WO2023284806A1 - 特异性结合cd38、bcma和cd3的抗原结合分子及其医药用途 - Google Patents

特异性结合cd38、bcma和cd3的抗原结合分子及其医药用途 Download PDF

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WO2023284806A1
WO2023284806A1 PCT/CN2022/105578 CN2022105578W WO2023284806A1 WO 2023284806 A1 WO2023284806 A1 WO 2023284806A1 CN 2022105578 W CN2022105578 W CN 2022105578W WO 2023284806 A1 WO2023284806 A1 WO 2023284806A1
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seq
bcma
amino acid
antigen
acid sequence
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WO2023284806A9 (zh
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叶鑫
陈雨潇
金薪盛
应华
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CN202280041322.3A priority Critical patent/CN117642427A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present disclosure belongs to the field of biotechnology, and more specifically, the present disclosure relates to antigen-binding molecules and applications thereof.
  • Multiple myeloma is the second most common blood cancer in the world. It is characterized by the uncontrolled proliferation of plasma cells in the bone marrow. Cancerous plasma cells proliferate and spread rapidly, which in turn leads to the massive production of monoclonal immunoglobulins. , causing immunosuppression, osteolysis, and end-organ damage. Worldwide, there are more than 138,500 newly diagnosed patients with MM. In the past few decades, due to the emergence of new treatments such as proteasome inhibitors, immunomodulators and CD38 antibodies, the clinical treatment effect of MM patients has been greatly improved, and the life expectancy of patients has increased from 3 to 4 years to 7 years. to 8 years.
  • Immunotherapy that remodels the antitumor activity of immune cells while targeting MM cells would be an excellent therapy for MM.
  • CD38 is a type II transmembrane glycoprotein, which can not only interact with the ligand CD31, transmit downstream signals to regulate the activation of lymphocytes, B cell differentiation and other processes, but also function through the action of exonucleases.
  • CD38 is widely expressed in plasma cells and multiple myeloma cells, and CD38 monoclonal antibody drugs Daratumumab (Daratumumab) and Isatuximab have a good treatment response rate and depth of response in clinical practice, fully confirming that CD38 is A better target for multiple myeloma.
  • BCMA Tumor necrosis factor receptor superfamily member 17
  • BCMA Tumor necrosis factor receptor superfamily member 17
  • BCMA binds to the ligands APRIL and BAFF, activates downstream ERK1/2, NFkB and other signaling pathways, and promotes the growth and proliferation of B cells.
  • APRIL and BAFF ligands that binds to the ligands APRIL and BAFF, activates downstream ERK1/2, NFkB and other signaling pathways, and promotes the growth and proliferation of B cells.
  • BCMA is mainly expressed in the late stage of B cell differentiation, that is, more mature B cells (Plasmablast, PC), and is not expressed in early B cells and other normal tissues.
  • BCMA is highly selectively expressed on the surface of multiple myeloma cells, and the expression level is much higher than that in normal B cells.
  • BCMA soluble BCMA
  • the sBCMA in plasma is significantly higher in patients with multiple myeloma and recurrent myeloma than in healthy people. It has been regarded as An important marker of multiple myeloma disease progression.
  • the bispecific antibody targeting CD3&TAA is a new type of immunotherapy, which can simultaneously bind T cells and tumor cells, mimic the interaction between MHC and TCR, and make T cells release perforin and granzyme specificity after forming lytic synapse Kill tumor cells.
  • Activated T cells can release cytokines, activate other immune cells and expand the immune response against tumors, eventually leading to the cascade reaction of T cell proliferation and killing tumor cells.
  • the present disclosure provides an antigen-binding molecule comprising at least one antigen-binding moiety that specifically binds BCMA, at least one antigen-binding moiety that specifically binds CD38, and at least one antigen-binding moiety that specifically binds CD3.
  • antigen-binding molecules can provide better therapeutic activity than BCMA/CD3 antibodies or CD38/CD3 antibodies, and cover a wider range of multiple myeloma patient populations.
  • the antigen binding moiety is a Fab or scFv.
  • the present disclosure provides an antigen binding molecule comprising two antigen binding moieties that specifically bind BCMA, two antigen binding moieties that specifically bind CD38, two antigen binding moieties that specifically bind CD3 Binding module and Fc region.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds BCMA is BCMA-Fab, the antigen-binding moiety that specifically binds CD3 is CD3-scFv and the specific The antigen binding moiety that specifically binds CD38 is CD38-scFv.
  • BCMA-Fab in the present disclosure means that the antigen-binding module has a Fab structure, and CD3-scFv and CD38-scFv mean that the antigen-binding module has a scFv structure.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds CD38 is CD38-Fab, the antigen-binding moiety that specifically binds CD3 is CD3-scFv and the specific The antigen binding moiety that specifically binds BCMA is BCMA-scFv.
  • CD38-Fab in the present disclosure means that the antigen-binding module has a Fab structure, and CD3-scFv and BCMA-scFv mean that the antigen-binding module has a scFv structure.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds BCMA is BCMA-Fab, the antigen-binding moiety that specifically binds CD3 is CD3-scFv and the specific The antigen-binding moiety that specifically binds CD38 is CD38-scFv; and a heavy chain of BCMA-Fab, a CD3-scFv, a subunit of the Fc region, and a CD38-scFv are directly or connected in the order from the N-terminal to the C-terminal child connection.
  • the heavy chain of another BCMA-Fab, another CD3-scFv, another subunit of the Fc region, and another CD38-scFv are also linked directly or via a linker in the order of N-terminus to C-terminus, respectively .
  • said antigen binding molecule has a structure as shown in Figure 1B.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds CD38 is CD38-Fab, the antigen-binding moiety that specifically binds CD3 is CD3-scFv and the specific The antigen-binding moiety that specifically binds BCMA is BCMA-scFv; and a heavy chain of CD38-Fab, a CD3-scFv, a subunit of the Fc region, and a BCMA-scFv are directly or connected in the order from N-terminal to C-terminal child connection.
  • the heavy chain of another CD38-Fab, another CD3-scFv, another subunit of the Fc region, and another BCMA-scFv are also connected directly or via a linker in the order of N-terminus to C-terminus .
  • said antigen binding molecule has a structure as shown in Figure 1D.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, the specific The antigen binding moiety that binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the antigen binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region Region CD38-VL; where:
  • the antigen-binding molecule comprises two first chains having a structure represented by formula (a) and two second chains having a structure represented by formula (b),
  • Linker 1, Linker 2, Linker 3 and Linker 4 in formula (a) are the same or different peptide linkers.
  • Linker 1, Linker 2, Linker 3, and Linker 4 in formula (a) are 3-15 amino acid residues in length.
  • the amino acid sequence of linker 1, linker 3 and linker 4 in formula (a) is shown in SEQ ID NO: 99, and the amino acid sequence of linker 2 is shown in SEQ ID NO: 96.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, the specific The antigen binding moiety that binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the antigen binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region Region CD38-VL; where:
  • the antigen-binding molecule comprises two first chains having a structure represented by formula (c) and two second chains having a structure represented by formula (d),
  • Linker 1, Linker 2, Linker 3 and Linker 4 in formula (c) are the same or different peptide linkers.
  • Linker 1, Linker 2, Linker 3, and Linker 4 in formula (c) are 3-15 amino acid residues in length.
  • the amino acid sequence of linker 1, linker 3 and linker 4 in formula (c) is shown in SEQ ID NO: 99, and the amino acid sequence of linker 2 is shown in SEQ ID NO: 96.
  • the present disclosure provides an antigen binding molecule comprising an antigen binding moiety that specifically binds BCMA, two antigen binding moieties that specifically bind CD38, an antigen binding moiety that specifically binds CD3 and Fc region.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds BCMA is BCMA-Fab, the antigen-binding moiety that specifically binds CD3 is CD3-scFv and the specific The antigen binding moiety that specifically binds CD38 is CD38-scFv.
  • said antigen binding molecule has a structure as shown in Figure 1A.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds CD38 is CD38-Fab, the antigen-binding moiety that specifically binds CD3 is CD3-scFv and the specific The antigen binding moiety that specifically binds BCMA is BCMA-scFv.
  • said antigen binding molecule has a structure as shown in Figure 1E.
  • the antigen-binding molecule comprises a first subunit Fc1 and a second subunit Fc2 capable of associating, and each of the Fc1 and Fc2 independently has one or more An amino acid substitution that reduces the homologous dimerization of the Fc region; and, the antigen binding moiety that specifically binds BCMA is BCMA-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the specific binding The antigen-binding moiety of CD38 is CD38-scFv; and the heavy chain of BCMA-Fab, Fc1 and one CD38-scFv are connected directly or through a linker in the order of N-terminal to C-terminal, respectively, and CD3-scFv, Fc2 and another CD38 - The scFvs are connected directly or via a linker in the order of N-terminal to C-terminal, respectively.
