the pharmaceutical 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CD28抗体或前述任一项所述的药物组合物在制备治疗或预防过度增殖性疾病的药物中的用途。In another aspect, the present disclosure also provides the use of the antigen-binding molecule described in any one of the foregoing, the anti-CD28 antibody described in any of the foregoing, or the pharmaceutical composition described in any of the foregoing in the preparation of the treatment or prevention of hyperproliferative diseases. uses in medicines.
在另一个方面，本披露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CD28抗体或前述任一项所述的药物组合物，其用于治疗或预防过度增殖性疾病。In another aspect, the present disclosure provides the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing or the pharmaceutical composition of any of the foregoing, which is used to treat or prevent hyperproliferative disease.
在另一个方面，本披露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CD28抗体或前述任一项所述的药物组合物，其用于治疗或预防过度增殖性疾病。在一些实施方案中，所述抗原结合分子和第二治疗剂同时施用、序贯施用或分开施用。在一些实施方案中，所述抗原结合分子和第二治疗剂在相同或不同的容器中。In another aspect, the present disclosure provides the antigen-binding molecule of any of the foregoing or the anti-CD28 antibody of any of the foregoing or the pharmaceutical composition of any of the foregoing, which is used to treat or prevent hyperproliferative disease. In some embodiments, the antigen-binding molecule and the second therapeutic agent are administered simultaneously, sequentially, or separately. In some embodiments, the antigen binding molecule and the second therapeutic agent are in the same or different containers.
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药物组合物。在一些实施方式中，所述药物用于治疗过度增殖性疾病。In another aspect, the present disclosure also provides the antigen-binding molecule according to any one of the foregoing items or the anti-CD28 antibody according to any one of the foregoing items or the pharmaceutical composition according to any one of the foregoing items for use as a medicament. In some embodiments, the drugs are used to treat hyperproliferative diseases.
在一些实施方式中，如前任一项所述的过度增殖性疾病是癌症。在一些实施方式中，如前任一项所述的癌症选自肺癌、肺腺癌、非小细胞肺癌、肺鳞状细胞癌、食管癌、宫颈鳞状细胞癌、子宫内膜癌、子宫内膜腺癌、膀胱癌、膀胱尿路上皮癌、结直肠癌(包括结肠癌和直肠癌)、头颈癌、头颈部鳞状细胞癌、神经胶质瘤、脑癌、多形性胶质母细胞瘤、胃癌、胃食管癌、胃食管腺癌、前列腺癌、卵巢癌、肾癌、肝癌、胰腺癌、黑色素瘤、骨肉瘤和皮肤癌。In some embodiments, the hyperproliferative disease of any one of the preceding items is cancer. In some embodiments, the cancer of any one of the preceding items is selected from the group consisting of lung cancer, lung adenocarcinoma, non-small cell lung cancer, lung squamous cell carcinoma, esophageal cancer, cervical squamous cell carcinoma, endometrial cancer, endometrium Adenocarcinoma, bladder cancer, bladder urothelial cancer, colorectal cancer (including colon and rectal cancer), head and neck cancer, head and neck squamous cell carcinoma, glioma, brain cancer, glioblastoma multiforme tumors, gastric cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, prostate cancer, ovarian cancer, kidney cancer, liver cancer, pancreatic cancer, melanoma, osteosarcoma and skin cancer.
在一些实施方式中，如前所述的所述的癌症是表达EGFR的癌症。In some embodiments, the cancer as described above is an EGFR-expressing cancer.
在一些实施方式中，如前所述的癌症是实体瘤。In some embodiments, the cancer as described above is a solid tumor.
在一些实施方式中，如前所述的癌症是肺癌。 In some embodiments, the cancer as described above is lung cancer.
在一些实施方式中，如前所述的癌症是非小细胞肺癌。In some embodiments, the cancer as described above is non-small cell lung cancer.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，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。In some embodiments, the method of treating a hyperproliferative disease is as described above, further comprising using a second therapeutic agent. In some embodiments, the second therapeutic agent includes an antineoplastic agent, radiation therapy, an antibody drug conjugate, a bispecific antibody, a bispecific antibody conjugated to an antineoplastic agent, an immune checkpoint inhibitor, or the like. combination.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，所述第二治疗剂选自PD-1抑制剂、抗PD-1抗体、PD-L1抑制剂、抗PD-L1抗体、与肿瘤靶抗原和CD3结合的双特异性抗体、铂抗癌化学治疗剂、紫杉醇、多西他赛(docetaxel)、长春新碱(vincristine)、顺铂(cisplatin)、卡铂(carboplatin)、奥沙利铂(oxaliplatin)、抗癌抗体、曲妥单抗(trastuzumab)、西妥昔单抗(cetuximab)、贝伐单抗(bevacizumab)和西米普利单抗(cemiplimab)。In some embodiments, the method of treating a hyperproliferative disease as described above, the second therapeutic agent is selected from the group consisting of a PD-1 inhibitor, an anti-PD-1 antibody, a PD-L1 inhibitor, an anti-PD-L1 antibody, Bispecific antibodies that bind to tumor target antigens and CD3, platinum anticancer chemotherapeutic agents, paclitaxel, docetaxel, vincristine, cisplatin, carboplatin, oroxin oxaliplatin, anti-cancer antibodies, trastuzumab, cetuximab, bevacizumab and cemiplimab.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，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, as described above in the method of treating a hyperproliferative disease, the second therapeutic agent and the antigen-binding molecule of any one of the present disclosure are administered simultaneously, sequentially, or separately.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，其中所述的第二治疗剂是免疫检查点抑制剂。In some embodiments, the method of treating a hyperproliferative disease is as described above, wherein the second therapeutic agent is an immune checkpoint inhibitor.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，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 hyperproliferative disease is as described above, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，其中所述的抗PD-1抗体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 method of treating a hyperproliferative disease as described above, wherein the anti-PD-1 antibody includes any known anti-PD-1 antibody with therapeutic activity, including but not limited to camrelizumab Camrelizumab, Keytruda, Opdivo and cemiplimab.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，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：In some embodiments, the method of treating a hyperproliferative disease as described above, wherein the anti-PD-1 antibody comprises a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, in:
所述PD-1-VH具有：包含SEQ ID NO：206的氨基酸序列的PD-1-HCDR1，包含SEQ ID NO：207的氨基酸序列的PD-1-HCDR2，和包含SEQ ID NO：208的氨基酸序列的PD-1-HCDR3；和所述PD-1-VL具有：包含SEQ ID NO：209的氨基酸序列的PD-1-LCDR1，包含SEQ ID NO：210的氨基酸序列的PD-1-LCDR2，和包含SEQ ID NO：211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: 206, PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO: 207, and comprising the amino acid sequence of SEQ ID NO: 208 the sequence of PD-1-HCDR3; and the PD-1-VL having: PD-1-LCDR1 comprising the amino acid sequence of SEQ ID NO: 209, PD-1-LCDR2 comprising the amino acid sequence of SEQ ID NO: 210, and PD-1-LCDR3 comprising the amino acid sequence of SEQ ID NO: 211.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，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：In some embodiments, the method of treating a hyperproliferative disease as described above, wherein the anti-PD-1 antibody comprises a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, in:
所述PD-1-VH包含如SEQ ID NO：206所示的的PD-1-HCDR1，包含如SEQ ID NO：207所示的PD-1-HCDR2，和如SEQ ID NO：208所示的PD-1-HCDR3；和所述PD-1-VL包含如SEQ ID NO：209所示的PD-1-LCDR1，包含如SEQ ID NO：210所示的PD-1-LCDR2，和如SEQ ID NO：211所示的PD-1-LCDR3。The PD-1-VH includes PD-1-HCDR1 as shown in SEQ ID NO: 206, includes PD-1-HCDR2 as shown in SEQ ID NO: 207, and as shown in SEQ ID NO: 208 PD-1-HCDR3; and said PD-1-VL comprising PD-1-LCDR1 as set forth in SEQ ID NO:209, comprising PD-1-LCDR2 as set forth in SEQ ID NO:210, and said PD-1-VL as set forth in SEQ ID NO:210 PD-1-LCDR3 shown in NO:211.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，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： In some embodiments, the method of treating a hyperproliferative disease as described above, wherein the anti-PD-1 antibody comprises a heavy chain variable region PD-1-VH and a light chain variable region PD-1-VL, in:
所述PD-1-VH包含SEQ ID NO：212的氨基酸序列，和所述PD-1-VL包含SEQ ID NO：213的氨基酸序列。The PD-1-VH includes the amino acid sequence of SEQ ID NO:212, and the PD-1-VL includes the amino acid sequence of SEQ ID NO:213.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，其中所述的抗PD-1抗体包含重链和轻链，其中：In some embodiments, the method of treating a hyperproliferative disease as described above, wherein the anti-PD-1 antibody comprises a heavy chain and a light chain, wherein:
所述抗PD-1抗体包含SEQ ID NO：215的氨基酸序列的重链，和SEQ ID NO：216的氨基酸序列的轻链。The anti-PD-1 antibody includes a heavy chain of the amino acid sequence of SEQ ID NO: 215, and a light chain of the amino acid sequence of SEQ ID NO: 216.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，所述的第二治疗剂是特异性结合MUC16和CD3的双特异性抗体。In some embodiments, as described above, the second therapeutic agent is a bispecific antibody that specifically binds to MUC16 and CD3.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，所述的第二治疗剂是特异性结合PSMA和CD3的双特异性抗体。In some embodiments, as described above, the second therapeutic agent is a bispecific antibody that specifically binds PSMA and CD3.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，所述的第二治疗剂是特异性结合STEAP2和CD3的双特异性抗体。In some embodiments, as described above, the second therapeutic agent is a bispecific antibody that specifically binds STEAP2 and CD3.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，所述的第二治疗剂是特异性结合MUC16和CD3的双特异性抗体，其包含至少一个特异性结合MUC16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 method of treating a hyperproliferative disease as described above, the second therapeutic agent is a bispecific antibody that specifically binds MUC16 and CD3, which includes at least one antigen-binding module that specifically binds MUC16. and at least one antigen-binding module that specifically binds CD3.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，所述的第二治疗剂是特异性结合MUC16和CD3的双特异性抗体，其包含至少一个特异性结合MUC16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MUC16的抗原结合模块包含重链可变区MUC16-VH和轻链可变区MUC16-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 method of treating a hyperproliferative disease as described above, the second therapeutic agent is a bispecific antibody that specifically binds MUC16 and CD3, which includes at least one antigen-binding module that specifically binds MUC16. and at least one antigen-binding module that specifically binds to CD3, the antigen-binding module that specifically binds to MUC16 includes the heavy chain variable region MUC16-VH and the light chain variable region MUC16-VL, and the antigen-binding module that specifically binds to CD3 The module contains the heavy chain variable domain CD3-VH and the light chain variable domain CD3-VL.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，其中：In some embodiments, a method of treating a hyperproliferative disease as described above, wherein:
所述MUC16-VH具有：包含SEQ ID NO：187的氨基酸序列的MUC16-HCDR1，包含SEQ ID NO：188的氨基酸序列的MUC16-HCDR2，和包含SEQ ID NO：189的氨基酸序列的MUC16-HCDR3；并且所述MUC16-VL具有：包含SEQ ID NO：190的氨基酸序列的MUC16-LCDR1，包含SEQ ID NO：191的氨基酸序列的MUC16-LCDR2，和包含SEQ ID NO：192的氨基酸序列的MUC16-LCDR3。The MUC16-VH has: MUC16-HCDR1 including the amino acid sequence of SEQ ID NO: 187, MUC16-HCDR2 including the amino acid sequence of SEQ ID NO: 188, and MUC16-HCDR3 including the amino acid sequence of SEQ ID NO: 189; And the MUC16-VL has: MUC16-LCDR1 including the amino acid sequence of SEQ ID NO: 190, MUC16-LCDR2 including the amino acid sequence of SEQ ID NO: 191, and MUC16-LCDR3 including the amino acid sequence of SEQ ID NO: 192 .
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，其中：In some embodiments, a method of treating a hyperproliferative disease as described above, wherein:
所述MUC16-VH包含如SEQ ID NO：187所示的MUC16-HCDR1，如SEQ ID NO：188所示的MUC16-HCDR2，和如SEQ ID NO：189所示的MUC16-HCDR3；并且所述MUC16-VL包含如SEQ ID NO：190所示的MUC16-LCDR1，如SEQ ID NO：191所示的MUC16-LCDR2，和如SEQ ID NO：192所示的MUC16-LCDR3。The MUC16-VH includes MUC16-HCDR1 as shown in SEQ ID NO: 187, MUC16-HCDR2 as shown in SEQ ID NO: 188, and MUC16-HCDR3 as shown in SEQ ID NO: 189; and the MUC16 -VL contains MUC16-LCDR1 as shown in SEQ ID NO:190, MUC16-LCDR2 as shown in SEQ ID NO:191, and MUC16-LCDR3 as shown in SEQ ID NO:192.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，其中所述MUC16-VH包含SEQ ID NO：199的氨基酸序列，和所述MUC16-VL包含SEQ ID NO：200的氨基酸序列。In some embodiments, the method of treating a hyperproliferative disease as described above, wherein the MUC16-VH comprises the amino acid sequence of SEQ ID NO: 199, and the MUC16-VL comprises the amino acid sequence of SEQ ID NO: 200.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，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：In some embodiments, a method of treating a hyperproliferative disease as described above, wherein the specific The antigen-binding module that binds CD3 contains the 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：193的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：194的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：195的氨基酸序列的CD3-HCDR3；并且所述CD3-VL具有：包含SEQ ID NO：196的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：197的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：198的氨基酸序列的CD3-LCDR3。The CD3-VH has: CD3-HCDR1 including the amino acid sequence of SEQ ID NO: 193, CD3-HCDR2 including the amino acid sequence of SEQ ID NO: 194, and CD3-HCDR3 including the amino acid sequence of SEQ ID NO: 195; And the CD3-VL has: CD3-LCDR1 including the amino acid sequence of SEQ ID NO: 196, CD3-LCDR2 including the amino acid sequence of SEQ ID NO: 197, and CD3-LCDR3 including the amino acid sequence of SEQ ID NO: 198 .
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，其中：In some embodiments, a method of treating a hyperproliferative disease as described above, wherein:
所述CD3-VH包含如SEQ ID NO：193所示的CD3-HCDR1，如SEQ ID NO：194所示的CD3-HCDR2，和如SEQ ID NO：195所示的CD3-HCDR3；并且所述CD3-VL包含如SEQ ID NO：196所示的CD3-LCDR1，如SEQ ID NO：197所示的CD3-LCDR2，和如SEQ ID NO：198所示的CD3-LCDR3。The CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 193, CD3-HCDR2 as shown in SEQ ID NO: 194, and CD3-HCDR3 as shown in SEQ ID NO: 195; and the CD3 -VL contains CD3-LCDR1 as set forth in SEQ ID NO: 196, CD3-LCDR2 as set forth in SEQ ID NO: 197, and CD3-LCDR3 as set forth in SEQ ID NO: 198.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，所述CD3-VH包含SEQ ID NO：201的氨基酸序列，和所述CD3-VL包含SEQ ID NO：202的氨基酸序列。In some embodiments, the method of treating a hyperproliferative disease as described above, the CD3-VH comprises the amino acid sequence of SEQ ID NO: 201, and the CD3-VL comprises the amino acid sequence of SEQ ID NO: 202.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，其中所述的特异性结合MUC16和CD3的双特异性抗体包含：In some embodiments, the method of treating a hyperproliferative disease as described above, wherein the bispecific antibody that specifically binds MUC16 and CD3 comprises:
所述MUC16-VH包含如SEQ ID NO：187所示的MUC16-HCDR1，如SEQ ID NO：188所示的MUC16-HCDR2，和如SEQ ID NO：189所示的MUC16-HCDR3；并且所述MUC16-VL包含如SEQ ID NO：190所示的MUC16-LCDR1，如SEQ ID NO：191所示的MUC16-LCDR2，和如SEQ ID NO：192所示的MUC16-LCDR3；且The MUC16-VH includes MUC16-HCDR1 as shown in SEQ ID NO: 187, MUC16-HCDR2 as shown in SEQ ID NO: 188, and MUC16-HCDR3 as shown in SEQ ID NO: 189; and the MUC16 -VL contains MUC16-LCDR1 as shown in SEQ ID NO:190, MUC16-LCDR2 as shown in SEQ ID NO:191, and MUC16-LCDR3 as shown in SEQ ID NO:192; and
所述CD3-VH包含如SEQ ID NO：193所示的CD3-HCDR1，如SEQ ID NO：194所示的CD3-HCDR2，和如SEQ ID NO：195所示的CD3-HCDR3；并且所述CD3-VL包含如SEQ ID NO：196所示的CD3-LCDR1，如SEQ ID NO：197所示的CD3-LCDR2，和如SEQ ID NO：198所示的CD3-LCDR3。The CD3-VH includes CD3-HCDR1 as shown in SEQ ID NO: 193, CD3-HCDR2 as shown in SEQ ID NO: 194, and CD3-HCDR3 as shown in SEQ ID NO: 195; and the CD3 -VL contains CD3-LCDR1 as set forth in SEQ ID NO: 196, CD3-LCDR2 as set forth in SEQ ID NO: 197, and CD3-LCDR3 as set forth in SEQ ID NO: 198.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，其中所述的特异性结合MUC16和CD3的双特异性抗体包含：In some embodiments, the method of treating a hyperproliferative disease as described above, wherein the bispecific antibody that specifically binds MUC16 and CD3 comprises:
所述MUC16-VH包含SEQ ID NO：199的氨基酸序列，和所述MUC16-VL包含SEQ ID NO：200的氨基酸序列；且The MUC16-VH comprises the amino acid sequence of SEQ ID NO: 199, and the MUC16-VL comprises the amino acid sequence of SEQ ID NO: 200; and
所述CD3-VH包含SEQ ID NO：201的氨基酸序列，和所述CD3-VL包含SEQ ID NO：202的氨基酸序列。The CD3-VH comprises the amino acid sequence of SEQ ID NO:201, and the CD3-VL comprises the amino acid sequence of SEQ ID NO:202.
在一些实施方案中，如前所述治疗过度增殖性疾病的方法，其中所述的特异性结合MUC16和CD3的双特异性抗体包含：一条包含SEQ ID NO：203的氨基酸序列的第一链、两条包含SEQ ID NO：204的氨基酸序列的第二链和一条包含SEQ ID NO：205的氨基酸序列的第三链。In some embodiments, the method for treating hyperproliferative diseases as described above, wherein the bispecific antibody that specifically binds MUC16 and CD3 comprises: a first chain comprising the amino acid sequence of SEQ ID NO: 203, Two second strands comprising the amino acid sequence of SEQ ID NO: 204 and one third strand comprising the amino acid sequence of SEQ ID NO: 205.
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 this disclosure have therapeutic activity, safety, and pharmacokinetic properties. and good drugability (such as stability).
本文中的序数词第一、第二、第三、连接子1、连接子2、Fc1、Fc2等仅用于区别不同的技术特征、要素、步骤，不意图对顺序、等级、水平、数量构成限制。The ordinal words first, second, third, connector 1, connector 2, Fc1, Fc2, etc. in this article are only used to distinguish different technical features, elements, steps, and are not intended to constitute sequence, grade, level, or quantity. limit.
附图说明Description of the drawings
图1A显示EGFR/CD28双特异性抗体的Format1的结构示意图；图1B显示EGFR/CD28双特异性抗体的Format2的结构示意图；其中Ob代表Obscurin。Figure 1A shows a schematic structural diagram of Format1 of EGFR/CD28 bispecific antibody; Figure 1B shows a schematic structural diagram of Format2 of EGFR/CD28 bispecific antibody; Ob represents Obscurin.
图2A显示固定MUC16/CD3双抗的浓度，联用EGFR/CD28双抗后PBMC细胞的IFN-γ的释放；图2B显示固定MUC16/CD3双抗的浓度，联用EGFR/CD28双抗后PBMC细胞的TNF-α的释放；图2C显示固定EGFR/CD28双抗的浓度，联用MUC16/CD3双抗后PBMC细胞的IFN-γ的释放；图2D显示固定EGFR/CD28双抗的浓度，联用MUC16/CD3双抗后PBMC细胞的TNF-α的释放。Figure 2A shows the release of IFN-γ from PBMC cells after fixing the concentration of MUC16/CD3 double antibody and combined with EGFR/CD28 double antibody; Figure 2B shows the concentration of fixed MUC16/CD3 double antibody and combined with EGFR/CD28 double antibody after PBMC The release of TNF-α from cells; Figure 2C shows the release of IFN-γ from PBMC cells after the concentration of fixed EGFR/CD28 double antibodies, combined with MUC16/CD3 double antibodies; Figure 2D shows the concentration of fixed EGFR/CD28 double antibodies, combined with MUC16/CD3 double antibodies Release of TNF-α from PBMC cells after using MUC16/CD3 double antibody.
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：19的氨基酸序列的CD28-HCDR1，其明确的涵盖氨基酸序列如SEQ ID NO：19所示的CD28-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 CD28-HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, it clearly covers CD28-HCDR1 with the amino acid sequence shown in SEQ ID NO: 19.
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是CD80(B7.1)蛋白和CD86(B7.2)蛋白的受体。示例性的人CD28的氨基酸序列显示在UniProt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 the receptor for CD80 (B7.1) protein and CD86 (B7.2) protein. The exemplary amino acid sequence of human CD28 is shown in UniProt accession number P10747.
技术人员应当理解，尽管本文给出了具体的登录号，但是靶分子不限于特定数据库中的编号，还意图涵盖现有技术中任何文献、书籍、数据库中的等同指代物。Skilled persons should understand that although specific accession numbers are given herein, the target molecules are not limited to numbers in specific databases, and are also intended to cover equivalent references in any documents, books, or databases in the prior art.
除非被指定为来自非人物种(例如，“小鼠EGFR”、“小鼠EGFR片段”、“猴 EGFR”、“猴EGFR片段”等)，否则如本文使用的“EGFR”和“EGFR片段”是指众所周知的人EGFR蛋白或其片段。Unless specified as being from a non-human species (e.g., "mouse EGFR", "mouse EGFR fragment", "monkey "EGFR", "monkey EGFR fragment", etc.), otherwise "EGFR" and "EGFR fragment" as used herein refer to the well-known human EGFR protein or fragments thereof.
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 refer to compounds that have the same basic chemical structure (i.e., alpha carbon bonded to hydrogen, carboxyl, amino, and R groups) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide , methionine methyl sulfonium. 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。应当理解，无论以T366W还是366W的方式进行限定，该位点处的原始残基都不对权利要求的范围构成限制。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. It should be understood that whether defined as T366W or 366W, the original residue at this position does not limit the scope of the claims.
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轻链和两条重链构成。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 light and two 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，片段型双特异性抗体，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、2:1 TCBs、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 can be divided into symmetric structure bispecific antibodies and asymmetric structure bispecific antibodies according to whether the structure is symmetrical or not. Among them, fragment-type bispecific antibodies, such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining two or more Fab fragments in one molecule, have lower immunogenicity and small molecular weight. , with high tumor tissue penetration. Typical antibody structures of this type include 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 to purify the antibody and improve its solubility and stability. The Fc part may also bind to the receptor FcRn. Increase antibody serum half-life, typical bispecific antibody structural models such as KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 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, pages585–608( 2019); Chen S1 et al., J Immunol Res. 2019Feb 11; 2019:4516041).
术语“可变区”或“可变域”指抗原结合分子中结合抗原/表位的域。本文中，特异性结合EGFR的抗原结合模块中的重链可变区标示为EGFR-VH，轻链可变区标示为EGFR-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，EGFR-VH中的3个CDR区分别标示为EGFR-HCDR1、EGFR-HCDR2和EGFR-HCDR3；EGFR-VL中的3个CDR区分别标示为EGFR-LCDR1、EGFR-LCDR2和EGFR-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/epitope. In this article, the heavy chain variable region in the antigen-binding module that specifically binds to EGFR is labeled EGFR-VH, and the light chain variable region is labeled EGFR-VL; the heavy-chain variable region in the antigen-binding module that specifically binds to CD28 is labeled EGFR-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 EGFR-VH are labeled EGFR-HCDR1, EGFR-HCDR2 and EGFR-HCDR3 respectively; the three CDR regions in EGFR-VL are labeled EGFR-LCDR1, EGFR-LCDR2 and EGFR-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, for example: the "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", pp. 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering scheme, "ABM" numbering scheme, "contact" numbering scheme (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [ J].2001) and ImMunoGenTics (IMGT) numbering rules (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; various numbering systems The corresponding relationship is well known to those skilled in the art. 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 of 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 Obscurin to form a dimerization complex. . 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 is capable of binding to Titin 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 part of the heavy and/or light chain is derived from a specific source. source or species, whereas the remainder 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人源化。The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, humanization can be achieved by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions and 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 interaction between the members of a binding pair (eg, antibody and antigen). 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)的选择性结合剂所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 bound by a selective binding agent of an antigen-binding molecule, such as 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 implementations In this manner, 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 an antibody binds an antigen to its epitope with an equilibrium dissociation constant (KD) of approximately 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。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.
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 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 the antigen-binding molecule refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), included in a single vector or separate vectors. such one or more nucleic acid molecules in the body, and such one or more nucleic acid molecules present at one or more locations in the 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及/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 (together with the host cell) operably linked thereto. 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 do not Restricted to mammalian cells, insect cell lines, 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, 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 genus, Saccharomycescerevisiae, Saccharomyces cerevisiae , Hansenula polymorpha, Kluyveromyces lactis, 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、稀释剂、缓冲剂或赋形剂”指药物制剂中与活性成分不同的，且对受试者无毒的成分。药学上可接受的载体、稀释剂、缓冲剂或赋形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, diluent, buffer or excipient" 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, diluents, buffers or excipients 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。"Administer" or "administer" when applied to animals, humans, experimental subjects, cells, tissues, organs or organisms By fluid, means the contact of an exogenous drug, therapeutic, diagnostic, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid.
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, abdominal cavity and other body cavities, fluid collected from bronchial lavage, synovial fluid, fluid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ conditions culture medium), 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.
“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 may 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, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5) and/or inhibitors of 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 the present disclosure may include, but are 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 .
靶分子target molecule
“CD28”应作广泛的理解，旨在涵盖CD28在哺乳动物体内各阶段中的各种形式的分子，例如但不限于CD28基因在扩增、复制、转录、剪接、加工、翻译、修饰过程中所产生的分子(例如前体CD28、成熟CD28、膜表达的CD28、CD28剪接变体、修饰的CD28、或其片段)；该术语也涵盖人工制备的或体外表达的CD28。"CD28" should be understood broadly and is intended to cover various forms of CD28 molecules at various stages in the mammalian body, such as but not limited to the amplification, replication, transcription, splicing, processing, translation, and modification of the CD28 gene. Produced molecules (eg, precursor CD28, mature CD28, membrane-expressed CD28, CD28 splice variants, modified CD28, or fragments thereof); this term also encompasses artificially prepared or in vitro-expressed CD28.
“EGFR”应作广泛的理解，旨在涵盖EGFR在哺乳动物体内各阶段中的各种形式的分子，例如但不限于EGFR基因在扩增、复制、转录、剪接、加工、翻译、修饰过程中所产生的分子(例如前体EGFR、成熟EGFR、膜表达的EGFR、EGFR剪接变体、修饰的EGFR、或其片段)；该术语也涵盖人工制备的或体外表达的EGFR。"EGFR" should be understood broadly and is intended to cover various forms of EGFR molecules at various stages in the mammalian body, such as but not limited to the amplification, replication, transcription, splicing, processing, translation, and modification processes of the EGFR gene. Produced molecules (eg, precursor EGFR, mature EGFR, membrane-expressed EGFR, EGFR splice variants, modified EGFR, or fragments thereof); the term also encompasses artificially prepared or in vitro-expressed EGFR.
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EGFR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或其组合：Antigen-binding molecules of the present disclosure include bispecific antigen-binding molecules (such as bispecific antibodies) that specifically bind to CD28 and EGFR and anti-CD28 antibodies. In particular, the antigen-binding molecules of the present disclosure have any one of the following properties or a combination thereof:
a.对CD28的高亲和力。在一些实施方式中(测试例3)，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 (Test Example 3), the anti-CD28 antibody binds human CD28 with an EC50 of less than 1 nM, as measured by ELISA.
b.对细胞(如CHO、NCI-H292细胞)表面的CD28和EGFR的高亲和力。在一些实施方式中(测试例5)，所述抗原结合分子以不超过24、23、22、21、20、18、15、13、10、9、8、7、6、5、4、3nM的EC50结合人CD28，所述抗原结合分子以不超过35、34、30、28、26、25、24、23、22、21、20、19、18、17、16、15、10、9、8、7、6、5nM的EC50结合食蟹猴CD28，所述抗原结合分子以不超过2、1.9、1.8、1.7、1.6、1.5、1.4、1.3、1.2、1.1、1.0、0.9、0.8nM的EC50结合人EGFR。b. High affinity to CD28 and EGFR on the surface of cells (such as CHO, NCI-H292 cells). In some embodiments (Test Example 5), the antigen-binding molecule is present at no more than 24, 23, 22, 21, 20, 18, 15, 13, 10, 9, 8, 7, 6, 5, 4, 3 nM The EC50 of the antigen-binding molecule binds to human CD28, and the antigen-binding molecule binds to human CD28 with an EC50 of no more than 35, 34, 30, 28, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 10, 9, The EC50 of 8, 7, 6, and 5 nM binds to cynomolgus monkey CD28, and the antigen-binding molecule binds to cynomolgus monkey CD28 at an EC50 of no more than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, and 0.8 nM. EC50 binds human EGFR.
c.对T细胞的体外激活活性。在一些实施方案中(如测试例7)，所述的抗原 结合分子在第一激活信号存在时，以小于0.15μg/mL(例如0.14、0.13、0.12、0.11、0.10、0.09、0.08、0.07、0.06、0.05μg/mL)的EC50激活表达IL-2的Jurkat细胞。c. In vitro activation activity on T cells. In some embodiments (such as Test Example 7), the antigen The binding molecule activates Jurkat expressing IL-2 with an EC50 of less than 0.15 μg/mL (e.g., 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05 μg/mL) in the presence of the first activation signal cell.