  • the antigen-binding molecule comprises a first subunit Fc1 and a second subunit Fc2 capable of associating, and each of the Fc1 and Fc2 independently has one or more An amino acid substitution that reduces the homologous dimerization of the Fc region; and, the antigen binding moiety that specifically binds CD38 is CD38-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the specific binding
  • the antigen-binding module of BCMA is BCMA-scFv; and the heavy chain, Fc1 and CD3-scFv of one CD38-Fab are connected directly or through a linker in the order of N-terminal to C-terminal, respectively, and BCMA-scFv, another CD38-Fab
  • the heavy chain and Fc2 are connected directly or through a linker in the order of N-terminus to C-terminus, respectively.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, the specific The antigen binding moiety that binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the antigen binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region Region CD38-VL; where:
  • the antigen-binding molecule comprises a first chain having a structure represented by formula (e), a second chain having a structure represented by formula (b) and a third chain having a structure represented by formula (f),
  • linker 1 and linker 2 in formula (e) are the same or different peptide linkers
  • linker 1, linker 2, linker 3 and linker 4 in formula (f) are the same or different peptide linker.
  • the peptide linker is a flexible peptide linker.
  • Linker 1, Linker 2 in formula (e), and Linker 1, Linker 2, Linker 3, and Linker 4 in formula (f) are 3-15 amino acids in length Residues.
  • amino acid sequence of linker 1 and linker 2 in formula (e) is as shown in SEQ ID NO: 99
  • amino acid sequence of linker 1, linker 3 and linker 4 in formula (f) The sequence is shown in SEQ ID NO: 99
  • amino acid sequence of linker 2 in formula (f) is shown in SEQ ID NO: 96.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, the specific The antigen binding moiety that binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the antigen binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region Region CD38-VL; where:
  • the antigen-binding molecule comprises a first chain having a structure represented by formula (g), a second chain having a structure represented by formula (d) and a third chain having a structure represented by formula (h),
  • Linker 1 and Linker 2 in formula (g) are the same or different peptide linkers, and Linker 1 and Linker 2 in formula (h) are the same or different peptide linkers.
  • the peptide linker is a flexible peptide linker.
  • Linker 1 , Linker 2 in formula (g), and Linker 1 and Linker 2 in formula (h) are 3-15 amino acid residues in length.
  • amino acid sequence of linker 1 and linker 2 in formula (g) is as shown in SEQ ID NO: 99
  • amino acid sequence of linker 1 in formula (h) is as shown in SEQ ID NO: 97
  • amino acid sequence of linker 2 in formula (h) is shown in SEQ ID NO:99.
  • the present disclosure provides an antigen binding molecule comprising an antigen binding moiety that specifically binds BCMA, an antigen binding moiety that specifically binds CD38, an antigen binding moiety that specifically binds CD3, and Fc region.
  • said antigen binding molecule has a structure as shown in Figure 1C.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds BCMA is BCMA-Fab, the antigen-binding moiety that specifically binds CD3 is CD3-scFv and the specific The antigen binding moiety that specifically binds CD38 is CD38-scFv.
  • the antigen-binding molecule comprises a first subunit Fc1 and a second subunit Fc2 capable of associating, and each of the Fc1 and Fc2 independently has one or more amino acid substitutions that reduce the homologous dimerization of the Fc region; and CD38-scFv, CD3-scFv and Fc1 are connected directly or via a linker in the order of N-terminal to C-terminal, respectively, and the heavy chain of BCMA-Fab and Fc2 are connected in N-terminal order The sequence from C-terminus to C-terminus is linked directly or via a linker, respectively.
  • the antigen-binding molecule according to any one of the preceding items, the antigen-binding moiety that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, the specific The antigen binding moiety that binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the antigen binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region Region CD38-VL; where:
  • the antigen-binding molecule comprises a first chain having a structure represented by formula (i), a second chain having a structure represented by formula (b) and a third chain having a structure represented by formula (j),
  • linker 1, linker 2, linker 3 and linker 4 in formula (j) are the same or different peptide linkers.
  • the peptide linker is a flexible peptide linker.
  • Linker 1, Linker 2, Linker 3, and Linker 4 in formula (j) are 3-15 amino acid residues in length.
  • the amino acid sequence of linker 1 and linker 3 in formula (j) is as shown in SEQ ID NO: 99
  • the amino acid sequence of linker 2 in formula (j) is as shown in SEQ ID NO: 97 Shown
  • the amino acid sequence of linker 4 in formula (j) is shown in SEQ ID NO:96.
  • the antigen-binding molecule of any one of the preceding items comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein:
  • the BCMA-VH comprises the amino acid sequence of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 23, and the BCMA-VL comprises BCMA-LCDR1, BCMA in SEQ ID NO: 24 - the amino acid sequence of LCDR2 and BCMA-LCDR3; or
  • the BCMA-VH comprises the amino acid sequence of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 25, and the BCMA-VL comprises BCMA-LCDR1, BCMA in SEQ ID NO: 26 - the amino acid sequence of LCDR2 and BCMA-LCDR3; or
  • the BCMA-VH comprises the amino acid sequence of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 27, and the BCMA-VL comprises BCMA-LCDR1, BCMA in SEQ ID NO: 28 - Amino acid sequences of LCDR2 and BCMA-LCDR3.
  • the antigen binding molecules as described above, said BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are obtained according to Kabat, IMGT, Chothia, AbM or Defined by the Contact numbering scheme.
  • the antigen binding molecule of any one of the preceding is any one of the preceding.
  • the BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:5, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:7 HCDR3; and the BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:8, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:10 LCDR3, or
  • the BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 13 HCDR3; and the BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 16 LCDR3, or
  • the BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 19 HCDR3; and the BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 22 LCDR3.
  • the antigen binding molecule as described above, said BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat numbering convention.
  • the antigen binding molecule as described above, wherein:
  • the BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:5, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and BCMA comprising the amino acid sequence of SEQ ID NO:7 -HCDR3; and the BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:8, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and comprising the amino acid sequence of SEQ ID NO:10 BCMA-LCDR3.
  • the BCMA-VH and/or the BCMA-VL are murine or humanized.
  • the BCMA-VH and/or the BCMA-VL are humanized.
  • the BCMA-VH has a heavy chain framework region derived from IGHV1-46*01 and is unsubstituted or has a group selected from the group consisting of 48I, 67A, 71A, 73K, 76T and 93V One or more amino acid substitutions in; and/or the BCMA-VL has a light chain framework region derived from IGKV1-39*01, and it is unsubstituted or has a composition selected from 43S, 45Q, 48V and 71Y One or more amino acid substitutions in the group.
  • the variable regions and CDRs described above are defined according to the Kabat numbering convention.
  • the antigen binding molecule as described above, wherein:
  • said BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 23, and said BCMA-VL comprises an amino acid sequence identical to SEQ ID NO: 23, SEQ ID NO: 24 An amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the BCMA-VH comprises the amino acid sequence of SEQ ID NO: 23, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO: 24.
  • the antigen binding molecule as described above, wherein:
  • said BCMA-VH comprises at least 90%, 95%, 96%, 97%, An amino acid sequence having 98% or 99% sequence identity
  • said BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35
  • Amino acid sequences are amino acid sequences having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, respectively.
  • the BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, and the BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: : 32, the amino acid sequence of the group consisting of SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.
  • the antigen binding moiety that specifically binds BCMA is functionally equivalent to an antigen binding moiety comprising SEQ ID NO: 29 and SEQ ID NO: 32.
  • the BCMA-VH comprises the amino acid sequence of SEQ ID NO:29
  • the BCMA-VL comprises the amino acid sequence of SEQ ID NO:32.
  • the antigen binding molecule as described above, wherein:
  • the BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 13 HCDR3; and the BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 16 LCDR3.
  • the BCMA-VH and/or the BCMA-VL are murine or humanized.
  • the BCMA-VH and/or the BCMA-VL are humanized.
  • the BCMA-VH has a heavy chain framework region derived from IGHV7-4-1*02, and it is unsubstituted or has One or more amino acid substitutions in the group consisting of 93L; and/or the BCMA-VL has a light chain framework region derived from IGKV1-27*01, and it is unsubstituted or has a composition selected from 43S and 66D One or more amino acid substitutions in the group.
  • the variable regions and CDRs described above are defined according to the Kabat numbering convention.
  • the antigen binding molecule as described above, wherein:
  • said BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 25, and said BCMA-VL comprises an amino acid sequence identical to SEQ ID NO: 25, SEQ ID NO: 26 An amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the BCMA-VH comprises the amino acid sequence of SEQ ID NO: 25, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO: 26.
  • the antigen binding molecule as described above, wherein:
  • the BCMA-VH comprises at least 90%, 95%, 96%, 97% of the amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively , an amino acid sequence with 98% or 99% sequence identity
  • said BCMA-VL comprises at least 90%, 95%, 96%, 97% respectively with the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40 , 98% or 99% sequence identity of amino acid sequences.
  • the BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, and the BCMA-VL comprises SEQ ID NO: 39 Or the amino acid sequence of SEQ ID NO:40.
  • the antigen binding moiety that specifically binds BCMA is functionally equivalent to an antigen binding moiety comprising SEQ ID NO: 36 and SEQ ID NO: 40.
  • the BCMA-VH comprises the amino acid sequence of SEQ ID NO:36
  • the BCMA-VL comprises the amino acid sequence of SEQ ID NO:40.
  • the antigen binding molecule as described above, wherein:
  • the BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 19 HCDR3; and the BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 22 LCDR3.
  • the BCMA-VH and/or the BCMA-VL are murine or humanized.
  • the BCMA-VH and/or the BCMA-VL are humanized.
  • the BCMA-VH has a heavy chain framework region derived from IGHV1-46*01 and is unsubstituted or has a composition selected from 38K, 48I, 66T, 67A, 71S, 73K and 78A One or more amino acid substitutions in the group; and/or the BCMA-VL has a light chain framework region derived from IGKV2-28*01, and it is unsubstituted or has a composition selected from 2V, 45K and 87F One or more amino acid substitutions in the group.
  • the variable regions and CDRs described above are defined according to the Kabat numbering convention.
  • the antigen binding molecule as described above, wherein:
  • said BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 27, and said BCMA-VL comprises an amino acid sequence identical to SEQ ID NO: 27, SEQ ID NO: 28 An amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the BCMA-VH comprises the amino acid sequence of SEQ ID NO:27
  • the BCMA-VL comprises the amino acid sequence of SEQ ID NO:28.
  • the antigen binding molecule as described above, wherein:
  • the BCMA-VH comprises at least 90%, 95% of the amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, respectively , 96%, 97%, 98% or 99% sequence identity amino acid sequence
  • said BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 45 or SEQ ID NO: 46 having at least Amino acid sequences having 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, and the BCMA-VL comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 45 or SEQ ID NO: 46.
  • the antigen binding moiety that specifically binds BCMA is functionally equivalent to an antigen binding moiety comprising SEQ ID NO: 43 and SEQ ID NO: 45.
  • the BCMA-VH comprises the amino acid sequence of SEQ ID NO:43
  • the BCMA-VL comprises the amino acid sequence of SEQ ID NO:45.
  • the antigen-binding molecule as described above, the antigen-binding moiety that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein:
  • said CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 respectively comprise the amino acid sequence of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 63; and said CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 respectively comprising the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO: 64, or
  • said CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 respectively comprise the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 65; and said CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 respectively comprise the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO:64.
  • the antigen-binding molecule as described above, said CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 is according to Kabat, IMGT, Chothia, AbM or Defined by the Contact numbering scheme.
  • the antigen binding molecule as described above, wherein:
  • the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:56, and CD3 comprising the amino acid sequence of SEQ ID NO:57 -HCDR3; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and comprising the amino acid sequence of SEQ ID NO:60 CD3-LCDR3, or
  • the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO: 55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO: 61, and CD3 comprising the amino acid sequence of SEQ ID NO: 62 -HCDR3; and the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and comprising the amino acid sequence of SEQ ID NO:60 CD3-LCDR3.
  • the antigen-binding molecule as described above, said CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 are defined according to the Kabat numbering convention.
  • the antigen binding molecule as described above, wherein:
  • said CD3-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 63
  • said CD3-VL comprises an amino acid sequence identical to SEQ ID NO: 63
  • SEQ ID NO: 64 is an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the antigen binding moiety that specifically binds CD3 is functionally equivalent to an antigen binding moiety comprising SEQ ID NO:63 and SEQ ID NO:64.
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO:63
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
  • the antigen binding molecule as described above, wherein:
  • said CD3-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 65
  • said CD3-VL comprises an amino acid sequence identical to SEQ ID NO: 65
  • SEQ ID NO: 64 is an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the antigen binding moiety that specifically binds CD3 is functionally equivalent to an antigen binding moiety comprising SEQ ID NO:65 and SEQ ID NO:64.
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO:65
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO:64.
  • the antigen-binding moiety that specifically binds to CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region CD38-VL, wherein the CD38-VH CD38-HCDR1, CD38-HCDR2 and CD38-HCDR3 in CD38-HCDR1, CD38-HCDR2 and CD38-HCDR3 respectively comprise the amino acid sequences of CD38-HCDR1, CD38-HCDR2 and CD38-HCDR3 in SEQ ID NO: 53, and CD38-LCDR1, CD38 in the CD38-VL -LCDR2 and CD38-LCDR3 comprise the amino acid sequences of CD38-LCDR1, CD38-LCDR2 and CD38-LCDR3 in SEQ ID NO:54, respectively.
  • the CD38-HCDR1, CD38-HCDR2, CD38-HCDR3, CD38-LCDR1, CD38-LCDR2, and CD38-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering conventions.
  • the antigen-binding molecule as described above has: CD38-HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, CD38-HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, and CD38-HCDR3 comprising the amino acid sequence of SEQ ID NO: 49; and the CD38-VL has: CD38-LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, CD38-LCDR2 comprising the amino acid sequence of SEQ ID NO: 51 , and CD38-LCDR3 comprising the amino acid sequence of SEQ ID NO:52.
  • the CD38-HCDR1, CD38-HCDR2, CD38-HCDR3, CD38-LCDR1, CD38-LCDR2, and CD38-LCDR3 are defined according to the Kabat numbering convention.
  • the antigen binding molecule as described above, wherein:
  • the CD38-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:53
  • the CD38-VL comprises an amino acid sequence identical to SEQ ID NO:53. :54 Amino acid sequences having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the antigen binding moiety that specifically binds CD38 is functionally equivalent to an antigen binding moiety comprising SEQ ID NO:53 and SEQ ID NO:54.
  • the CD38-VH comprises the amino acid sequence of SEQ ID NO:53
  • the CD38-VL comprises the amino acid sequence of SEQ ID NO:54.
  • the antigen binding molecule of any one of the preceding wherein the linker is a peptide linker.
  • the peptide linker is 3-15 amino acid residues in length.
  • each of the peptide linkers independently has an amino acid sequence as shown in L 1 -(GGGGS) n -L 2 , wherein L 1 is a bond, A, GS, GGS or GGGS (SEQ ID NO: 102), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L 2 is a bond, G, GG, GGG or GGGG (SEQ ID NO: 103), and the Connectors are not keys.
  • the peptide linker sequence is shown in SEQ ID NO:96 to SEQ ID NO:99.
  • the antigen binding molecule of any one of the preceding comprising an Fc region.
  • the Fc region is an IgG Fc region, preferably an IgG 1 Fc region.
  • the Fc region comprises one or more amino acid substitutions that reduce binding of the Fc region to an Fc receptor.
  • the amino acid substitutions reduce binding to Fc ⁇ receptors.
  • the Fc region is a human IgG 1 Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is based on the EU index.
  • the Fcl has the amino acid sequence of SEQ ID NO:68.
  • the antigen-binding molecule of any one of the preceding items comprising an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, each of Fc1 and Fc2 Independently having one or more amino acid substitutions that reduce homodimerization of the Fc region.
  • the Fc1 and Fc2 each independently have one or more amino acid substitutions according to the pestle-and-hole technique.
  • the Fc1 has a convex structure according to the knob-and-hole technique
  • the Fc2 has a pore structure according to the knob-and-hole technique.
  • the amino acid residue at position 366 of the Fc1 is W; and the amino acid residue at position 366 of the Fc2 is S, the amino acid residue at position 368 is A, and the amino acid residue at position 407 is The base is V, and the numbering basis is the EU index.
  • the Fc1 has the amino acid sequence of SEQ ID NO: 100, and the Fc2 has the amino acid sequence of SEQ ID NO: 101.
  • the antigen binding molecule of any one of the preceding is any one of the preceding.