d.与MUC16/CD3双特异性抗体联用(测试例11)，诱导低水平的细胞因子(IL6和IFNγ)释放。d. Combined with MUC16/CD3 bispecific antibody (Test Example 11) to induce the release of low-level cytokines (IL6 and IFNγ).
e.更强的体内治疗活性。在一些实施方案中，与MUC16/CD3双特异性抗体或抗PD-1抗体(测试例8)联用，具有协同效应，表现出更好的体内治疗活性。e. Stronger therapeutic activity in vivo. In some embodiments, combination with MUC16/CD3 bispecific antibody or anti-PD-1 antibody (Test Example 8) has a synergistic effect and shows better in vivo therapeutic activity.
f.与MUC16/CD3双特异性抗体联用(测试例10)，可显著激活EGFR/CD3双特异性抗体对肿瘤细胞的杀伤作用。f. Combined with MUC16/CD3 bispecific antibody (Test Example 10), the killing effect of EGFR/CD3 bispecific antibody on tumor cells can be significantly activated.
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EGFR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EGFR的抗原结合模块包含EGFR-VH和EGFR-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基于克隆81、94、97和129的抗体系列。以下以抗体克隆97和94为例描述本文的抗体或抗原结合分子。The present disclosure provides an antigen-binding molecule comprising at least one antigen-binding module that specifically binds CD28 and at least one antigen-binding module that specifically binds EGFR, wherein the antigen-binding module that specifically binds CD28 includes CD28-VH and CD28 -VL, the antigen-binding module that specifically binds to EGFR includes EGFR-VH and EGFR-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 based on clones 81, 94, 97, and 129. The antibodies or antigen-binding molecules herein are described below by taking antibody clones 97 and 94 as examples.
特异性结合CD28和EGFR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 EGFR, 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：62、17、54、55、56、57、58、59、60或61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, CD28-LCDR2 as shown in SEQ ID NO: 62, 17, 54, 55, 56, 57, 58, 59, 60 or 61 and CD28-LCDR3 shown in SEQ ID NO:18.
特异性结合CD28和EGFR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 EGFR, 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、66K、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, FR2, FR3 derived from IGHV1-3*01 and FR4 derived from IGHJ1*01, and is unsubstituted or has FR4 derived from IGHJ1*01 , one or more amino acid substitutions in the group consisting of 28S, 66K, 69L, 71V, 73K and 94S; and/or the humanized CD28-VL has FR1, FR2, FR3 derived from IGKV1-12*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 43S and 70K. 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、66K、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 in the FR region of SEQ ID NO: 69 selected from the group consisting of 28S, 66K, 69L, 71V, 73K, and 94S. 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，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。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:106 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 above, wherein the amino acid sequence of CD28-VH is consistent with SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69 Having 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 amino acid sequence of CD28-VH is set forth in SEQ ID NO: 68, 33, 63, 64, 65, 66, 67 or 69, and the amino acid sequence of CD28-VL is 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 CD28-VH is shown in SEQ ID NO: 63, and the amino acid sequence of CD28-VL As shown in SEQ ID NO: 70 or 71, 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:
所述EGFR-VH具有：包含SEQ ID NO：160的氨基酸序列的EGFR-HCDR1，包含SEQ ID NO：161的氨基酸序列的EGFR-HCDR2，和包含SEQ ID NO：162的氨基酸序列的EGFR-HCDR3；和所述EGFR-VL具有：包含SEQ ID NO：163的氨基酸序列的EGFR-LCDR1，包含SEQ ID NO：164的氨基酸序列的EGFR-LCDR2，和包含SEQ ID NO：165的氨基酸序列的EGFR-LCDR3。The EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and the EGFR-VL having: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR3 comprising the amino acid sequence of SEQ ID NO: 165 .
在一些实施方案中，如前任一项所述的抗原结合分子，所述EGFR-VH和/或所述EGFR-VL是鼠源的或人源化的。在一些实施方案中，所述EGFR-VH和/或所述EGFR-VL是人源化的。In some embodiments, the antigen-binding molecule of any one of the preceding items, the EGFR-VH and/or the EGFR-VL is murine or humanized. In some embodiments, the EGFR-VH and/or the EGFR-VL are humanized.
在一些实施方案中，如前任一项所述的抗原结合分子，其中所述EGFR-VH的氨基酸序列如SEQ ID NO：158所示，和所述EGFR-VL的氨基酸序列SEQ ID NO：159所示。In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the amino acid sequence of the EGFR-VH is shown in SEQ ID NO: 158, and the amino acid sequence of the EGFR-VL is shown in SEQ ID NO: 159 Show.
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.
在一些实施方案中，如前任一项所述的抗原结合分子，其包含：In some embodiments, the antigen-binding molecule as described in any one of the preceding items, comprising:
如SEQ ID NO：174所示的第一链、如SEQ ID NO：176所示的第二链、如SEQ ID NO：180所示的第三链和如SEQ ID NO：182所示的第四链；或The first strand as shown in SEQ ID NO: 174, the second strand as shown in SEQ ID NO: 176, the third strand as shown in SEQ ID NO: 180 and the fourth strand as shown in SEQ ID NO: 182 chain; or
如SEQ ID NO：178所示的第一链、如SEQ ID NO：179所示的第二链、如SEQ ID NO：180所示的第三链和如SEQ ID NO：182所示的第四链。The first strand as shown in SEQ ID NO: 178, the second strand as shown in SEQ ID NO: 179, the third strand as shown in SEQ ID NO: 180 and the fourth strand as shown in SEQ ID NO: 182 chain.
在一些实施方案中，如前任一项所述的抗原结合分子，其包含：In some embodiments, the antigen-binding molecule as described in any one of the preceding items, comprising:
如SEQ ID NO：172所示的第一链、如SEQ ID NO：173所示的第二链、如SEQ ID NO：181所示的第三链和如SEQ ID NO：183所示的第四链；或The first strand as shown in SEQ ID NO: 172, the second strand as shown in SEQ ID NO: 173, the third strand as shown in SEQ ID NO: 181 and the fourth strand as shown in SEQ ID NO: 183 chain; or
如SEQ ID NO：170所示的第一链、如SEQ ID NO：171所示的第二链、如 SEQ ID NO：181所示的第三链和如SEQ ID NO：183所示的第四链；或The first strand as shown in SEQ ID NO: 170, the second strand as shown in SEQ ID NO: 171, as The third strand as shown in SEQ ID NO: 181 and the fourth strand as shown in SEQ ID NO: 183; or
如SEQ ID NO：175所示的第一链、如SEQ ID NO：177所示的第二链、如SEQ ID NO：181所示的第三链和如SEQ ID NO：183所示的第四链。The first strand as shown in SEQ ID NO: 175, the second strand as shown in SEQ ID NO: 177, the third strand as shown in SEQ ID NO: 181 and the fourth strand as shown in SEQ ID NO: 183 chain.
根据本文实施例所披露的包含抗体系列81和129的抗原结合分子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 antigen-binding molecules comprising antibody series 81 and 129 disclosed according to the embodiments herein have a similar scope of technical solutions as the above-described antibody series 97 and 94.
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4)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：284)、GGGS(SEQ ID NO：285)、SGGGGS(SEQ ID NO：286)、GGGTKLTVLGGG(SEQ ID NO：287)，n是0、1、2、3、4、5、6、7、8、9或10，L2是键、G、GG、GGG(SEQ ID NO：288)或GGGG(SEQ ID NO：289)，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：169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 4) 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: 284), GGGS (SEQ ID NO: 285), SGGGGS (SEQ ID NO: 286), GGGTKLTVLGGG (SEQ ID NO: 287), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L2 is bond, G, GG, GGG (SEQ ID NO:288) or GGGG (SEQ ID NO:289), 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: 169.
示例性的，本披露的所述抗原结合分子具有式(a-1)所示结构的第一链、一条具有式(b-1)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，Illustratively, the antigen-binding molecule of the present disclosure has a first chain having a structure represented by formula (a-1), a second chain having a structure represented by formula (b-1), and a second chain having a structure represented by formula (c). a third chain having the structure shown and a fourth chain having the structure shown in formula (d),
(a-1)[CD28-VH]-[GGGGS]-[Titin链]-[Fc1]，(a-1)[CD28-VH]-[GGGGS]-[Titin chain]-[Fc1],
(b-1)[CD28-VL]-[GGGGS]-[Obscurin链]，(b-1)[CD28-VL]-[GGGGS]-[Obscurin chain],
(c)[EGFR-VH]-[CH1]-[Fc2]，(c)[EGFR-VH]-[CH1]-[Fc2],
(d)[EGFR-VL]-[CL]，(d)[EGFR-VL]-[CL],
式(a-1)、(b-1)、(c)和(d)所示的结构是从N端至C端排列的。The structures shown in formulas (a-1), (b-1), (c) and (d) 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：174的氨基酸序列的第一链、一条包含SEQ ID NO：176的氨基酸序列的第二链、一条包含SEQ ID NO：180的氨基酸序列的第三链和一条包含SEQ ID NO：182的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 174, a second strand comprising the amino acid sequence of SEQ ID NO: 176, a third strand comprising the amino acid sequence of SEQ ID NO: 180 and a third strand comprising the amino acid sequence of SEQ ID NO: 174 : The fourth strand of the amino acid sequence 182; or
一条包含SEQ ID NO：178的氨基酸序列的第一链、一条包含SEQ ID NO：179的氨基酸序列的第二链、一条包含SEQ ID NO：180的氨基酸序列的第三链和一条包含SEQ ID NO：182的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 178, a second strand comprising the amino acid sequence of SEQ ID NO: 179, a third strand comprising the amino acid sequence of SEQ ID NO: 180 and a third strand comprising the amino acid sequence of SEQ ID NO: 179 : The fourth strand of the amino acid sequence of 182.
示例性的，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-1)所示结构的第三链和一条具有式(h-1)所示结构的第四链，Exemplarily, the antigen-binding molecule includes a first chain having a structure shown in formula (e), a first chain having There is a second chain having the structure shown in formula (f), a third chain having the structure shown in formula (g-1) and a fourth chain having the structure shown in formula (h-1),
(e)[CD28-VH]-[CH1]-[Fc1]，(e)[CD28-VH]-[CH1]-[Fc1],
(f)[CD28-VL]-[CL]，(f)[CD28-VL]-[CL],
(g-1)[EGFR-VH]-[GGGGS]-[Obscurin链]-[Fc2]，(g-1)[EGFR-VH]-[GGGGS]-[Obscurin chain]-[Fc2],
(h-1)[EGFR-VL]-[GGGGS]-[Titin链]，(h-1)[EGFR-VL]-[GGGGS]-[Titin chain],
式(e)、(f)、(g-1)和(h-1)所示的结构是从N端至C端排列的。The structures shown in formulas (e), (f), (g-1) and (h-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：172的氨基酸序列的第一链、一条包含SEQ ID NO：173的氨基酸序列的第二链、一条包含SEQ ID NO：181的氨基酸序列的第三链和一条包含SEQ ID NO：183的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 172, a second strand comprising the amino acid sequence of SEQ ID NO: 173, a third strand comprising the amino acid sequence of SEQ ID NO: 181 and a third strand comprising the amino acid sequence of SEQ ID NO: 172 : The fourth strand of the amino acid sequence 183; or
一条包含SEQ ID NO：170的氨基酸序列的第一链、一条包含SEQ ID NO：171的氨基酸序列的第二链、一条包含SEQ ID NO：181的氨基酸序列的第三链和一条包含SEQ ID NO：183的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 170, a second strand comprising the amino acid sequence of SEQ ID NO: 171, a third strand comprising the amino acid sequence of SEQ ID NO: 181 and a third strand comprising the amino acid sequence of SEQ ID NO: 171 : The fourth strand of the amino acid sequence 183; or
一条包含SEQ ID NO：175的氨基酸序列的第一链、一条包含SEQ ID NO：177的氨基酸序列的第二链、一条包含SEQ ID NO：181的氨基酸序列的第三链和一条包含SEQ ID NO：183的氨基酸序列的第四链。A first strand comprising the amino acid sequence of SEQ ID NO: 175, a second strand comprising the amino acid sequence of SEQ ID NO: 177, a third strand comprising the amino acid sequence of SEQ ID NO: 181 and a third strand comprising the amino acid sequence of SEQ ID NO: 175 : The fourth strand of the amino acid sequence of 183.
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. Such changes can be made to CDR "hotspots," residues encoded by codons that undergo mutations at high frequency during the somatic maturation process, and/or residues that contact the antigen, while simultaneously modifying the resulting variant VH or VL tests 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, where several CDR residues (e.g. 4-6 residues at a time) are randomized change. 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EGFR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 EGFR 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 IgG1 Fc region, substitution of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 can reduce effector function. In some embodiments, the Fc region is a human IgGl 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, WO2011143545, 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 readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes capable of binding specifically to genes encoding 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 used for vectors encoding antibodies. Suitable cloning or expression hosts include fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of 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, US6040498, US6420548, US7125978 and US6417429; Vertebrate animal cells can also be used as hosts, for example mammalian cell lines adapted for 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 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, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (Eds.), Humana Press, Totowa, NJ (2004) , pp. 255-268.
免疫缀合物Immunoconjugate
本披露还提供免疫缀合物，其包含与一种或多种细胞毒性剂缀合的抗原结合分子，所述一种或多种细胞毒性剂为诸如化学治疗剂或药物、生长抑制剂、毒素(例如细菌、真菌、植物或动物来源的蛋白质毒素、酶活性毒素，或它们的片段)、或放射性同位素。The present disclosure also provides immunoconjugates comprising an antigen-binding molecule conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (such as protein toxins, enzymatically active toxins, or fragments thereof of bacterial, fungal, plant or animal origin), or radioactive isotopes.
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EGFR和/或CD28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 EGFR and/or CD28 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EGFR和/或CD28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EGFR和/或CD28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 EGFR and/or CD28 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, EGFR and/or CD28 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. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent, chromogenic, electron dense, chemiluminescent, and radioactive labels), and indirectly detected moieties (e.g., indirectly detected via enzymatic reactions or molecular interactions). module, such as enzymes or ligands).
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MUC16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 MUCl6 and CD3.
抗原结合分子与另外的治疗剂可以同时施用、或贯序、分开施用。施用的时间间隔可以由本领域技术人员确定，只要使得二者能够产生协同作用即可。The antigen-binding molecule and the additional therapeutic agent can be administered simultaneously, sequentially, or separately. The time interval of administration can be determined by those skilled in the art, as long as the two can produce a synergistic effect.
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：169)、ASTKG(SEQ ID NO：278)或RTVAS(SEQ ID NO：279)，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 specification), 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 includes GGGGS (SEQ ID NO: 169), ASTKG (SEQ ID NO: 278) or RTVAS (SEQ ID NO: 279). The amino acid sequences of Fc1, Fc2 and CH1 in this example are as follows.
>实施例1-Fc1(knob，SEQ ID NO：280)
>Example 1-Fc1 (knob, SEQ ID NO: 280)
>实施例1-Fc2(hole，SEQ ID NO：281)
>Example 1-Fc2 (hole, SEQ ID NO: 281)
>实施例1-CH1(SEQ ID NO：166)。>Example 1-CH1 (SEQ ID NO: 166).
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