  • the antigen binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO: 76 and two second strands comprising the amino acid sequence of SEQ ID NO: 74; or
  • the antigen binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO: 75 and two second strands comprising the amino acid sequence of SEQ ID NO: 74; or
  • the antigen binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO: 82 and two second strands comprising the amino acid sequence of SEQ ID NO: 81; or
  • the antigen binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO: 83 and two second strands comprising the amino acid sequence of SEQ ID NO: 81; or
  • the antigen binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO:90 and two second strands comprising the amino acid sequence of SEQ ID NO:89; or
  • the antigen binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO: 91 and two second strands comprising the amino acid sequence of SEQ ID NO: 89; or
  • the antigen binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO: 77, two second strands comprising the amino acid sequence of SEQ ID NO: 71; or
  • the antigen-binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO: 78, two second strands comprising the amino acid sequence of SEQ ID NO: 71; or
  • the antigen binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO: 85, two second strands comprising the amino acid sequence of SEQ ID NO: 71; or
  • the antigen binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO: 86, two second strands comprising the amino acid sequence of SEQ ID NO: 71; or
  • the antigen binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO: 93, two second strands comprising the amino acid sequence of SEQ ID NO: 71; or
  • the antigen binding molecule has: two first strands comprising the amino acid sequence of SEQ ID NO:94, two second strands comprising the amino acid sequence of SEQ ID NO:71; or
  • the antigen-binding molecule has: a first strand comprising the amino acid sequence of SEQ ID NO: 73, a second strand comprising the amino acid sequence of SEQ ID NO: 74 and a third strand comprising the amino acid sequence of SEQ ID NO: 66 chain; or
  • the antigen-binding molecule has: a first strand comprising the amino acid sequence of SEQ ID NO:73, a second strand comprising the amino acid sequence of SEQ ID NO:74 and a third strand comprising the amino acid sequence of SEQ ID NO:67 chain; or
  • the antigen-binding molecule has: a first strand comprising the amino acid sequence of SEQ ID NO: 80, a second strand comprising the amino acid sequence of SEQ ID NO: 81 and a third strand comprising the amino acid sequence of SEQ ID NO: 66 chain; or
  • the antigen-binding molecule has: a first strand comprising the amino acid sequence of SEQ ID NO: 80, a second strand comprising the amino acid sequence of SEQ ID NO: 81 and a third strand comprising the amino acid sequence of SEQ ID NO: 67 chain; or
  • the antigen-binding molecule has: a first strand comprising the amino acid sequence of SEQ ID NO: 88, a second strand comprising the amino acid sequence of SEQ ID NO: 89 and a third strand comprising the amino acid sequence of SEQ ID NO: 66 chain; or
  • the antigen-binding molecule has: a first strand comprising the amino acid sequence of SEQ ID NO: 88, a second strand comprising the amino acid sequence of SEQ ID NO: 89 and a third strand comprising the amino acid sequence of SEQ ID NO: 67 chain; or
  • the antigen-binding molecule has: a first strand comprising the amino acid sequence of SEQ ID NO: 84, a second strand comprising the amino acid sequence of SEQ ID NO: 81 and a third strand comprising the amino acid sequence of SEQ ID NO: 69 chain; or
  • the antigen-binding molecule has: a first strand comprising the amino acid sequence of SEQ ID NO: 84, a second strand comprising the amino acid sequence of SEQ ID NO: 81 and a third strand comprising the amino acid sequence of SEQ ID NO: 70 chain; or
  • the antigen-binding molecule has: a first strand comprising the amino acid sequence of SEQ ID NO: 92, a second strand comprising the amino acid sequence of SEQ ID NO: 89, and a third strand comprising the amino acid sequence of SEQ ID NO: 69 chain; or
  • the antigen-binding molecule has: a first strand comprising the amino acid sequence of SEQ ID NO:92, a second strand comprising the amino acid sequence of SEQ ID NO:89 and a third strand comprising the amino acid sequence of SEQ ID NO:70 chain; or
  • the antigen-binding molecule has: a first chain comprising the amino acid sequence of SEQ ID NO: 79, two second chains comprising the amino acid sequence of SEQ ID NO: 71 and a second chain comprising the amino acid sequence of SEQ ID NO: 72 triple chain; or
  • the antigen-binding molecule has: a first chain comprising the amino acid sequence of SEQ ID NO: 87, two second chains comprising the amino acid sequence of SEQ ID NO: 71 and a second chain comprising the amino acid sequence of SEQ ID NO: 72 triple chain; or
  • the antigen-binding molecule has: a first chain comprising the amino acid sequence of SEQ ID NO: 95, two second chains comprising the amino acid sequence of SEQ ID NO: 71 and a second chain comprising the amino acid sequence of SEQ ID NO: 72 Three strands.
  • the antigen-binding molecule of any one of the preceding items has: two first strands comprising the amino acid sequence of SEQ ID NO: 76 and two second strands comprising the amino acid sequence of SEQ ID NO: 74 chain.
  • the antigen-binding molecule of any one of the preceding items has: two first strands comprising the amino acid sequence of SEQ ID NO: 82 and two second strands comprising the amino acid sequence of SEQ ID NO: 81 chain.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a therapeutically effective amount of the antigen-binding molecule described in any one of the foregoing, and one or more pharmaceutically acceptable carriers, diluents, buffer or excipient.
  • the pharmaceutical composition further comprises at least one second therapeutic agent.
  • the present disclosure also provides an isolated nucleic acid encoding the antigen-binding molecule of any one of the foregoing.
  • the present disclosure also provides a host cell comprising the aforementioned isolated nucleic acid.
  • the present disclosure also provides a method for treating a disease, the method comprising administering to a subject a therapeutically effective amount of the antigen-binding molecule or composition described in any one of the foregoing.
  • the present disclosure also provides the use of the antigen-binding molecule or composition described in any one of the foregoing in the preparation of a medicament for treating or preventing a disease.
  • the present disclosure also provides the antigen-binding molecule or composition of any one of the foregoing for use as a medicament.
  • the medicament is used to treat a disease.
  • the disease is a hematopoietic tumor of the lymphoid or myeloid lineage or an autoimmune disease.
  • the hematopoietic tumor of the lymphoid or myeloid lineage is selected from the group consisting of multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, Solitary plasmacytoma, extramedullary plasmacytoma, sclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma, B-cell lymphoma, Burkitt Lymphoma, Hodgkin's lymphoma, and hairy cell lymphoma; said autoimmune disease selected from the group consisting of: rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, multiple sclerosis
  • the antigen-binding molecule provided by the present disclosure has the characteristics of good therapeutic activity, safety, pharmacokinetic properties and druggability (such as stability).
  • Fig. 1A Schematic structure of Format5
  • Fig. 1B Schematic structure of Format12
  • Fig. 1C Schematic structure of Format20
  • Fig. 1D Schematic structure of Format21
  • Fig. 1E Schematic structure of Format24.
  • Figure 2 Ability of antibodies to bind RPMI-8226 cells.
  • Figure 3 Cytotoxic activity of antibodies against CHO K1 cells.
  • 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 amino acids that are later modified, eg, hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine.
  • Amino acid analogs are compounds that have the same basic chemical structure (i.e., the alpha carbon bonded to a hydrogen, carboxyl, amino group, and R group) as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide , Methylsulfonium methionine.
  • Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • An amino acid mimetic refers to a chemical compound that has a structure that differs from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.
  • 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 so long as the final construct possesses the desired properties, such as reduced or binding to Fc receptors.
  • Amino acid sequence deletions and insertions include deletions and insertions at the amino and/or carboxyl termini of the polypeptide chain.
  • Specific amino acid mutations may be amino acid substitutions.
  • the amino acid mutation is a non-conservative amino acid substitution, that is, replacing one amino acid with another amino acid having different structural and/or chemical properties.
  • Amino acid substitutions include substitutions with non-naturally occurring amino acids or with 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, and the like. It is anticipated 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.
  • amino acid residue at a specific position can be expressed in the form of 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 the 366th position is mutated from the original T to W.
  • antigen-binding molecule is used in the broadest sense and covers various molecules that specifically bind to an antigen, including but not limited to antibodies, other polypeptides with antigen-binding activity, and antibody fusion proteins fused thereto.
  • the antigen-binding molecules herein are trispecific antigen-binding molecules (eg, trispecific antibodies).
  • trispecific antigen binding molecule refers to an antigen binding molecule capable of specifically binding three different antigens.
  • antibody is used in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as 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.
  • Native antibody refers to a naturally occurring immunoglobulin molecule. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains joined by disulfide bonds.
  • each heavy chain has a variable region (VH), also called variable heavy domain, heavy chain variable region, followed by three constant domains (CH1, CH2 and CH3).
  • VH variable region
  • VL variable light domain
  • CL constant light domain
  • variable region refers to the antigen-binding domain of an antigen-binding molecule.
  • the heavy chain variable region in the antigen-binding module that specifically binds BCMA is marked as BCMA-VH, and the light chain variable region is marked as BCMA-VL;
  • the heavy-chain variable region in the antigen-binding module that specifically binds CD38 The variable region of the light chain is marked as CD38-VH, and the variable region of the light chain is marked as CD38-VL;
  • the variable region of the heavy chain in the antigen-binding module that specifically binds to CD3 is marked as CD3-VH, and the variable region of the light chain is marked as CD3-VL.
  • VH and VL each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • CDR complementarity determining region
  • VH contains 3 CDR regions: HCDR1, HCDR2 and HCDR3
  • VL contains 3 CDR regions: LCDR1, LCDR2 and LCDR3.
  • the three CDR regions in BCMA-VH are marked as BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3; the three CDR regions in BCMA-VL are marked as BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 ;
  • the three CDR regions in CD38-VH are respectively marked as CD38-HCDR1, CD38-HCDR2 and CD38-HCDR3; the three CDR regions in CD38-VL are respectively marked as CD38-LCDR1, CD38-LCDR2 and CD38-LCDR3;
  • CD3 -The three CDR regions in VH are marked as CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3; the three CDR regions in CD3-VL are marked as CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 respectively.
  • Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a single VH or VL may be sufficient to confer antigen binding specificity.
  • amino acid sequence boundaries of CDRs can be determined by various known schemes, for example: “Kabat” numbering convention (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health , Bethesda, MD), “Chothia” numbering sequence, “ABM” numbering sequence, "contact” numbering sequence (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J].
  • Kabat numbering convention is applicable to the variable regions and CDR sequences in the examples of the present disclosure.
  • antibody fragment refers to a molecule other than an intact antibody that comprises the portion of an intact antibody that binds to the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , single domain antibody, single chain Fab (scFab), diabody, linear antibody, single chain antibody molecule (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • Fab refers to a protein consisting of the VH and CH1 (Fab heavy chain) and VL and CL (Fab light chain) of an immunoglobulin.
  • BCMA-Fab means an antigen-binding module specifically binding to BCMA having a Fab structure
  • BCMA-scFv means an antigen-binding module specifically binding to BCMA having a scFv structure, and so on.
  • scFv refers to a single-chain antibody (single chain antibody fragment, scFv), which is composed of an antibody heavy chain variable region (VH) and a light chain variable region (VL) connected by a short peptide linker of 15 to 20 amino acids. made antibodies.
  • Fc region or “fragment crystallizable region” are used to define the C-terminal region of an antibody heavy chain, including native and engineered Fc regions.
  • the Fc region comprises the same or different two subunits.
  • 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 carboxyl terminus.
  • Suitable native sequence Fc regions for 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.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chains is derived from a particular source or species, while the remaining portion of the heavy and/or light chains is derived from a different source or species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions and the framework portion of the variable regions).
  • affinity refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • binding affinity refers to internal binding affinity, which reflects a 1:1 interaction between 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 (ie, kd/ka) and is expressed as molarity (M).
  • M molarity
  • the KD value of an antibody can be determined using methods known in the art, for example: methods for determining the KD of an antibody include measuring surface plasmon resonance using a biosensing system such as a system, or measuring K in solution by solution equilibrium titration (SET). affinity.
  • the term “capable of specifically binding”, “specifically binds” or “binds” means that an antibody is capable of binding to a certain antigen or an epitope within the antigen with a higher affinity than to other antigens or epitopes.
  • the antibody binds the antigen or a surface within the antigen with an equilibrium dissociation constant (KD) of about 1 ⁇ 10 ⁇ 6 M or less (e.g., about 1 ⁇ 10 ⁇ 7 M, about 1 ⁇ 10 ⁇ 8 M or less). bit.
  • KD equilibrium dissociation constant
  • the antibody binds an antigen with a KD that is 10% or less (eg, 1%) of the antibody's KD for binding to a non-specific antigen (eg, BSA, casein).
  • KD can be measured using known methods, such as by FACS or Measured by surface plasmon resonance assay.
  • antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, e.g. (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (commonmarmoset, marmoset) are cross-reactive.
  • antigen binding moiety refers to a polypeptide molecule that specifically binds an antigen of interest.
  • Antigen binding moieties include antibodies and fragments thereof as defined herein. Particular antigen binding moieties include an antigen binding domain of an antibody comprising an antibody heavy chain variable region and an antibody light chain variable region.
  • antigen binding moiety that specifically binds BCMA refers to a moiety that is capable of binding BCMA with sufficient affinity.
  • an antigen binding moiety that specifically binds BCMA has an equilibrium dissociation constant (KD) of ⁇ about 1 ⁇ M, ⁇ about 100 nM, or ⁇ about 10 nM, as measured by FACS, surface plasmon resonance, etc. .
  • KD equilibrium dissociation constant
  • an antigen binding moiety that specifically binds BCMA binds a conserved epitope in BCMA from a different species.
  • the term "antigen-binding moiety that specifically binds CD38” refers to a moiety that is capable of binding CD38 with sufficient affinity.
  • an antigen binding moiety that specifically binds CD38 has an equilibrium dissociation constant (KD) of ⁇ about 1 ⁇ M, ⁇ about 100 nM, or ⁇ about 10 nM, as measured by FACS, surface plasmon resonance, etc. .
  • KD equilibrium dissociation constant
  • an antigen binding moiety that specifically binds CD38 binds a conserved epitope in CD38 from a different species.
  • antigen-binding moiety that specifically binds CD3 refers to a moiety that is capable of binding CD3 with sufficient affinity.
  • the antigen binding moiety that specifically binds CD3 has an equilibrium dissociation constant (KD) of ⁇ about 1 ⁇ M, ⁇ about 200 nM, ⁇ about 100 nM, and/or >50 nM, as determined by FACS, surface measured by plasmon resonance.
  • the anti-CD3 antibody binds a conserved epitope in CD3 from different species.
  • Antigen binding moieties include antibody fragments as defined herein, eg Fab or scFv.
  • effector functions refers to those biological activities attributable to an antibody Fc region (either native sequence Fc region or amino acid sequence variant Fc region) and which vary with antibody isotype.
  • antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; ; and B cell activation.
  • the term “monoclonal antibody” refers to a population of substantially homogeneous antibodies, ie, the antibody molecules comprised in the population are identical in amino acid sequence, except for natural mutations that may be present in minor amounts.
  • polyclonal antibody preparations typically comprise multiple different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes.
  • “Monoclonal” denotes 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.
  • 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, such as an antigen binding protein (eg, an antibody), and which can additionally be used in an animal to generate antibodies capable of binding the antigen.
  • a selective binding agent such as an antigen binding protein (eg, an antibody)
  • An antigen may have one or more epitopes capable of interacting with different antigen binding proteins (eg antibodies).
  • linker refers to a linking unit that joins two polypeptide fragments.
  • linkers appearing in the same formula may be the same or different.
  • the linker may be a peptide linker comprising 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.
  • “-" appears in the structural formula it means that the units on both sides are directly connected by covalent bonds.
  • bond appears in a structural unit, it means that the unit has no amino acids, and the units on either side of the unit are directly connected.
  • nucleic acid is used herein interchangeably 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, synthetic, naturally occurring and non-naturally occurring, having similar binding properties to the reference nucleic acid, and defined in Metabolized in a manner similar to the reference nucleotide.
  • nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • An isolated nucleic acid encoding the antigen-binding molecule refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such one or more nucleic acids in a single vector or in separate vectors molecule, and such one or more nucleic acid molecules present at one or more locations in the host cell.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence.
  • degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is mixed with bases and/or deoxygenated Inosine 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 an artificial chemical mimetic of the corresponding naturally occurring amino acid, and to both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • sequence identity means that when two sequences are optimally aligned, gaps are introduced as necessary to obtain the maximum percent sequence identity and any conservative substitutions are not considered part of the sequence identity, two The degree (percentage) to which amino acids/nucleic acids of a sequence are identical at equivalent positions.
  • alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • fused or “linked” refers to the covalent linking of components, such as an antigen binding module and an Fc domain, directly or via a linker.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop 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 and episomal mammalian vectors with a bacterial origin of replication).
  • vectors can integrate into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome.
  • expression vector or "expression construct” refers to a vector suitable for transforming a host cell and containing the expression of one or more heterologous coding regions operatively linked thereto and/or controlling (along with the host cell).
  • Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when an intron is present, RNA splicing of the coding region to which it is operably linked.
  • host cell refers to a cell 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. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells.