注：表格中Titin链和Obscurin链的编号见表3-1和表3-2。Table 5. Correspondence table of Obscurin chain/Titin chain and linker in DI bispecific antibodies


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.
>VH2-L(SEQ ID NO：282)
>VH2-L(SEQ ID NO: 282)
>VL2-L(SEQ ID NO：283)
>VL2-L(SEQ ID NO: 283)
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

注：表格中Titin链和Obscurin链的编号见表3-1和表3-2。Table 9. Correspondence table of Obscurin chain/Titin chain and linker in PL bispecific antibodies

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-APC-hEGFR，CHO-K1-hCD28，HEK293-hCD28，CHO-K1-cyno CD28，CHO-APC-hEGFR-PDL1和Jurkat-PD1细胞，使用的相关蛋白序列如下：To construct CHO-APC-hEGFR, CHO-K1-hCD28, HEK293-hCD28, CHO-K1-cyno CD28, CHO-APC-hEGFR-PDL1 and Jurkat-PD1 cells, the relevant protein sequences used are as follows:
人EGFR蛋白序列(UniProtKB-P00533)：
Human EGFR protein sequence (UniProtKB-P00533):
人CD28蛋白(UniProt P10747)：
Human CD28 protein (UniProt P10747):
cyno CD28蛋白序列(UniProt Q0PDN3)：

cyno CD28 protein sequence (UniProt Q0PDN3):

人PD1蛋白(UniProt Q15116)：
Human PD1 protein (UniProt Q15116):
人PDL1蛋白(UniProt Q9NZQ7)：
Human PDL1 protein (UniProt Q9NZQ7):
将编码人EGFR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-h EGFR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EGFR或CD28的CHO-APC-hEGFR，CHO-K1-hCD28，HEK293-hCD28和CHO-K1-cyno CD28细胞株。Clone the full-length gene encoding human EGFR 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-h EGFR. Packaging viruses. 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-APC-hEGFR, CHO-K1-hCD28, HEK293-hCD28 and CHO-K1-cyno CD28 cell lines with high expression of EGFR 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-hEGFR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-hEGFR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-hEGFR-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-hEGFR ( CRL-11268) packaged virus, 48 hours after transfection, collect the virus to infect CHO-APC-hEGFR 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-hEGFR-PDL1 and Jurkat PD1 cell lines.
2.2抗人CD28杂交瘤抗体的筛选和鉴定2.2 Screening and identification of anti-human CD28 hybridoma antibodies
本披露通过杂交瘤技术制备针对人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，并且与食蟹猴CD28有交叉结合，与细胞表面的人CD28，食蟹猴CD28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 high affinity, cross-bind to cynomolgus CD28, and bind to human CD28 and cynomolgus CD28 on the cell surface. Has better 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:
人CD28-his：
Human CD28-his:
人CD28-Fc：
Human CD28-Fc:
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。分别收集对数生长期单克隆杂交瘤细胞，用NucleoZol(MN)提取RNA(按照试剂盒说明书步骤)，并进行反转录(PrimeScriptTMReverse Transcriptase，Takara,cat#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen，TB326Rev.B 0503)进行PCR扩增后测序。鼠源抗体的CDR和可变区的氨基酸序列如下：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 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, TB326Rev.B 0503). The amino acid sequences of the CDR and variable regions of the mouse antibody are as follows:
表14.抗CD28抗体的CDR

Table 14. CDRs of anti-CD28 antibodies

>m81VH：
>m81VH：
>m81VL：
>m81VL：
>m94VH：
>m94VH：
>m94VL：
>m94VL：
>m97VH：
>m97VH：
>m97VL：
>m97VL：
>m129VH：
>m129VH：
>m129VL：

注：下划线部分为根据Kabat编号规则获得的CDR区。>m129VL：

Note: The underlined part is the CDR area obtained according to Kabat numbering rules.
鼠源抗CD28抗体的可变区序列与SEQ ID NO：144和SEQ ID NO：145所示的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SEQ ID NO：144和SEQ ID NO：145所示的恒定区的嵌合抗体。Chi94、Chi97和Chi129以此类推。The variable region sequence of the mouse anti-CD28 antibody was combined with the constant region 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 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germline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germline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 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. Sort the FR homology 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 combine the mouse The constant region of the source antibody is replaced with a human constant region, resulting in the final humanized molecule.
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.m81人源化抗体的CDR
Table 16. CDRs of m81 humanized antibody
人源化抗体h81可变区具体序列如下：The specific sequence of the variable region of humanized antibody h81 is as follows:
>h81VH1：
>h81VH1：
>h81VH2：
>h81VH2：
>h81VH3：
>h81VH3：
>h81VH4：
>h81VH4：
>h81VH5：

>h81VH5：

>h81VH6：
>h81VH6：
>h81VL1：
>h81VL1：
>h81VL2：
>h81VL2：
>h81VL3：
>h81VL3:
>h81VH7：

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区。>h81VH7：

Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are
FR zone.
3-2.m94抗体的人源化3-2. Humanization of m94 antibody
m94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IGHV1-3*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 antibody 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 IGHJ1*01 FR4 serves as the 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18.94的人源化抗体的CDR
CDRs of humanized antibodies in Table 18.94
人源化抗体h94可变区序列如下：The sequence of the humanized antibody h94 variable region is as follows:
>h94VH1：
>h94VH1：
>h94VH2：
>h94VH2：
>h94VH3：

>h94VH3:

>h94VH4：
>h94VH4：
>h94VH5：
>h94VH5：
>h94VH6：
>h94VH6：
>h94VH7：
>h94VH7：
>h94VL1：
>h94VL1：
>h94VL2：
>h94VL2:
>h94VL3：
>h94VL3:
>h94VL4：
>h94VL4:
>h94VL5：
>h94VL5：
>h94VL6：
>h94VL6:
>h94VL7：
>h94VL7:
>h94VL8：
>h94VL8：
>h94VL9：
>h94VL9:
>h94VL10：
>h94VL10:
>h94VL12：

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区。>h94VL12:

Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are
FR zone.
3-3.m97抗体的人源化 3-3.Humanization of m97 antibody
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. 97的人源化抗体的CDR

Table 20. CDRs of 97 humanized antibodies

人源化抗体h97可变区序列如下：The sequence of the variable region of humanized antibody h97 is as follows:
>h97VH1：
>h97VH1：
>h97VH2：
>h97VH2：
>h97VH3：
>h97VH3：
>h97VH4：
>h97VH4：
>h97VH5：
>h97VH5：
>h97VL1：
>h97VL1：
>h97VL2：
>h97VL2：
>h97VL3：
>h97VL3:
>h97VL4：
>h97VL4:
>h97VL1-1：
>h97VL1-1：
>h97VL1-2：
>h97VL1-2：
>h97VL1-3：
>h97VL1-3:
>h97VL1-4：
>h97VL1-4：
>h97VL1-5：
>h97VL1-5：
>h97VL1-6：
>h97VL1-6：
>h97VL2-1：
>h97VL2-1：
>h97VL2-2：
>h97VL2-2：
>h97VL2-3：
>h97VL2-3：
>h97VL2-4：
>h97VL2-4：
>h97VL2-5：
>h97VL2-5：
>h97VL2-6：
>h97VL2-6：
>h97VL2-7：

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区。>h97VL2-7：

Note: The single underlined part is the CDR, the double underlined part is the amino acid substitution site, and the remaining parts are
FR zone.
3-4.m129抗体的人源化3-4. Humanization of m129 antibody
鼠源抗体129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。For the humanized antibody of mouse antibody 129, 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, 53, 54, 55 and/or 73 of the light chain variable region of the humanized antibody are substituted; and/or the humanized antibody The amino acid residues at positions 1, 25, 30, 34, 52, 71 and/or 78 of the heavy chain variable region of the 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. 129人源化抗体的CDR

Table 22. CDRs of 129 humanized antibodies

m129的人源化抗体的可变区序列如下：The variable region sequence of the humanized antibody of m129 is as follows:
>h129VH1：
>h129VH1：
>h129VH2：
>h129VH2：
>h129VH3：
>h129VH3：
>h129VH4：
>h129VH4：
>h129VH5：
>h129VH5：
>h129VH6：
>h129VH6：
>h129VH7：
>h129VH7：
>h129VH8：
>h129VH8：
>h129VH9：
>h129VH9：
>h129VH10：
>h129VH10：
>h129VH11：
>h129VH11：
>h129VL1：
>h129VL1：
>h129VL2：
>h129VL2：
>h129VL3：

>h129VL3:

>h129VL4：
>h129VL4:
>h129VL5：
>h129VL5：
>h129VL6：
>h129VL6:
>h129VL7：
>h129VL7：
>h129VL8：
>h129VL8：
>h129VL9：
>h129VL9:
>h129VL10：
>h129VL10：
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.
>hIgG1:CH1-Fc：

>hIgG1:CH1-Fc:

>CL：
>CL:
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:
81H3L3重链：

81H3L3 heavy chain:

81H3L3轻链：
81H3L3 light chain:
94H6L12重链：
94H6L12 heavy chain:
94H6L12轻链：
94H6L12 light chain:
97H2L3重链：

97H2L3 heavy chain:

97H2L3轻链：
97H2L3 light chain:
129H4L1重链：
129H4L1 heavy chain:
129H4L1轻链：
129H4L1 light chain:
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:
>REGN-hIgG1重链：

>REGN-hlgG1 heavy chain:

>REGN-hIgG1轻链：
>REGN-hIgG1 light chain:
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:
>TGN1412轻链：
>TGN1412 light chain:
>TGN1412重链：
>TGN1412 heavy chain:
实施例4.抗EGFR/CD28双特异性抗体的制备Example 4. Preparation of anti-EGFR/CD28 bispecific antibodies
本披露EGFR/CD28双特异性抗体的EGFR臂可以来源于任意适宜的抗EGFR抗体。示例性的，本披露双特异性抗体中的抗EGFR臂的CDR和可变区序列参考WO2002100348专利中的zalutumumab 2F8制备，其可变区及CDR序列如下：The EGFR arm of the EGFR/CD28 bispecific antibodies of the present disclosure can be derived from any suitable anti-EGFR antibody. Exemplarily, the CDR and variable region sequences of the anti-EGFR arm in the bispecific antibody of the present disclosure are prepared with reference to zalutumumab 2F8 in the WO2002100348 patent, and its variable region and CDR sequences are as follows:
>EGFR-VH：
>EGFR-VH：
>EGFR-VL：
>EGFR-VL:
表24. 2F8的CDR
Table 24. CDR of 2F8
本披露的EGFR-CD28双特异性抗体分子的结构如下：The structure of the disclosed EGFR-CD28 bispecific antibody molecule is as follows:
Format1为非对称结构分子，包含四条链，四条链均不相同，具体如下：Format1 is an asymmetric structure molecule, containing four chains, all of which are different. The details are 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：EGFR-VH-IgG1(CH1)-IgG1Fc(Hole)；Chain 3: EGFR-VH-IgG 1 (CH1)-IgG1Fc(Hole);
链4：EGFR-VL-CL；Chain 4: EGFR-VL-CL;
其示意图如图1A所示，其中Ob代表Obscurin。Its schematic diagram is shown in Figure 1A, where Ob represents Obscurin.
Format 2为非对称结构分子，包含四条链，四条链均不相同，具体如下：Format 2 is an asymmetric structure molecule, containing four chains, all of which are different. The details are 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：EGFR-VH-连接子1a-Obscurin-IgG1Fc(Hole)；和Chain 3: EGFR-VH-linker 1a-Obscurin-IgG1Fc (Hole); and
链4：EGFR-VL-连接子1a-Titin；Chain 4: EGFR-VL-linker 1a-Titin;
其示意图如图1B所示，其中Ob代表Obscurin。Its schematic diagram is shown in Figure 1B, where Ob represents Obscurin.
相关序列如下：The relevant sequences are as follows:
>IgG1(CH1)：
>IgG1(CH1)：
>IgG1Fc-Knob(L234A/L235A/S354C/T366W)：

>IgG1Fc-Knob(L234A/L235A/S354C/T366W):

>IgG1Fc-Hole(L234A/L235A/Y349C/T366S/L368A/Y407V)
>IgG1Fc-Hole(L234A/L235A/Y349C/T366S/L368A/Y407V)
>CL：
>CL：
>连接子1a：
>Connector 1a:
>Titin(T.16):
>Titin(T.16):
>Obscurin(O.28)
>Obscurin(O.28)
表25.本披露的EGFR-CD28双特异性抗体

Table 25. EGFR-CD28 bispecific antibodies of the present disclosure

本披露示例性的双特异性抗体的序列如下：The sequences of exemplary bispecific antibodies of the present disclosure are as follows:
>81H3-CH1-knob：
>81H3-CH1-knob：
>81L3-CL：
>81L3-CL：
>94H6-CH1-knob：
>94H6-CH1-knob：
>94L12-CL：
>94L12-CL：
>97H2-Titin-knob：
>97H2-Titin-knob：
>97H2-CH1-knob
>97H2-CH1-knob
>97L3-Ob：
>97L3-Ob：
>97L1-4-CL：
>97L1-4-CL:
>129H4-Titin-knob：

>129H4-Titin-knob：

>129L1-Ob
>129L1-Ob
>ZAL-CH1-hole：
>ZAL-CH1-hole：
>ZAL-Ob-hole
>ZAL-Ob-hole
>ZAL-VL-CL
>ZAL-VL-CL
>ZAL-VL-Titin