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster cells 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 puntiae, Pichia thermotolerans, Pichia willow salictaria), Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia, Saccharomycescerevisiae, Saccharomyces cerevisiae , Hansenula polymorpha, Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fus
  • Pichia any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Luke Mold (Chrysosporium lucknowense), any Fusarium species, Yarrowia lipolytica, and Neurospora crassa.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include primary subject cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that not all progeny will have the exact same DNA content due to deliberate or unintentional mutations. Mutant progeny having the same function or biological activity as the original transformed cell from which they were screened are included.
  • composition means a mixture comprising one or more antigen binding molecules described herein together with other chemical components such as physiologically/pharmaceutically acceptable carriers and excipients.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is nontoxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • subject or “individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles.
  • patient or “subject” are used interchangeably herein unless otherwise indicated.
  • cyno or “cynomolgus” refers to Macaca fascicularis.
  • the individual or subject is a human.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the interaction of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human , subjects, cells, tissues, organs or biological fluids.
  • sample refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject.
  • exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tears, faeces, sputum, mucous membrane secretions of secretory tissues and organs, vaginal secretions, ascites , pleura, pericardium, peritoneum, peritoneal and other body cavity fluids, fluid collected from bronchial lavage, synovial fluid, liquid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ condition culture medium), lavage fluid, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.
  • biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tears, faeces, sputum, mucous membrane
  • Treatment refers to clinical intervention that attempts to alter the natural course of the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, alleviation/reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of rate of disease progression, amelioration or palliation of disease state, and regression or amelioration of prognosis.
  • the antibodies of the disclosure are used to delay the development of a disease or slow the progression of a disease.
  • an “effective amount” is generally sufficient to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or underlying causes, prevent the occurrence of symptoms and/or their underlying causes, and/or ameliorate or ameliorate the impairment caused by or associated with the disease state (e.g. lung disease).
  • the effective amount is a therapeutically or prophylactically effective amount.
  • a “therapeutically effective amount” is sufficient to treat a disease state or symptom, especially a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other adverse effect in any way related to the disease state. The amount of progression of the desired symptoms.
  • 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.
  • a therapeutically or prophylactically effective amount may be administered in one or more administrations.
  • “Therapeutically effective amount” and “prophylactically effective amount” can vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual.
  • Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of a patient.
  • Antigen binding molecules of the present disclosure are provided.
  • antigen-binding molecules with favorable properties such as affinity, specificity for cell surface antigens, activity to specifically activate T cells in the presence of antigens, therapeutic activity, safety (e.g. lower cytokine Release), pharmacokinetic properties and druggability (such as yield, purity and stability, etc.).
  • the present disclosure provides an antigen binding molecule comprising at least one antigen binding moiety that specifically binds BCMA, at least one antigen binding moiety that specifically binds CD38, and at least one antigen binding moiety that specifically binds CD3.
  • the antigen-binding molecules of the present disclosure have at least one of the following functional activities:
  • the antigen binding molecule binds to CD38/BCMA/CD3d & CD3e with a KD of less than 200 nM. In some embodiments, the antigen binding molecule binds to CD38 or BCMA with a KD of less than 10 nM, and binds to CD3d & CD3e with a KD of less than 200 nM. The KD is determined by surface plasmon resonance.
  • the antigen-binding molecule can specifically kill cells expressing BCMA and/or CD38, but cannot kill cells not expressing BCMA and CD38. See Test Example 4 for specific test methods.
  • the present disclosure provides an antigen binding molecule comprising at least one antigen binding moiety that specifically binds BCMA, at least one antigen binding moiety that specifically binds CD38, and at least one antigen binding moiety that specifically binds CD3.
  • the antigen-binding module that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein:
  • the BCMA-VH has: BCMA-HCDR1 with amino acid sequence as shown in SEQ ID NO:5, BCMA-HCDR2 with amino acid sequence as shown in SEQ ID NO:6 and amino acid sequence as shown in SEQ ID NO:7 BCMA-HCDR3; and the BCMA-VL has: amino acid sequence such as BCMA-LCDR1 shown in SEQ ID NO: 8, amino acid sequence such as BCMA-LCDR2 shown in SEQ ID NO: 9 and amino acid sequence such as SEQ ID NO: BCMA-LCDR3 as shown in 10; or
  • the BCMA-VH has: BCMA-HCDR1 with amino acid sequence as shown in SEQ ID NO: 11, BCMA-HCDR2 with amino acid sequence as shown in SEQ ID NO: 12 and amino acid sequence as shown in SEQ ID NO: 13 BCMA-HCDR3; and the BCMA-VL has: amino acid sequence such as BCMA-LCDR1 shown in SEQ ID NO: 14, amino acid sequence such as BCMA-LCDR2 shown in SEQ ID NO: 15 and amino acid sequence such as SEQ ID NO: BCMA-LCDR3 indicated in 16; or
  • the BCMA-VH has: BCMA-HCDR1 with amino acid sequence as shown in SEQ ID NO: 17, BCMA-HCDR2 with amino acid sequence as shown in SEQ ID NO: 18 and amino acid sequence as shown in SEQ ID NO: 19 BCMA-HCDR3; and the BCMA-VL has: amino acid sequence such as BCMA-LCDR1 shown in SEQ ID NO: 20, amino acid sequence such as BCMA-LCDR2 shown in SEQ ID NO: 21 and amino acid sequence such as SEQ ID NO: BCMA-LCDR3 shown in 22;
  • BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat numbering rules.
  • the antigen binding molecule as described above, wherein:
  • amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 29
  • amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 32, or
  • amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 36
  • amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 40, or
  • amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 43
  • amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 45
  • the antigen-binding molecule of any one of the preceding, wherein the antigen-binding moiety 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 with amino acid sequence as shown in SEQ ID NO:55, CD3-HCDR2 with amino acid sequence as shown in SEQ ID NO:56 and amino acid sequence as shown in SEQ ID NO:57 CD3-HCDR3; and the CD3-VL has: the amino acid sequence of CD3-LCDR1 shown in SEQ ID NO: 58, the amino acid sequence of CD3-LCDR2 shown in SEQ ID NO: 59 and the amino acid sequence of such as SEQ ID NO: CD3-LCDR3 indicated at 60; or
  • the CD3-VH has: CD3-HCDR1 with amino acid sequence as shown in SEQ ID NO:55, CD3-HCDR2 with amino acid sequence as shown in SEQ ID NO:61 and amino acid sequence as shown in SEQ ID NO:62 CD3-HCDR3; and the CD3-VL has: the amino acid sequence of CD3-LCDR1 shown in SEQ ID NO: 58, the amino acid sequence of CD3-LCDR2 shown in SEQ ID NO: 59 and the amino acid sequence of such as SEQ ID NO: CD3-LCDR3 shown in 60;
  • CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 are defined according to the Kabat numbering rules.
  • the antigen-binding molecule according to any one of the preceding items, the amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO: 63, and the light chain variable region CD3-VL
  • the amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO: 64; or the amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO: 65, and the amino acid sequence of the light chain variable region CD3-VL is shown in Shown in SEQ ID NO: 64.
  • the antigen-binding molecule of any one of the preceding, wherein the antigen-binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region CD38-VL, wherein:
  • the CD38-VH has: CD38-HCDR1 with amino acid sequence as shown in SEQ ID NO:47, CD38-HCDR2 with amino acid sequence as shown in SEQ ID NO:48 and CD38-HCDR2 with amino acid sequence as shown in SEQ ID NO:49 HCDR3; and the CD38-VL has: the amino acid sequence of CD38-LCDR1 as shown in SEQ ID NO:50, the amino acid sequence of CD38-LCDR2 as shown in SEQ ID NO:51 and the amino acid sequence as shown in SEQ ID NO:52 CD38-LCDR3;
  • CD38-HCDR1, CD38-HCDR2, CD38-HCDR3, CD38-LCDR1, CD38-LCDR2 and CD38-LCDR3 are defined according to the Kabat numbering rules.
  • the antigen-binding molecule according to any one of the preceding items, the amino acid sequence of the heavy chain variable region CD38-VH is as shown in SEQ ID NO: 53, and the light chain variable region CD38-VL The amino acid sequence is shown in SEQ ID NO: 54.
  • the antigen binding molecule of any one of the preceding wherein
  • the BCMA-VH has: BCMA-HCDR1 with amino acid sequence as shown in SEQ ID NO:5, BCMA-HCDR2 with amino acid sequence as shown in SEQ ID NO:6 and amino acid sequence as shown in SEQ ID NO:7 BCMA-HCDR3; and the BCMA-VL has: amino acid sequence such as BCMA-LCDR1 shown in SEQ ID NO: 8, amino acid sequence such as BCMA-LCDR2 shown in SEQ ID NO: 9 and amino acid sequence such as SEQ ID NO: BCMA-LCDR3 shown in 10, and
  • the CD3-VH has: CD3-HCDR1 with amino acid sequence as shown in SEQ ID NO:55, CD3-HCDR2 with amino acid sequence as shown in SEQ ID NO:61 and CD3-HCDR2 with amino acid sequence as shown in SEQ ID NO:62 HCDR3; and the CD3-VL has: the amino acid sequence of CD3-LCDR1 as shown in SEQ ID NO: 58, the amino acid sequence of CD3-LCDR2 as shown in SEQ ID NO: 59 and the amino acid sequence as shown in SEQ ID NO: 60 CD3-LCDR3, and
  • the CD38-VH has: CD38-HCDR1 with amino acid sequence as shown in SEQ ID NO:47, CD38-HCDR2 with amino acid sequence as shown in SEQ ID NO:48 and CD38-HCDR2 with amino acid sequence as shown in SEQ ID NO:49 HCDR3; and the CD38-VL has: the amino acid sequence of CD38-LCDR1 as shown in SEQ ID NO:50, the amino acid sequence of CD38-LCDR2 as shown in SEQ ID NO:51 and the amino acid sequence as shown in SEQ ID NO:52 CD38-LCDR3; or
  • the BCMA-VH has: BCMA-HCDR1 with amino acid sequence as shown in SEQ ID NO: 11, BCMA-HCDR2 with amino acid sequence as shown in SEQ ID NO: 12 and amino acid sequence as shown in SEQ ID NO: 13 BCMA-HCDR3; and the BCMA-VL has: amino acid sequence such as BCMA-LCDR1 shown in SEQ ID NO: 14, amino acid sequence such as BCMA-LCDR2 shown in SEQ ID NO: 15 and amino acid sequence such as SEQ ID NO: BCMA-LCDR3 shown in 16, and
  • the CD3-VH has: CD3-HCDR1 with amino acid sequence as shown in SEQ ID NO:55, CD3-HCDR2 with amino acid sequence as shown in SEQ ID NO:56 and CD3-HCDR2 with amino acid sequence as shown in SEQ ID NO:57 HCDR3; and the CD3-VL has: the amino acid sequence of CD3-LCDR1 as shown in SEQ ID NO: 58, the amino acid sequence of CD3-LCDR2 as shown in SEQ ID NO: 59 and the amino acid sequence as shown in SEQ ID NO: 60 CD3-LCDR3, and
  • the CD38-VH has: CD38-HCDR1 with amino acid sequence as shown in SEQ ID NO:47, CD38-HCDR2 with amino acid sequence as shown in SEQ ID NO:48 and CD38-HCDR2 with amino acid sequence as shown in SEQ ID NO:49 HCDR3; and the CD38-VL has: the amino acid sequence of CD38-LCDR1 as shown in SEQ ID NO:50, the amino acid sequence of CD38-LCDR2 as shown in SEQ ID NO:51 and the amino acid sequence as shown in SEQ ID NO:52 CD38-LCDR3;
  • BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2, BCMA-LCDR3, CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2, CD3-LCDR3, CD38-HCDR1, CD38-HCDR2, CD38-HCDR3, CD38-LCDR1, CD38-LCDR2 and CD38-LCDR3 are defined according to the Kabat numbering convention.
  • the antigen-binding molecule according to any one of the preceding items, the amino acid sequence of the heavy chain variable region BCMA-VH is as shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region BCMA-VL is The amino acid sequence is shown in SEQ ID NO: 32, the amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO: 65, and the amino acid sequence of the light chain variable region CD3-VL is shown in SEQ ID NO : 64, the amino acid sequence of the heavy chain variable region CD38-VH is shown in SEQ ID NO: 53, and the amino acid sequence of the light chain variable region CD38-VL is shown in SEQ ID NO: 54.
  • the antigen-binding molecule according to any one of the preceding items, the amino acid sequence of the heavy chain variable region BCMA-VH is as shown in SEQ ID NO: 36, the light chain variable region BCMA-VL
  • the amino acid sequence is shown in SEQ ID NO: 40
  • the amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO: 63
  • the amino acid sequence of the light chain variable region CD3-VL is shown in SEQ ID NO : 64
  • the amino acid sequence of the heavy chain variable region CD38-VH is shown in SEQ ID NO: 53
  • the amino acid sequence of the light chain variable region CD38-VL is shown in SEQ ID NO: 54.
  • the amino acid sequences of the two subunits are shown in SEQ ID NO: 68.
  • the amino acid sequences of the two subunits are shown in SEQ ID NO: 100 and SEQ ID NO: 101, respectively.
  • the present disclosure provides a trispecific antigen binding molecule, which may be trivalent, tetravalent, pentavalent, hexavalent and more.
  • the antigen-binding molecule comprises an antigen-binding moiety, which may be any polypeptide molecule capable of binding to a target antigen, such as an antibody fragment.
  • the antigen binding moiety refers to an antibody fragment comprising a heavy chain variable region and a light chain variable region that together constitute a domain that binds an antigen of interest .
  • the antigen binding moiety is a Fab or scFv.
  • the antigen-binding moieties in the antigen-binding molecules of the present disclosure can be linked in any manner that does not affect the binding activity. Exemplarily, the schematic diagram of the structure of the antigen-binding molecule is shown in Fig. 1A, Fig. 1B, Fig. 1C, Fig. 1D or Fig. 1E.
  • 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 arrive at 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.
  • Sites of interest for substitution mutagenesis include CDRs and FRs.
  • 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 amino acid side chain classes.
  • Amino acid substitutions can be introduced into an antibody of interest, and the products screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • Trp(W) Tyr Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
  • amino acids can be grouped as follows:
  • Non-conservative substitutions would entail replacing a member of one of these classes for a member of another class.
  • substitutional variant involves substituting 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 variant selected for further study will have an altered (e.g. improved) certain biological property (e.g. increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will be substantially Some of the biological properties of the parental antibody are retained.
  • An exemplary substitution variant is an affinity matured antibody, which can 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 (eg, binding affinity).
  • Alterations can be made to the CDRs, eg, to improve antibody affinity. Such changes can be made to CDR "hot spots", i.e. residues encoded by codons that undergo mutation at high frequency during the somatic maturation process, and/or residues that contact antigen, while making changes to the resulting variant VH or VL test for 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 residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling.
  • HCDR3 and LCDR3 are frequently targeted.
  • substitutions, insertions or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen.
  • conservative changes eg, conservative substitutions, as provided herein
  • Such changes may eg be outside antigen contacting residues in the CDRs.
  • 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 of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis".
  • a residue or group of target residues e.g. charged residues such as Arg, Asp, His, Lys and Glu
  • neutral or negatively charged amino acids e.g. Ala or polyalanine
  • Further substitutions can be introduced at amino acid positions showing functional sensitivity to the initial substitution.
  • contact points between antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and neighboring residues can be targeted or eliminated as candidates for substitution.
  • Variants can be screened to determine whether they contain desired properties.
  • Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, 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 of the N- or C-terminus of the antibody to enzymes or polypeptides that extend the serum half-life of the antibody.
  • the Fc region of an antigen binding molecule of the disclosure comprises 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.
  • a native IgG Fc region specifically an IgG 1 Fc region or an IgG 4 Fc region, may result in the targeting of an antigen binding molecule of the present disclosure to cells expressing Fc receptors, rather than cells expressing antigen.
  • the engineered Fc regions of the present disclosure exhibit reduced binding affinity to Fc receptors and/or reduced effector functions.
  • the engineered Fc region has a binding affinity for Fc receptors that is reduced by more than 50%, 80%, 90%, or 95% compared to a native Fc region.
  • the Fc receptor is an Fc gamma receptor.
  • the Fc receptor is a human Fc ⁇ receptor, eg, Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIB, Fc ⁇ RIIIa.
  • the engineered Fc region also has reduced binding affinity for complement, such as C1q, compared to a native Fc region.
  • the engineered Fc region has no reduced binding affinity for neonatal Fc receptor (FcRn) compared to a native Fc region.
  • the engineered Fc region has reduced effector function, which may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced Antibody-dependent cell-mediated cytotoxicity (ADCC), decreased antibody-dependent cellular phagocytosis (ADCP), decreased cytokine secretion, decreased immune complex-mediated antigen uptake by antigen-presenting cells, decreased interaction with NK cells decreased binding to macrophages, decreased binding to monocytes, decreased binding to polymorphonuclear cells, decreased direct signaling-induced apoptosis, decreased dendritic cell maturation, or decreased T cells primed.