>ZAL-VL-Titin

本披露的EGFR/CD28双特异性抗体的对照分子为REGN7075，参考WO2020198009构建，将其Fc换成本披露IgG1Fc(Knob)和IgG1Fc(Hole)，得到REGN7075V，其具有common light chain的结构。具体序列如下：The control molecule of the disclosed EGFR/CD28 bispecific antibody is REGN7075, which was constructed with reference to WO2020198009. Its Fc was replaced with the disclosed IgG1 Fc (Knob) and IgG 1 Fc (Hole) to obtain REGN7075V, which has a common light chain structure. The specific sequence is as follows:
>REGN7075V第一链：
>REGN7075V first chain:
>REGN7075V第二链：
>REGN7075V second chain:
>REGN7075V第三链(common light chain)：
>REGN7075V third chain (common light chain):
此外，本披露也使用了MUC16/CD3双特异性抗体，参考Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3affinity tuning,Scientific Reports,11,1,(2021).制备，具体序列如下： In addition, this disclosure also uses MUC16/CD3 bispecific antibodies, prepared with reference to Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3affinity tuning, Scientific Reports, 11, 1, (2021). The specific sequence is as follows:
表26.MUC16/CD3双特异性抗体的CDR
Table 26. CDRs of MUC16/CD3 bispecific antibodies
>MUC16VH：
>MUC16VH：
>MUC16VL：
>MUC16VL：
>CD3VH：
>CD3VH：
>CD3VL：
>CD3VL:
MUC16/CD3双特异性抗体的全长序列：Full-length sequence of MUC16/CD3 bispecific antibody:
>链1：

>Chain 1:

>链2：
>Chain 2:
>链3：
>Chain 3:
>链4：序列同链2(SEQ ID NO：204)。>Chain 4: The sequence is the same as chain 2 (SEQ ID NO: 204).
本披露还涉及EGFR/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 EGFR/CD28 bispecific antibodies and anti-PD-1 antibodies. The anti-PD-1 antibodies used are prepared with reference to patent WO2020156509A1. The specific sequence is as follows:
表27.抗PD-1抗体的CDR区
Table 27. CDR regions of anti-PD-1 antibodies
>PD-1 VH：
>PD-1 VH：
>PD-1 VL：
>PD-1 VL：
>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 co-stimulatory 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2-3次备用，将抗体用1640+10％FBS的培养基梯度稀释，50μL每孔加入96孔的3590酶标板中。Jurkat-NFAT lum细胞离心收集，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28-1至表28-4。Jurkat-NFAT lum cells were subcultured 2-3 times in complete culture medium for later use. The antibody was serially diluted with 1640+10% FBS culture medium, and 50 μL was added to each well of a 96-well 3590 microplate. Jurkat-NFAT lum cells are collected by centrifugation, use 1640+10% FBS culture medium, resuspend and count, adjust the number of cells to 1E6 cells/mL, add 50μL (5E4/well) to each well of a 96-well 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 functional microplate reader (BMG Cat.#PHERAstar). The results are shown in Table 28-1 to Table 28-4 below.
表28-1.抗体81的T细胞激活作用(生物发光值)
Table 28-1. T cell activation effect of antibody 81 (bioluminescence value)
表28-2.抗体94的T细胞激活作用(生物发光值)
Table 28-2. T cell activation effect of antibody 94 (bioluminescence value)
表28-3.抗体97的T细胞激活作用(生物发光值)

Table 28-3. T cell activation effect of antibody 97 (bioluminescence value)

表28-4.抗体129的T细胞激活作用(生物发光值)
Table 28-4. T cell activation effect of antibody 129 (bioluminescence value)
结果显示，基于81，129，97和129克隆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 based on clones 81, 129, 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 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。实验时使用，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/mLTNB383B-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-IL2lum cells were cultured in 1640 complete medium and passaged every 2 days. For use in experiments, pipette 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. Use 2μg/mL Anti-hu (H+L) to coat the high-adsorption cell culture plate, and the cell culture box is coated for 2 hours; discard the supernatant, add 30ng/mLLTNB383B-H, and the cell culture box is coated for 0.5 to 1 hour; discard Supernatant, add diluted CD28 humanized antibody (starting from 3 μg/mL, diluted 3 times to 8 concentrations), cover the cell culture box for 1 hour; discard the supernatant, add Jurkat-IL2-lum cells, and adjust the density to 3E4 cells/50μL/well, culture overnight. Read the plate: add 50μL/well firefly-glo luciferase reporter gene detection reagent (Meilun BioCat.#MA0519-2), incubate at room temperature for 10 minutes in the dark, and then PheraStar performs lumin signal Read.
结果显示，TNB383B-H单用时IL2激活信号较弱，CD28单抗联用可以增强IL2激活信号，基于81，129，97，129克隆的所有抗体均可激活T细胞的IL2信号通路。The results showed that the IL2 activation signal was weak when TNB383B-H was used alone, but the combination of CD28 monoclonal antibody could enhance the IL2 activation signal. All antibodies based on the 81, 129, 97, and 129 clones could activate the IL2 signaling pathway of T cells.
表29.抗体对T细胞IL2信号通路的激活结果


Table 29. Results of antibody activation of T cell IL2 signaling pathway


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anti human 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30和31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 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. TMB After color development, the reaction was terminated with 0.1M sulfuric acid. The ELISA results are shown in Tables 30 and 31 below.
表30.CD28人源化单抗与人CD28抗原的结合作用
Table 30. Binding effect of CD28 humanized monoclonal antibody and human CD28 antigen
表31.CD28人源化单抗与猴CD28抗原的结合作用

Table 31. 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.双特异性抗体的Biacore结合作用Test Example 4. Biacore binding of bispecific antibodies
为测试本披露EGFR/CD28双特异性抗体与人CD28、cyno CD28和人EGFR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人CD28-his、cyno CD28-his(90182-C08H，sino biological inc)、EGFR-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32和33所示。In order to test the binding ability of the disclosed EGFR/CD28 bispecific antibody to human CD28, cyno CD28 and human EGFR, Protein A biosensor chip affinity capture antibody was used, and then human CD28-his, cyno CD28- was flowed on the chip surface. his (90182-C08H, sino biological inc), EGFR-his antigen, use Biacore T200 instrument 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 32 and 33 below.
EGFR-his序列：
EGFR-his sequence:
表32.双特异性抗体与CD28的Biacore结合结果
Table 32. Biacore binding results of bispecific antibodies and CD28
表33.双特异性抗体与EGFR的Biacore结合结果

Table 33. Biacore binding results of bispecific antibodies and EGFR

结果显示，本披露双特异性抗体与CD28和EGFR均具有良好结合作用。The results show that the bispecific antibody of the present disclosure has good binding effect on both CD28 and EGFR.
测试例5.双特异性抗体与细胞的结合能力Test Example 5. Binding ability of bispecific antibodies to cells
为测试本披露EGFR/CD28双特异性抗体与EGFR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表达人EGFR的NCI-H292细胞(中科院细胞库)、过表达人CD28的CHO-K1-hCD28细胞和过表达cyno CD28的CHOK1-cyno CD28细胞的结合能力。具体如下：In order to test the binding ability of the disclosed EGFR/CD28 bispecific antibody to EGFR and CD28, this test example used flow cytometry to detect the bispecific antibody and NCI-H292 cells expressing human EGFR (Cell Bank of the Chinese Academy of Sciences). Binding ability of CHO-K1-hCD28 cells expressing human CD28 and CHOK1-cyno CD28 cells overexpressing cyno CD28. details as follows:
上述细胞培养于10％FBS的F12或1640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 APC-Goat Anti-human IgG Fc荧光二抗(BioLegend Way,410712)稀释液(1:200)，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-K1-hCD28、CHO-K1-cyno CD28、NCI-H292细胞的结合情况如下表34至36所示。The above cells were cultured in F12 or 1640 culture 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 the number of 1× 10 cells per well. Centrifuge at 300g for 5 minutes and wash once 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 diluted with 100 μL APC-Goat Anti-human IgG Fc fluorescent secondary antibody (BioLegend Way, 410712) added to each well. solution (1:200) and incubated 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 status of each antibody to CHO-K1-hCD28, CHO-K1-cyno CD28, and NCI-H292 cells is shown in Tables 34 to 36 below.
表34.EGFR/CD28双抗与CHO-K1-hCD28细胞的结合作用
Table 34. Binding effect of EGFR/CD28 double antibodies on CHO-K1-hCD28 cells
表35.EGFR/CD28双抗与CHO-K1-cyno CD28细胞的结合作用
Table 35. Binding effect of EGFR/CD28 double antibodies on CHO-K1-cyno CD28 cells
表36.EGFR/CD28双抗与NCI-H292细胞的结合作用
Table 36. Binding effect of EGFR/CD28 double antibodies on NCI-H292 cells
结果显示，本披露双特异性抗体与表达CD28和EGFR的细胞均具有良好结合作用。 The results show that the bispecific antibody of the present disclosure has good binding effect on cells expressing CD28 and EGFR.
测试例6.双特异性抗体抑制EGF与EGFR结合的能力Test Example 6. Ability of bispecific antibodies to inhibit the binding of EGF and EGFR
为测试本披露EGFR/CD28双特异性抗体抑制EGF与EGFR结合的能力，本测试例用ELISA的方法进行了检测。具体如下：In order to test the ability of the disclosed EGFR/CD28 bispecific antibody to inhibit the binding of EGF and EGFR, this test example was tested using ELISA. details as follows:
过夜包被anti-his后封闭ELISA板，封闭结束后加入2μg/mLEGFR-his，孵育1小时后洗板，再加入20μg/mL待测抗体与EGF-mFc(Acro)2μg/mL的混合液，3倍梯度稀释，共7个梯度，加入酶标板中，37℃孵育1小时后，加入100μL/孔用PBS稀释(1:4000)的二抗HRP-mFc(115-035-003，Jackson Immunoresearch)，37℃孵育40分钟。用PBST洗板5次后，加入100μL/孔TMB显色底物，于室温孵育3-5分钟，加入100μL/孔1M H2SO4终止反应，用NOVOStar酶标仪在450nm处读取吸收值，计算抗体对抗原的结合IC50值。各抗体抑制EGF与EGFR结合的情况如下表37所示：After coating anti-his overnight, block the ELISA plate. After blocking, add 2 μg/mL EGFR-his, incubate for 1 hour, wash the plate, and then add a mixture of 20 μg/mL test antibody and 2 μg/mL EGF-mFc (Acro). 3-fold gradient dilution, a total of 7 gradients, was added to the enzyme plate. After incubation at 37°C for 1 hour, 100 μL/well of secondary antibody HRP-mFc (115-035-003, Jackson Immunoresearch) diluted in PBS (1:4000) was added. ), incubate at 37°C for 40 minutes. After washing the plate 5 times with PBST, add 100 μL/well TMB chromogenic substrate, incubate at room temperature for 3-5 minutes, add 100 μL/well 1M H 2 SO 4 to terminate the reaction, and read the absorbance value at 450 nm with a NOVOStar microplate reader. , calculate the IC50 value of the antibody binding to the antigen. The ability of each antibody to inhibit the binding of EGF to EGFR is shown in Table 37 below:
表37.EGFR/CD28双抗抑制EGF与EGFR结合的能力
Table 37. The ability of EGFR/CD28 dual antibodies to inhibit the binding of EGF and EGFR
结果显示，本披露双特异性抗体均能抑制EGF与EGFR的结合。The results show that the bispecific antibodies of the present disclosure can inhibit the binding of EGF and EGFR.
测试例7.双特异性抗体的激活作用Test Example 7. Activation of bispecific antibodies
为测试本披露EGFR-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-IL2细胞的激活作用。计数仪计数CHO-APC-hEGFR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-IL2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。各双抗对Jurkat-IL2lum激活作用见下表38。In order to test the activation effect of the EGFR-CD28 bispecific antibody of the present disclosure on T cells, this test example detects the activation effect of the bispecific antibody on Jurkat-IL2 cells in the presence of the first activation signal of T cells. Use a counter to count CHO-APC-hEGFR cells. Dilute to 2E5/mL with non-resistant medium 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-IL2 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 effects of each dual antibody on Jurkat-IL2lum activation are shown in Table 38 below.
表38.双特异性抗体对Jurkat-IL2lum细胞的激活作用
Table 38. 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激活T细胞的 IL2信号通路。The results show that the bispecific antibody of the present disclosure can effectively activate T cells in the presence of the first signal. IL2 signaling pathway.
测试例8.双特异性抗体与PD1抗体联用的激活作用Test Example 8. Activation effect of bispecific antibody combined with PD1 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EGFR/CD28双特异性抗体与PD1/PDL1抗体联用在MHC-peptide/TCR信号存在时对T细胞的激活作用，本测试例检测了双特异性抗体在MHC/peptide复合物存在时对Jurkat-PD1细胞的激活作用。具体如下：Since the PD1/PDL1 signaling pathway blocks the costimulatory signal of CD28, in order to test the activation effect of the EGFR/CD28 bispecific antibody and PD1/PDL1 antibody disclosed in the present disclosure on T cells in the presence of MHC-peptide/TCR signals, this The test example detects the activation effect of bispecific antibodies on Jurkat-PD1 cells in the presence of MHC/peptide complexes. details as follows:
计数仪计数CHO-APC-EGFR-PDL1细胞，用没有抗性的培养基稀释到4E5/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5μg/mL的起始浓度，4倍稀释，共稀释8个点。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培养基稀释Jurkat-PD1细胞到5E5/mL，并加入10μg/mL的PD1抗体。吸掉细胞板中的抗体，加入100μL/孔稀释好的Jurkat-PD1细胞-PD1抗体混合物。37℃,CO2培养箱孵育5小时。加入50μL/孔荧光素酶底物，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-IL2-PD1的激活作用见下表39。Count CHO-APC-EGFR-PDL1 cells with a counter, dilute them to 4E5/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 antibody to a starting concentration of 5 μg/mL with culture medium supplemented with 10% FBS, 4-fold dilution, for a total of 8 dilutions. Discard the culture medium in the cell plate, then add 50 μL/well of the diluted antibody, and incubate for 1 hour in a 37°C CO2 incubator. Dilute Jurkat-PD1 cells to 5E5/mL with antibiotic-free 1640 medium, and add 10 μg/mL of PD1 antibody. Aspirate the antibodies in the cell plate and add 100 μL/well of the diluted Jurkat-PD1 cell-PD1 antibody mixture. Incubate in a 37°C, CO2 incubator for 5 hours. Add 50 μL/well of luciferase substrate, 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 on Jurkat-IL2-PD1 are shown in Table 39 below.
表39.双特异性抗体对Jurkat-PD1细胞的激活作用

注：N/A表示无激活作用。Table 39. Activation effect of bispecific antibodies on Jurkat-PD1 cells

Note: N/A means no activation.
结果显示，本披露双特异性抗体与PD1抗体联用能有效激活T细胞。The results show that the combination of the bispecific antibody of the present disclosure and the PD1 antibody can effectively activate T cells.
测试例9.双特异性抗体的在MHC-肽-TCR第一信号存在下的杀伤作用Test Example 9. Killing effect of bispecific antibodies in the presence of MHC-peptide-TCR first signal
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EGFR的细胞系CHO-APC-hEGFR细胞的作为靶细胞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 killing effect of the disclosed bispecific antibody in the presence of the first T cell stimulation signal. The target-specific cytotoxic activity of the bispecific antibodies of the present disclosure was tested using the EGFR-expressing cell line CHO-APC-hEGFR cells as target cells.
CHO-APC-hEGFR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(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-hEGFR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200nM，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)。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：
杀伤百分比％＝100×实验组LDH释放量/100％裂解值。CHO-APC-hEGFR 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, resuspended in phenol red-free 1640 (Gibco, 11835-030) supplemented with 4% FBS, centrifuged again, resuspended, counted, and adjusted. The cell number is 7.5E5 cells/mL, and 50 μL is added to each well. Digest CHO-APC-hEGFR 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 200 nM with phenol red-free 1640+4% FBS medium, diluted 5 times, and 50 μL was added 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). 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:
Killing percentage % = 100 × LDH release amount of experimental group / 100% lysis value.
各抗体联用的杀伤作用如下表。The killing effect of each antibody combination is shown in the table below.
表40.EGFR/CD28双抗对细胞的杀伤作用
Table 40. Killing effect of EGFR/CD28 double antibodies on cells
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EGFR的细胞表现出较强杀伤作用。The results showed that in the presence of the first TCR activation signal, the bispecific antibody of the present disclosure showed a strong killing effect on cells expressing EGFR.
测试例10.双特异性抗体与MUC16/CD3联用的体外杀伤作用Test Example 10. In vitro killing effect of bispecific antibody combined with MUC16/CD3
本测试例研究了本披露的双特异性抗体与MUC16/CD3双抗联用介导的杀伤作用。用表达MUC16的细胞系OVCAR3细胞的作为靶细胞来检测本披露的双特异性抗体与MUC16/CD3联用的细胞毒活性。This test case studies the killing effect mediated by the combination of the disclosed bispecific antibody and the MUC16/CD3 dual antibody. The MUC16-expressing cell line OVCAR3 cells were used as target cells to detect the cytotoxic activity of the bispecific antibody of the present disclosure in combination with MUC16/CD3.
OVCAR3细胞(ATCC)培养在1640完全培养基中，冻存PBMC复苏后用1640+10％FBS完全培养基重悬，置于T75培养瓶培养4小时(密度为3E6细胞/mL)。PBMC收集离心后用无酚红1640+4％FBS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。OVCAR3细胞消化，1000rpm离心3分钟，重悬，计数，调整细胞数为3E5细胞/mL，每孔加入50μL，确保E:T Ratio为10:1。抗体用无酚红1640+4％FBS培养基稀释。OVCAR3 cells (ATCC) were cultured in 1640 complete medium. After recovery, the frozen PBMC were resuspended in 1640+10% FBS complete medium and placed in a T75 culture flask for 4 hours (density: 3E6 cells/mL). Collect PBMC and resuspend them in phenol red-free 1640+4% FBS after centrifugation. Centrifuge again and resuspend and count. Adjust the cell number to 1.5E6 cells/mL and add 50 μL to each well. Digest OVCAR3 cells, centrifuge at 1000 rpm for 3 minutes, resuspend, count, adjust the number of cells to 3E5 cells/mL, add 50 μL to each well, and ensure that the E:T Ratio is 10:1. Antibodies were diluted in phenol red-free 1640 + 4% FBS medium.
固定MUC16/CD3抗体浓度为20pM，EGFR/CD28双抗起始浓度为200nM，6倍稀释，9个剂量点。每孔加入MUC16/CD3及EGFR/CD28各25μL。The fixed MUC16/CD3 antibody concentration is 20pM, the starting concentration of EGFR/CD28 double antibody is 200nM, 6-fold dilution, and 9 dose points. Add 25 μL each of MUC16/CD3 and EGFR/CD28 to each well.
固定EGFR/CD28抗体浓度为400pM，MUC16/CD3双抗起始浓度为60nM，6倍稀释，9个剂量点。每孔加入MUC16/CD3及EGFR/CD28各25μL。The fixed EGFR/CD28 antibody concentration is 400pM, the starting concentration of MUC16/CD3 double antibody is 60nM, 6-fold dilution, and 9 dose points. Add 25 μL each of MUC16/CD3 and EGFR/CD28 to each well.
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裂解缓冲液(10X)，裂 解45分钟。取出培养板，吸取90μL上清于新的96孔板，可使用CTG检测PBMC的增殖。在原来含有细胞的板子中，加入50μL基础培养基(1640+10％FBS)，按1:1的比例加入50μL one-Glo，室温孵育5分钟，检测发光的数值。各抗体联用的杀伤作用如下表。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 buffer (10X), and lyse. Solution 45 minutes. Take out the culture plate and pipet 90 μL of supernatant into a new 96-well plate. CTG can be used to detect the proliferation of PBMC. In the original plate containing cells, add 50 μL of basic medium (1640+10% FBS), add 50 μL of one-Glo at a ratio of 1:1, incubate at room temperature for 5 minutes, and detect the luminescence value. The killing effect of each antibody combination is shown in the table below.
表41.固定MUC16/CD3，与EGFR/CD28联用的细胞杀伤作用

注：N/A表示无杀伤作用。Table 41. Cell killing effect of fixed MUC16/CD3 and combination with EGFR/CD28

Note: N/A means no killing effect.
表42.固定EGFR/CD28，与MUC16/CD3联用的细胞杀伤作用

注：N/A表示无杀伤作用。Table 42. Cell killing effect of fixed EGFR/CD28 in combination with MUC16/CD3

Note: N/A means no killing effect.
结果显示，本披露EGFR/CD28双特异性抗体能协同低剂量的MUC16/CD3双抗发挥对表达MUC16的细胞更强的杀伤作用。The results show that the disclosed EGFR/CD28 bispecific antibody can cooperate with a low-dose MUC16/CD3 bispecific antibody to exert a stronger killing effect on MUC16-expressing cells.
测试例11.双特异性抗体与MUC16/CD3联用的细胞因子释放Test Example 11. Cytokine release using bispecific antibody in combination with MUC16/CD3
本测试例研究了本披露的双特异性抗体在MUC16/CD3双抗、EGFR/CD28双抗和OVCAR3细胞的共同作用下，PBMC中细胞因子TNF-α及IFN-γ分泌的变化。取出测试例10的细胞杀伤实验中收集的冻存的上清，常温解冻，震荡混匀，抗体使用1640+4％FBS培养基稀释20倍备用。使用无菌水溶解TNF-α及IFNγ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，平衡TNF-α试剂盒(62HTNFAPEG，cisbio)和IFNγ试剂盒(62HIFNGPEG，cisbio)至常温，使用检测缓冲液稀释试剂盒中的两个检测抗体(20倍稀释)。取出孵育过夜的抗体，1000rpm离心1分钟，使用PHERAstar多功能酶标仪读取665nm和620nm的吸收值。结果图2A-图2D所示。This test case studies the changes in the secretion of cytokines TNF-α and IFN-γ in PBMCs under the combined action of the disclosed bispecific antibodies, MUC16/CD3 double antibodies, EGFR/CD28 double antibodies and OVCAR3 cells. Take out the frozen supernatant collected in the cell killing experiment of Test Example 10, thaw it at room temperature, shake and mix, and dilute the antibody 20 times with 1640+4% FBS culture medium for later use. Use sterile water to dissolve TNF-α and IFNγ standard powder, and use 1640+4% FBS culture medium to dilute the standards to the required concentration. Take out the kit of the cytokine to be detected, balance the TNF-α kit (62HTNFAPEG, cisbio) and IFNγ kit (62HIFNGPEG, cisbio) to room temperature, and use the detection buffer to dilute the two detection antibodies in the kit (20-fold dilution ). Remove the antibody 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 Figure 2A-Figure 2D.
结果显示，本披露的双特异性抗体与MUC16/CD3双抗联用后，PBMC的细胞因子TNF-α和IFNγ的释放水平较低。The results showed that after the bispecific antibody of the present disclosure was combined with the MUC16/CD3 dual antibody, the release levels of cytokines TNF-α and IFNγ from PBMC were lower.
体内活性生物学评价Biological evaluation of in vivo activity
测试例12.双特异性抗体在A431皮下移植瘤模型中的药效 Test Example 12. The efficacy of bispecific antibodies in the A431 subcutaneous xenograft tumor model
本测试例使用人皮肤癌A431细胞人PBMC NOG小鼠异种移植瘤模型，对EGFR/CD28双抗单用在抗肿瘤活性方面进行评价。This test example uses the human skin cancer A431 cell human PBMC NOG mouse xenograft tumor model to evaluate the anti-tumor activity of the EGFR/CD28 dual antibody alone.
50只雌性NOG小鼠，6-8周龄，购自北京维通利华。PBMC购于妙顺商业。50 female NOG mice, 6-8 weeks old, were purchased from Beijing Vitong Lever. PBMC was purchased from Miaoshun Commercial.
将A431细胞和PBMC细胞以1.5:1的比例在体外混合均匀，按照(3E6A431+2E6PBMC)/200μL/只(含50％人工基底膜)的接种量接种于50只NOG小鼠右肋部皮下，将接种当天定义为该实验Day 0(第零天)，接种后Day1将小鼠按照体重随机分为4组，每组10只，并按照表43腹腔给药各抗体药物，每周2次，共给药7次。肿瘤接种7天后，开始每周两次监测肿瘤体积、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。
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。A431 cells and PBMC cells were mixed evenly in vitro at a ratio of 1.5:1, and inoculated subcutaneously into the right ribs of 50 NOG mice at an inoculation volume of (3E6A431+2E6PBMC)/200 μL/mouse (containing 50% artificial basement membrane). The day of vaccination was defined as Day 0 of the experiment. On Day 1 after vaccination, the mice were randomly divided into 4 groups according to body weight, with 10 mice in each group. Each antibody drug was administered intraperitoneally according to Table 43, twice a week. A total of 7 doses were administered. Seven days after tumor inoculation, 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.
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。
抑瘤率TGI(％)＝1-T/C(％)。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.
Tumor inhibition rate TGI(%)=1-T/C(%).
表43.EGFR/CD28双抗单用的抗肿瘤活性
Table 43. Anti-tumor activity of EGFR/CD28 dual antibodies used alone
结果显示，本披露的双特异性抗体在单独使用时有较强的抑制肿瘤生长的作用。The results show that the bispecific antibodies disclosed herein have a strong inhibitory effect on tumor growth when used alone.
测试例13.双特异性抗体与PD-1抗体联用在A431皮下移植瘤模型中的药效Test Example 13. The efficacy of the combination of bispecific antibody and PD-1 antibody in the A431 subcutaneous xenograft tumor model
本测试例使用人皮肤癌A431细胞人PBMC NOG小鼠异种移植瘤模型，对EGFR/CD28双抗与PD1抗体联用在抗肿瘤活性方面进行评价。This test example uses the human skin cancer A431 cell human PBMC NOG mouse xenograft tumor model to evaluate the anti-tumor activity of the combination of EGFR/CD28 dual antibodies and PD1 antibodies.
90只雌性NOG小鼠，6-8周龄，购自北京维通利华。PBMC购于妙顺商业。90 female NOG mice, 6-8 weeks old, were purchased from Beijing Vitong Lever. PBMC was purchased from Miaoshun Commercial.
将A431细胞和PBMC细胞以1.5:1的比例在体外混合均匀，按照(3E6A431+2E6PBMC)/200μL/只(含50％人工基底膜)的接种量接种于90只NOG小鼠右肋部皮下，将接种当天定义为该实验Day0(第零天)，接种后Day3将小鼠按照体重随机分为8组，每组10只，并按照表44腹腔给药各抗体，每周2次，共给药7次。肿瘤接种7天后，开始每周两次监测肿瘤体积、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。A431 cells and PBMC cells were mixed evenly in vitro at a ratio of 1.5:1, and inoculated subcutaneously into the right ribs of 90 NOG mice at an inoculation volume of (3E6A431+2E6PBMC)/200 μL/mouse (containing 50% artificial basement membrane). The day of vaccination was defined as Day 0 (day zero) of the experiment. On Day 3 after vaccination, the mice were randomly divided into 8 groups according to body weight, with 10 mice in each group. Each antibody was administered intraperitoneally according to Table 44, twice a week, for a total of Take medicine 7 times. Seven days after tumor inoculation, 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。
抑瘤率TGI(％)＝1-T/C(％)。Relative tumor proliferation rate T/C (%) = (T-T0)/(C-C0)×100%, where T and C are the treatments at the end of the experiment The tumor volumes of the group and the control group; T0 and C0 are the tumor volumes at the beginning of the experiment.
Tumor inhibition rate TGI(%)=1-T/C(%).
表44.EGFR/CD28双抗与PD1抗体联用的抗肿瘤活性
Table 44. Anti-tumor activity of EGFR/CD28 dual antibody combined with PD1 antibody
结果显示，与对照组相比，EGFR/CD28双特异性抗体单独给药时，97H2L3OT-ZAL和94H6L12-ZALOT均显示出了较好的抑瘤效果，优于REGN7075V。PD-1抗体单独给药在该模型上没有抑瘤作用，但其与EGFR/CD28双抗分子联合给药时显示出了较好的协同作用，所有联用组的药效均明显优于其单独给药组。The results showed that compared with the control group, when the EGFR/CD28 bispecific antibodies were administered alone, both 97H2L3OT-ZAL and 94H6L12-ZALOT showed better tumor inhibition effects, which were better than REGN7075V. The PD-1 antibody alone has no anti-tumor effect in this model, but it shows a good synergistic effect when combined with the EGFR/CD28 dual-antibody molecule. The efficacy of all combination groups is significantly better than that of the other two groups. Alone dosing group.
测试例14.双特异性抗体在NCI-H292皮下移植瘤模型中的药效Test Example 14. The efficacy of bispecific antibodies in the NCI-H292 subcutaneous xenograft tumor model
本披露使用人肺癌NCI-H292细胞人PBMC NOG小鼠异种移植瘤模型，对EGFR/CD28双抗与PD1抗体联用在抗肿瘤活性方面进行评价。This disclosure uses the human lung cancer NCI-H292 cell human PBMC NOG mouse xenograft tumor model to evaluate the anti-tumor activity of the combination of EGFR/CD28 dual antibodies and PD1 antibodies.
84只雌性NOG小鼠，8-10周龄，购自北京维通利华。84 female NOG mice, 8-10 weeks old, were purchased from Beijing Vitong Lever.
NCI-H292细胞培养于含10％FBS的RPMI-1640培养基，维持在5％CO2的37℃饱和湿度培养箱中。收集对数生长期NCI-H292细胞，计数后备用。复苏液氮冻存的hPBMC，培养于含10％HIFBS(FBS，56℃30分钟热灭活)的PRMI-1640培养基中，在含5％CO2的37℃培养箱中孵育6小时。收集孵育后hPBMC，计数后按E/T比值(2:4)加入相应数量hPBMC至NCI-H292细胞悬液中。离心后重悬于含50％人工基底膜的PRMI-1640基础培养基中，调整NCI-H292细胞浓度至4E7/mL。在无菌条件下，接种0.1mL细胞悬液至小鼠右侧背部皮下，接种细胞浓度为4E6/0.1mL/只。接种后7天，将动物按体重随机分组，使各组动物体重差异小于均值的10％。分组当日记为Day 0(第0天)，并按照动物体重开始给药。给药期间，个别动物体重与Day 0相比下降超过15％(BWL≥15％)，将做停药处理，直至动物体重恢复后(BWL<15％)，恢复给药。NCI-H292 cells were cultured in RPMI-1640 medium containing 10% FBS and maintained in a 37°C saturated humidity incubator with 5% CO2 . Collect NCI-H292 cells in the logarithmic growth phase and count them for later use. The hPBMC frozen in liquid nitrogen were resuscitated, cultured in PRMI-1640 medium containing 10% HIFBS (FBS, heat inactivated at 56°C for 30 minutes), and incubated for 6 hours in a 37°C incubator containing 5% CO2 . Collect hPBMC after incubation. After counting, add the corresponding number of hPBMC to the NCI-H292 cell suspension according to the E/T ratio (2:4). After centrifugation, the cells were resuspended in PRMI-1640 basic medium containing 50% artificial basement membrane, and the NCI-H292 cell concentration was adjusted to 4E7/mL. Under sterile conditions, inoculate 0.1 mL of cell suspension subcutaneously on the right back of the mouse at a concentration of 4E6/0.1 mL/mouse. Seven days after inoculation, the animals were randomly divided into groups according to body weight, so that the difference in body weight of animals in each group was less than 10% of the mean. The day of grouping was recorded as Day 0 (Day 0), and administration was started according to the animal's body weight. During the dosing period, if the body weight of individual animals decreases by more than 15% compared with Day 0 (BWL ≥ 15%), the drug will be discontinued until the animal's body weight recovers (BWL < 15%), and the dosing will be resumed.
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。
抑瘤率TGI(％)＝1-T/C(％)。
CR率(％)＝(同一组中完全缓解小鼠只数/小鼠总数)×100％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.
Tumor inhibition rate TGI(%)=1-T/C(%).
CR rate (%) = (number of mice with complete remission in the same group/total number of mice) × 100%
表45.EGFR/CD28双抗与PD1抗体联用的抗肿瘤活性
Table 45. Anti-tumor activity of EGFR/CD28 dual antibody combined with PD1 antibody
结果显示，EGFR/CD28双特异性抗体单独给药时，97H2L3OT-ZAL与REGN7075V显示出了较弱的抑瘤效果，两个抗体之间无统计学差异。PD-1抗体单独给药有两只小鼠出现了肿瘤消退，63天的TGI为47％。PD-1抗体与EGFR/CD28双抗联合给药时显示出了较好的协同作用，97H2L3OT-ZAL联用组TGI为87.2％，优于REGN7075V联用组；CR率显示出更显著的差异，REGN7075V联用组CR率为43％，97H2L3OT-ZAL联用组CR率高达86％，7只小鼠中有6只出现了肿瘤消退。The results showed that when EGFR/CD28 bispecific antibodies were administered alone, 97H2L3OT-ZAL and REGN7075V showed weak tumor inhibitory effects, and there was no statistical difference between the two antibodies. Two mice given the PD-1 antibody alone experienced tumor regression, with a TGI of 47% at 63 days. The combined administration of PD-1 antibodies and EGFR/CD28 dual antibodies showed good synergy. The TGI of the 97H2L3OT-ZAL combination group was 87.2%, which was better than the REGN7075V combination group; the CR rate showed a more significant difference. The CR rate of the REGN7075V combination group was 43%, and the CR rate of the 97H2L3OT-ZAL combination group was as high as 86%. Tumor regression occurred in 6 out of 7 mice.
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 (24)

  1. 一种抗原结合分子，其特异性结合CD28和EGFR，其中所述的抗原结合分子包含：An antigen-binding molecule that specifically binds CD28 and EGFR, wherein the antigen-binding molecule includes:
    至少一个特异性结合CD28的抗原结合模块、和at least one antigen-binding module that specifically binds CD28, and
    至少一个特异性结合EGFR的抗原结合模块，at least one antigen-binding module that specifically binds EGFR,
    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，The antigen-binding module that specifically binds to CD28 includes a heavy chain variable region CD28-VH and a light chain variable region CD28-VL,
    所述特异性结合EGFR的抗原结合模块包含重链可变区EGFR-VH和轻链可变区EGFR-VL；The antigen-binding module that specifically binds to EGFR includes a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL;
    其中：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
    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, 91, CD28-LCDR2 with the amino acid sequence 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
    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The CD28-VL has: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, CD28 comprising the amino acid sequence of SEQ ID NO: 62, 17, 54, 55, 56, 57, 58, 59, 60 or 61 -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 sequence of CD28-HCDR3; and
    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-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 comprising SEQ ID NO : CD28-LCDR3 with an amino acid sequence of 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：39、9、40、41或42的氨基酸序列的CD28-HCDR3；和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: 39, 9, 40, 41 or CD28-HCDR3 having an amino acid sequence of 42; and
    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； 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 or 11, and CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 12 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
    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：84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；The CD28-VH has: CD28-HCDR1 including the amino acid sequence of SEQ ID NO: 19, CD28-HCDR2 including the amino acid sequence of SEQ ID NO: 20, and CD28-HCDR3 including the amino acid sequence of SEQ ID NO: 21; and the CD28-VL having: CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, CD28-LCDR2 comprising the amino acid sequence of SEQ ID NO: 84, and CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 24 ;
    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：39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: 39 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
    所述EGFR-VH具有：包含SEQ ID NO：160的氨基酸序列的EGFR-HCDR1，包含SEQ ID NO：161的氨基酸序列的EGFR-HCDR2，和包含SEQ ID NO：162的氨基酸序列的EGFR-HCDR3；和The EGFR-VH has: EGFR-HCDR1 comprising the amino acid sequence of SEQ ID NO: 160, EGFR-HCDR2 comprising the amino acid sequence of SEQ ID NO: 161, and EGFR-HCDR3 comprising the amino acid sequence of SEQ ID NO: 162; and
    所述EGFR-VL具有：包含SEQ ID NO：163的氨基酸序列的EGFR-LCDR1，包含SEQ ID NO：164的氨基酸序列的EGFR-LCDR2，和包含SEQ ID NO：165 的氨基酸序列的EGFR-LCDR3。The EGFR-VL has: EGFR-LCDR1 comprising the amino acid sequence of SEQ ID NO: 163, EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 164, and EGFR-LCDR2 comprising the amino acid sequence of SEQ ID NO: 165 Amino acid sequence of EGFR-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：46、44、31、45、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: 46, 44, 31, 45, 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，i) said CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98, and said CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102,
    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 SEQ ID NO: 70, 71, 72, 74, 75, 76 or 79 amino acid sequence, 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：46的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52或50的氨基酸序列，或iv) 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 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、50或51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, 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：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
    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
    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：46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: 46, 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，其中所述EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述EGFR-VL包含SEQ ID NO：159的氨基酸序列。The antigen-binding molecule according to any one of claims 1 to 3, wherein the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 158, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159.
  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EGFR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 EGFR 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：218至SEQ ID NO：236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: 218 to SEQ ID NO: 236,
    所述Obscurin链包含选自由SEQ ID NO：237至SEQ ID NO：277组成的组的氨基酸序列；The Obscurin chain includes an amino acid sequence selected from the group consisting of SEQ ID NO: 237 to SEQ ID NO: 277;
    更优选地，More preferably,
    所述Titin链包含SEQ ID NO：234的氨基酸序列，和所述Obscurin链包含SEQ ID NO：272的氨基酸序列。The Titin chain includes the amino acid sequence of SEQ ID NO: 234, and the Obscurin chain includes the amino acid sequence of SEQ ID NO: 272.
  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包含Fc区，所述Fc区优选为人IgG Fc区，进一步优选为人IgG1 Fc区；The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule comprises an Fc region, and the Fc region is preferably a human IgG Fc region, and further preferably a human 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位置的氨基酸残基分别为A，编号依据为EU索引。Most preferably, the Fc region is a human IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A respectively, and the numbering basis is the EU index.
  7. 根据权利要求1至6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；The antigen-binding molecule according to any one of claims 1 to 6, wherein the antigen-binding molecule includes an Fc region, the Fc region includes a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region;
    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 The amino acid 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：167的氨基酸序列；和所述Fc2包含SEQ ID NO：168的氨基酸序列。Most preferably, said Fc1 comprises the amino acid sequence of SEQ ID NO: 167; and said Fc2 comprises the amino acid sequence of SEQ ID NO: 168.
  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EGFR的抗原结合模块，所述特异性结合EGFR的抗原结合模块是Fab；The antigen-binding molecule according to claim 7, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds CD28 and an antigen-binding module that specifically binds EGFR, and the antigen-binding module that specifically binds EGFR is Fab. ;
    所述特异性结合CD28的抗原结合模块是经替换的Fab，并且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 replaced Fab and 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 second chain having a structure represented by formula (b), a third chain having a structure represented by formula (c), and a third chain having a structure represented by formula (c). The fourth chain of the structure shown in formula (d),
    式(a)[CD28-VH]-[连接子1]-[Titin链]-[Fc1]，Formula (a) [CD28-VH]-[linker 1]-[Titin chain]-[Fc1],
    式(b)[CD28-VL]-[连接子2]-[Obscurin链]，Formula (b) [CD28-VL]-[linker 2]-[Obscurin chain],
    式(c)[EGFR-VH]-[CH1]-[Fc2]，Formula (c) [EGFR-VH]-[CH1]-[Fc2],
    式(d)[EGFR-VL]-[CL]，Formula (d)[EGFR-VL]-[CL],
    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；或所述连接子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;
    所述Fc1和所述Fc2可互换；The Fc1 and the Fc2 are interchangeable;
    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：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EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述的EGFR-VL包含SEQ ID NO：159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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159; or
    ii)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EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述的EGFR-VL包含SEQ ID NO：159的氨基酸序列；ii) 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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL includes the amino acid sequence of SEQ ID NO: 159;
    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：174的氨基酸序列的第一链、一条包含SEQ ID NO：176的氨基酸序列的第二链、一条包含SEQ ID NO：180的氨基酸序列的第三链和一条包含SEQ ID NO：182的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 174, a second strand comprising the amino acid sequence of SEQ ID NO: 176, a third strand comprising the amino acid sequence of SEQ ID NO: 180 and a third strand comprising the amino acid sequence of SEQ ID NO: 174 : The fourth strand of the amino acid sequence 182; or
    一条包含SEQ ID NO：178的氨基酸序列的第一链、一条包含SEQ ID NO：179的氨基酸序列的第二链、一条包含SEQ ID NO：180的氨基酸序列的第三链和一条包含SEQ ID NO：182的氨基酸序列的第四链。 A first strand comprising the amino acid sequence of SEQ ID NO: 178, a second strand comprising the amino acid sequence of SEQ ID NO: 179, a third strand comprising the amino acid sequence of SEQ ID NO: 180 and a third strand comprising the amino acid sequence of SEQ ID NO : The fourth strand of the amino acid sequence of 182.
  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EGFR的抗原结合模块，所述特异性结合EGFR的抗原结合模块是经替换的Fab，并且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 CD28 and an antigen-binding module that specifically binds EGFR, and the antigen-binding module that specifically binds EGFR is Replaced Fab and contains Titin chain and Obscurin chain capable of forming dimers;
    所述特异性结合CD28的抗原结合模块是Fab；The antigen-binding module that specifically binds CD28 is Fab;
    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),
    式(e)[CD28-VH]-[CH1]-[Fc1]，Formula (e)[CD28-VH]-[CH1]-[Fc1],
    式(f)[CD28-VL]-[CL]，Formula (f)[CD28-VL]-[CL],
    式(g)[EGFR-VH]-[连接子3]-[Obscurin链]-[Fc2]，Formula (g) [EGFR-VH]-[linker 3]-[Obscurin chain]-[Fc2],
    式(h)[EGFR-VL]-[连接子4]-[Titin链]，Formula (h) [EGFR-VL]-[linker 4]-[Titin chain],
    其中：所述连接子3和所述连接子4是相同或不同的肽连接子；或所述连接子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;
    所述Fc1和所述Fc2可互换；The Fc1 and the Fc2 are interchangeable;
    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：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EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述的EGFR-VL包含SEQ ID NO：159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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159; or
    ii)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EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述的EGFR-VL包含SEQ ID NO：159的氨基酸序列；或ii) 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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 159; 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EGFR-VH包含SEQ ID NO：158的氨基酸序列，和所述的EGFR-VL包含SEQ ID NO：159的氨基酸序列；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 EGFR-VH includes the amino acid sequence of SEQ ID NO: 158 sequence, and the EGFR-VL includes the amino acid sequence of SEQ ID NO: 159;
    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：172的氨基酸序列的第一链、一条包含SEQ ID NO：173的氨基酸序列的第二链、一条包含SEQ ID NO：181的氨基酸序列的第三链和一条包含SEQ ID NO：183的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 172, a second strand comprising the amino acid sequence of SEQ ID NO: 173, a third strand comprising the amino acid sequence of SEQ ID NO: 181 and a third strand comprising the amino acid sequence of SEQ ID NO: 172 : The fourth strand of the amino acid sequence 183; or
    一条包含SEQ ID NO：170的氨基酸序列的第一链、一条包含SEQ ID NO：171的氨基酸序列的第二链、一条包含SEQ ID NO：181的氨基酸序列的第三链和一条包含SEQ ID NO：183的氨基酸序列的第四链；或A first strand comprising the amino acid sequence of SEQ ID NO: 170, a second strand comprising the amino acid sequence of SEQ ID NO: 171, a third strand comprising the amino acid sequence of SEQ ID NO: 181 and a third strand comprising the amino acid sequence of SEQ ID NO: 171 : The fourth strand of the amino acid sequence 183; or
    一条包含SEQ ID NO：175的氨基酸序列的第一链、一条包含SEQ ID NO： 177的氨基酸序列的第二链、一条包含SEQ ID NO：181的氨基酸序列的第三链和一条包含SEQ ID NO：183的氨基酸序列的第四链。One contains the first strand of the amino acid sequence of SEQ ID NO: 175, and one contains the first strand of the amino acid sequence of SEQ ID NO: 175. A second strand comprising the amino acid sequence of SEQ ID NO: 177, a third strand comprising the amino acid sequence of SEQ ID NO: 181 and a fourth strand comprising the amino acid sequence of SEQ ID NO: 183.
  10. 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：An anti-CD28 antibody that can specifically bind to CD28. The anti-CD28 antibody includes a heavy chain variable region CD28-VH and a light chain variable region CD28-VL, 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, comprising SEQ ID NO: 23, 81, 82, 83, 84, 85, 86, 87, 88, 89, CD28-LCDR2 with 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 has: a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 16, a CD28-LCDR1 comprising the amino acid sequence of SEQ ID NO: 62, 17, 54, 55, 56, 57, 58, 59, 60 or 61 The amino acid 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 CD28-HCDR2 comprising the amino acid sequence of SEQ ID NO: 27 the amino acid sequence of CD28-HCDR3; and the CD28-VL having: 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：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, a CD28-HCDR3 having an amino acid sequence of 41 or 42; and the CD28-VL having: 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 CD28-LCDR3 comprising the amino acid sequence of SEQ ID NO: 12;
    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-HCDR2 comprising the amino acid sequence of SEQ ID NO: 15 the amino acid sequence of CD28-HCDR3; and the CD28-VL having: 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-LCDR2 comprising the amino acid sequence of SEQ ID NO: 18 The amino acid sequence of CD28-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CD28抗体在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 anti-CD28 antibody binds human CD28 with a KD of less than 2×10 -8 M at 25°C, as measured by surface plasmon resonance.
  11. 根据权利要求10所述的抗CD28抗体，其中：The anti-CD28 antibody according to claim 10, 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：46、44、31、45、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: 46, 44, 31, 45, 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，i) said CD28-VH comprises the amino acid sequence of SEQ ID NO: 95, 94, 96, 97 or 98, and said CD28-VL comprises the amino acid sequence of SEQ ID NO: 101, 99, 100 or 102,
    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 comprises the amino acid sequence of SEQ ID NO: 94, and the CD28-VL comprises SEQ The amino acid sequence of ID NO: 99, 100, 101, 103, 104, 105, 106, 107, 108, 109, 110, 111, 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：46的氨基酸序列，和所述CD28-VL包含SEQ ID NO：52或50的氨基酸序列，或iv) 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 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、50或51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, 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：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：46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: 46, and the CD28-VL comprises the amino acid sequence of SEQ ID NO: 52.
  12. 根据权利要求10或11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；优选地，所述重链恒定区为人IgG1恒定区。The anti-CD28 antibody according to claim 10 or 11, wherein the anti-CD28 antibody comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is a human IgG1 constant region.
  13. 根据权利要求12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：The anti-CD28 antibody according to claim 12, wherein the anti-CD28 antibody comprises a heavy chain and a light chain, wherein:
    所述重链包含与SEQ ID NO：150具有至少85％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％序列同一性的氨基酸序列；或The heavy chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 150, and the light chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 151; or
    所述重链包含与SEQ ID NO：146具有至少85％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％序列同一性的氨基酸序列；或The heavy chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 146, and the light chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 147; or
    所述重链包含与SEQ ID NO：148具有至少85％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％序列同一性的氨基酸序列；或The heavy chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 148, and the light chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 149; or
    所述重链包含与SEQ ID NO：152具有至少85％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％序列同一性的氨基酸序列；The heavy chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 152, and the light chain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 153;
    优选地，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Preferably, 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：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 comprises the amino acid sequence of SEQ ID NO: 146, and the light chain comprises SEQ ID NO: Amino acid sequence of 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：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.
  14. 根据权利要求10至11中任一项所述的抗CD28抗体，其中所述的抗CD28抗体是抗体片段；优选地，所述的抗体片段为Fab、Fab’、F(ab')2、Fd、Fv、scFv、dsFv或dAb。The anti-CD28 antibody according to any one of claims 10 to 11, wherein the anti-CD28 antibody is an antibody fragment; preferably, the antibody fragment is Fab, Fab', F(ab')2, Fd , Fv, scFv, dsFv or dAb.
  15. 根据权利要求10至14中任一项所述的抗CD28抗体，其中所述的抗CD28抗体是双特异性抗体；The anti-CD28 antibody according to any one of claims 10 to 14, wherein the anti-CD28 antibody is a bispecific antibody;
    优选地，所述双特异性抗体特异性结合CD28和EGFR。Preferably, the bispecific antibody specifically binds CD28 and EGFR.
  16. 一种药物组合物，其含有：A pharmaceutical composition containing:
    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至15中任一项所述的抗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；A therapeutically effective amount of the antigen-binding molecule of any one of claims 1 to 9, or the anti-CD28 antibody of any one of claims 10 to 15, 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至少一种第二治疗剂；Preferably, the pharmaceutical composition also contains at least one second therapeutic agent;
    更优选地，所述第二治疗剂是能够特异性结合CD3的抗体或抗PD-1抗体。More preferably, the second therapeutic agent is an antibody capable of specifically binding CD3 or an anti-PD-1 antibody.
  17. 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或权利要求10至15中任一项所述的抗CD28抗体。An isolated nucleic acid encoding the antigen-binding molecule of any one of claims 1 to 9 or the anti-CD28 antibody of any one of claims 10 to 15.
  18. 一种宿主细胞，其包含如权利要求17所述分离的核酸。A host cell comprising the isolated nucleic acid of claim 17.
  19. 一种治疗过度增殖性疾病的方法，所述方法包括：A method of treating hyperproliferative diseases, the method comprising:
    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至15中任一项所述的抗CD28抗体，或权利要求16所述的药物组合物；Administering to a subject a therapeutically effective amount of the antigen-binding molecule of any one of claims 1 to 9, or the anti-CD28 antibody of any one of claims 10 to 15, or the medicament of claim 16 combination;
    优选地，所述过度增殖性疾病是癌症；Preferably, the hyperproliferative disease is cancer;
    更优选地，所述的癌症选自以下任一项：肺癌、肺腺癌、非小细胞肺癌、肺鳞状细胞癌、食管癌、宫颈鳞状细胞癌、子宫内膜癌、子宫内膜腺癌、膀胱癌、膀胱尿路上皮癌、结直肠癌、头颈癌、头颈部鳞状细胞癌、神经胶质瘤、脑癌、多形性胶质母细胞瘤、乳腺癌、胃癌、胃食管癌、胃食管腺癌、前列腺癌、卵巢 癌、肾癌、肝癌、胰腺癌、黑色素瘤、骨肉瘤和皮肤癌；More preferably, the cancer is selected from any one of the following: lung cancer, lung adenocarcinoma, non-small cell lung cancer, lung squamous cell carcinoma, esophageal cancer, cervical squamous cell carcinoma, endometrial cancer, endometrial gland Cancer, bladder cancer, bladder urothelial cancer, colorectal cancer, head and neck cancer, head and neck squamous cell carcinoma, glioma, brain cancer, glioblastoma multiforme, breast cancer, gastric cancer, gastroesophageal cancer Cancer, gastroesophageal adenocarcinoma, prostate cancer, ovary cancer, kidney cancer, liver cancer, pancreatic cancer, melanoma, osteosarcoma, and skin cancer;
    最优选地，其中所述的癌症是表达EGFR的癌症。Most preferably, wherein said cancer is an EGFR-expressing cancer.
  20. 根据权利要求19所述的治疗过度增殖性疾病的方法，所述方法进一步包括施用第二治疗剂；The method of treating a hyperproliferative disease according to claim 19, further comprising administering a second therapeutic agent;
    所述的抗原结合分子和所述第二治疗剂同时施用、序贯施用或分开施用；The antigen-binding molecule and the second therapeutic agent are administered simultaneously, sequentially, or separately;
    优选地，所述第二治疗剂选自以下任一项：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；Preferably, the second therapeutic agent is selected from any one of the following: anti-tumor agents, radiotherapy, antibody-drug conjugates, bispecific antibodies, bispecific antibodies conjugated with anti-tumor agents, immune checkpoint inhibition agent or combination thereof;
    更优选地，所述的第二治疗剂选自以下任一项：PD-1抑制剂、抗PD-1抗体、PD-L1抑制剂、抗PD-L1抗体、与肿瘤靶抗原和CD3结合的双特异性抗体、铂抗癌化学治疗剂、紫杉醇、多西他赛(docetaxel)、长春新碱(vincristine)、顺铂(cisplatin)、卡铂(carboplatin)、奥沙利铂(oxaliplatin)、抗癌抗体、曲妥单抗(trastuzumab)、西妥昔单抗(cetuximab)、贝伐单抗(bevacizumab)和西米普利单抗(cemiplimab)。More preferably, the second therapeutic agent is selected from any one of the following: PD-1 inhibitors, anti-PD-1 antibodies, PD-L1 inhibitors, anti-PD-L1 antibodies, and tumor target antigens and CD3. Bispecific antibodies, platinum anticancer chemotherapeutics, paclitaxel, docetaxel, vincristine, cisplatin, carboplatin, oxaliplatin, anti- cancer antibodies, trastuzumab, cetuximab, bevacizumab and cemiplimab.
  21. 根据权利要求20所述的治疗过度增殖性疾病的方法，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 treating hyperproliferative diseases according to claim 20, 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：206的氨基酸序列的PD-1-HCDR1，包含SEQ ID NO：207的氨基酸序列的PD-1-HCDR2，和包含SEQ ID NO：208的氨基酸序列的PD-1-HCDR3；和所述PD-1-VL具有：包含SEQ ID NO：209的氨基酸序列的PD-1-LCDR1，包含SEQ ID NO：210的氨基酸序列的PD-1-LCDR2，和包含SEQ ID NO：211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: 206, PD-1-HCDR2 comprising the amino acid sequence of SEQ ID NO: 207, and comprising the amino acid sequence of SEQ ID NO: 208 the sequence of PD-1-HCDR3; and the PD-1-VL having: PD-1-LCDR1 comprising the amino acid sequence of SEQ ID NO: 209, PD-1-LCDR2 comprising the amino acid sequence of SEQ ID NO: 210, and PD-1-LCDR3 comprising the amino acid sequence of SEQ ID NO: 211;
    优选地，Preferably,
    所述PD-1-VH包含SEQ ID NO：212的氨基酸序列，和所述PD-1-VL包含SEQ ID NO：213的氨基酸序列；The PD-1-VH includes the amino acid sequence of SEQ ID NO: 212, and the PD-1-VL includes the amino acid sequence of SEQ ID NO: 213;
    更优选地，More preferably,
    所述抗PD-1抗体包含SEQ ID NO：215的氨基酸序列的重链，和SEQ ID NO：216的氨基酸序列的轻链。The anti-PD-1 antibody includes a heavy chain of the amino acid sequence of SEQ ID NO: 215, and a light chain of the amino acid sequence of SEQ ID NO: 216.
  22. 根据权利要求20所述的治疗过度增殖性疾病的方法，所述的第二治疗剂是选自以下任一项：特异性结合MUC16和CD3的双特异性抗体、特异性结合PSMA和CD3的双特异性抗体或特异性结合STEAP2和CD3的双特异性抗体；The method of treating hyperproliferative diseases according to claim 20, wherein the second therapeutic agent is selected from any one of the following: a bispecific antibody that specifically binds to MUC16 and CD3, a bispecific antibody that specifically binds to PSMA and CD3. Specific antibodies or bispecific antibodies that specifically bind STEAP2 and CD3;
    优选地，所述的第二治疗剂是特异性结合MUC16和CD3的双特异性抗体，其包含至少一个特异性结合MUC16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MUC16的抗原结合模块包含重链可变区MUC16-VH和轻链可变区MUC16-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；Preferably, the second therapeutic agent is a bispecific antibody that specifically binds to MUC16 and CD3, which contains at least one antigen-binding module that specifically binds to MUC16 and at least one antigen-binding module that specifically binds to CD3, and the specific The antigen-binding module that specifically binds to MUC16 includes the heavy chain variable region MUC16-VH and the light chain variable region MUC16-VL. The antigen-binding module that specifically binds to CD3 includes Contains heavy chain variable region CD3-VH and light chain variable region CD3-VL;
    更优选地，More preferably,
    所述MUC16-VH具有：包含SEQ ID NO：187的氨基酸序列的MUC16-HCDR1，包含SEQ ID NO：188的氨基酸序列的MUC16-HCDR2，和包含SEQ ID NO：189的氨基酸序列的MUC16-HCDR3；并且所述MUC16-VL具有：包含SEQ ID NO：190的氨基酸序列的MUC16-LCDR1，包含SEQ ID NO：191的氨基酸序列的MUC16-LCDR2，和包含SEQ ID NO：192的氨基酸序列的MUC16-LCDR3；The MUC16-VH has: MUC16-HCDR1 including the amino acid sequence of SEQ ID NO: 187, MUC16-HCDR2 including the amino acid sequence of SEQ ID NO: 188, and MUC16-HCDR3 including the amino acid sequence of SEQ ID NO: 189; And the MUC16-VL has: MUC16-LCDR1 including the amino acid sequence of SEQ ID NO: 190, MUC16-LCDR2 including the amino acid sequence of SEQ ID NO: 191, and MUC16-LCDR3 including the amino acid sequence of SEQ ID NO: 192 ;
    最优选地，Most preferably,
    所述MUC16-VH包含SEQ ID NO：199的氨基酸序列，和所述MUC16-VL包含SEQ ID NO：200的氨基酸序列。The MUC16-VH comprises the amino acid sequence of SEQ ID NO: 199, and the MUC16-VL comprises the amino acid sequence of SEQ ID NO: 200.
  23. 根据权利要求22所述的治疗增殖性疾病的方法，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 treating proliferative diseases according to claim 22, 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:
    所述CD3-VH具有：包含SEQ ID NO：193的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：194的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：195的氨基酸序列的CD3-HCDR3；并且所述CD3-VL具有：包含SEQ ID NO：196的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：197的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：198的氨基酸序列的CD3-LCDR3；The CD3-VH has: CD3-HCDR1 including the amino acid sequence of SEQ ID NO: 193, CD3-HCDR2 including the amino acid sequence of SEQ ID NO: 194, and CD3-HCDR3 including the amino acid sequence of SEQ ID NO: 195; And the CD3-VL has: CD3-LCDR1 including the amino acid sequence of SEQ ID NO: 196, CD3-LCDR2 including the amino acid sequence of SEQ ID NO: 197, and CD3-LCDR3 including the amino acid sequence of SEQ ID NO: 198 ;
    优选地，Preferably,
    所述CD3-VH包含SEQ ID NO：201的氨基酸序列，和所述CD3-VL包含SEQ ID NO：202的氨基酸序列。The CD3-VH comprises the amino acid sequence of SEQ ID NO:201, and the CD3-VL comprises the amino acid sequence of SEQ ID NO:202.
  24. 根据权利要求23所述的治疗过度增殖性疾病的方法，其中所述的特异性结合MUC16和CD3的双特异性抗体具有：The method of treating hyperproliferative diseases according to claim 23, wherein the bispecific antibody that specifically binds MUC16 and CD3 has:
    一条包含SEQ ID NO：203的氨基酸序列的第一链、A first strand containing the amino acid sequence of SEQ ID NO: 203,
    两条包含SEQ ID NO：204的氨基酸序列的第二链、和Two second strands comprising the amino acid sequence of SEQ ID NO: 204, and
    一条包含SEQ ID NO：205的氨基酸序列的第三链。 A third strand comprising the amino acid sequence of SEQ ID NO:205.
PCT/CN2023/087574 2022-04-11 2023-04-11 Antigen-binding molecule specifically binding to egfr and cd28, and medical use thereof WO2023198042A1 (en)

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