  • CDC complement-dependent cytotoxicity
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • cytokine secretion decreased immune complex-mediated antigen uptake by antigen-presenting cells
  • decreased interaction with NK cells decreased binding to macrophages
  • monocytes decreased binding to monocytes
  • polymorphonuclear cells
  • amino acid residue substitutions at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function.
  • the Fc region is a human IgG 1 Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is based on the EU index.
  • amino acid residue substitutions at positions such as 228 may reduce effector function.
  • Antigen binding molecules may also comprise disulfide bond engineering, eg, 354C of the first subunit and 349C of the second subunit.
  • disulfide bond engineering eg, 354C of the first subunit and 349C of the second subunit.
  • the 356th amino acid residue of the Fc region may be E or D, and the 358th amino acid residue may be M or L.
  • the 356th amino acid residue of the Fc region may be E, and the 358th amino acid residue may be M.
  • the 356th amino acid residue of the Fc region may be D, and the 358th amino acid residue may be L.
  • Antigen binding molecules may comprise different antigen binding moieties fused to the two subunits of the Fc region, thus potentially leading to undesired homodimerization.
  • the Fc region of the present disclosure comprises modifications according to the knob-into-hole (KIH) technique, which involves the introduction of a knob at the interface of the first subunit and the introduction of a knob at the interface of the second subunit.
  • KH knob-into-hole
  • a hole structure is introduced at the interface of the base.
  • the bulge structure is constructed by replacing small amino acid side chains from the interface of the first subunit with larger side chains such as tyrosine or tryptophan. Instead, the pore structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller ones, such as alanine or threonine.
  • Protrusion structures and hole structures are prepared by changing the nucleic acid encoding the polypeptide, and the optional amino acid substitutions are shown in the table below:
  • the specific amino acid sequence of the Fc region is as follows:
  • knob-and-hole technique In addition to the knob-and-hole technique, other techniques for modifying the CH3 domain of the heavy chain of a multispecific antibody to achieve heterodimerization are known in the art, for example WO96/27011, WO98/050431, EP1870459, WO2007/ 110205, WO 007/147901, WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954 and WO013/096291.
  • the C-terminus of the Fc region may be a complete C-terminus ending with the amino acid residue PGK; it may also be a shortened C-terminus in which, for example, one or two C-terminal amino acid residues have been removed.
  • the C-terminus of the heavy chain is a shortened C-terminus ending in PG.
  • a composition of intact antibodies can include a population of antibodies from which all K447 residues and/or G446+K447 residues have been removed.
  • a composition of intact antibodies can include a population of antibodies in which the K447 residue and/or the G446+K447 residues have not been removed.
  • the composition of whole antibodies has a population of antibodies with and without a K447 residue and/or a mixture of antibodies with 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 molecule are provided.
  • nucleic acids In the case of native antibodies, native antibody fragments or bispecific antigen binding molecules with homodimeric heavy chains, two nucleic acids are required, one for the light chain or fragment thereof and one for the heavy chain or fragment 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 and/or heavy chains 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 heteromonomeric Fc region polypeptide, One for the second light chain and one for the second heavy chain comprising a second heteromeric monomeric Fc region polypeptide.
  • These four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors, usually these nucleic acids are located on two or three expression vectors, ie one vector may contain more than one of these nucleic acids.
  • the present disclosure provides an isolated nucleic acid encoding an antigen binding molecule as previously described. Such nucleic acid may be derived from an independent polypeptide chain encoding any of the foregoing.
  • the present disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids.
  • the disclosure provides host cells comprising such nucleic acids.
  • an antigen-binding molecule comprising, under conditions suitable for expression of the antigen-binding molecule, culturing a host cell comprising a nucleic acid encoding said antigen-binding molecule, as provided above, and optionally recovering said antigen binding molecule 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 (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antigen-binding molecule), or produced recombinantly or by chemical synthesis. get.
  • Suitable host cells for cloning or expressing vectors encoding antigen-binding molecules include prokaryotic or eukaryotic cells as described herein.
  • antigen binding molecules can be produced in bacteria, especially when the antigen binding molecules do not require glycosylation and Fc effector functions. After expression, antigen binding molecules can be isolated from bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antigen-binding molecules, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in Antigen binding molecules are produced with partially or fully human glycosylation patterns.
  • Suitable host cells suitable for expressing (glycosylated) antigen binding molecules may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells.
  • a number of baculovirus strains have been identified for use in combination with insect cells, particularly for the transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, e.g. US5959177, US6040498, US6420548 , US7125978 and US6417429; vertebrate cells can also be used as hosts, eg 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); 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 hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells.
  • Suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as YO, NSO and Sp2/0.
  • CHO Chinese Hamster Ovary
  • myeloma cell lines such as YO, NSO and Sp2/0.
  • the present disclosure also provides immunoconjugates comprising an antigen binding molecule conjugated to one or more cytotoxic agents such as chemotherapeutics 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 chemotherapeutics 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 antigen binding molecules provided by the present disclosure can be used to detect the presence of an antigen of interest in a biological sample.
  • the term “detection” encompasses quantitative or qualitative detection.
  • the biological sample comprises cells or tissue, such as tumor tissue.
  • an antigen binding molecule for use in a diagnostic or detection method is provided.
  • methods of detecting the presence of an antigen of interest in a biological sample are provided.
  • the method comprises contacting a biological sample with an antigen-binding molecule under suitable 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 BCMA, CD38 or CD3 are biomarkers used to select patients.
  • Exemplary disorders that can be diagnosed using the antigen binding molecules of the disclosure such as a B cell disorder or an autoimmune disease selected from the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia amyloidosis, WM, solitary plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma; the autoimmune disease is systemic lupus erythematosus.
  • Labeled antigen binding molecules include, but are not limited to, labels or moieties for direct detection (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties for indirect detection (e.g., indirect detection via enzymatic reactions or molecular interactions).
  • modules 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 comprises any of the antigen binding molecules provided herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises any of the antigen binding molecules provided herein and at least one additional therapeutic agent.
  • compositions of antigen-binding molecules described in the present disclosure are prepared by mixing such antigen-binding molecules having the desired purity with one or more optional pharmaceutically acceptable carriers, the pharmaceutical composition In the form of a lyophilized composition or an aqueous solution.
  • Formulations for in vivo administration are generally sterile. Sterility is readily achieved, for example, by filtration through sterile filters.
  • antigen binding molecules Any of the antigen binding molecules provided herein can be used in methods of treatment.
  • the present disclosure provides the use of an antigen binding molecule in the manufacture or preparation of a medicament.
  • the medicament is for the treatment of a B-cell disorder or an autoimmune disease selected from the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloid Degeneration, WM, solitary plasmacytoma, extramedullary plasmacytoma, sclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma;
  • the autoimmune disease is systemic lupus erythematosus.
  • the drug is in the form of an effective amount for the above 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 therapeutic agents agent).
  • additional therapeutic agent e.g., one, two, three, four, five, or six additional therapeutic agents agent.
  • a "subject" according to any of the above embodiments may be a human.
  • a pharmaceutical composition comprising said antigen binding molecule, eg, for any of the above pharmaceutical uses or methods of treatment.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • antigen binding molecules of the present disclosure can be used alone or in combination with other agents for therapy.
  • an antigen binding molecule of the disclosure can be co-administered with at least one additional therapeutic agent.
  • the antigen binding molecules of the present disclosure can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if local treatment is desired, intralesional.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic.
  • a variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • antigen binding molecules of the present disclosure will be formulated, dosed and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular 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.
  • Antigen binding molecules need not, but are optionally, formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents depends on the amount of antigen-binding molecule present in the pharmaceutical composition, the type of disorder 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 any route empirically/clinically determined to be suitable.
  • 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 amount of the therapeutic molecule Type, severity and course of disease, whether administered for prophylactic or therapeutic purposes, previous therapy, patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician.
  • the therapeutic molecule is suitably administered to the patient at one time 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 dosage might 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 comprising materials useful for the treatment, prevention and/or diagnosis of the disorders described above.
  • the article comprises 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 various materials such as glass or plastic.
  • the container contains a composition effective, alone or in combination with another composition, for the treatment, prophylaxis and/or diagnosis of a condition, and may have a sterile access opening (e.g., the container may have a stopper pierceable by a hypodermic needle). IV solution bag or vial).
  • At least one active agent in the composition is an antigen binding molecule of the present disclosure.
  • the label or package insert indicates that the composition is used to treat the condition of choice.
  • the 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 combination
  • the drug contains an additional cytotoxic or other therapeutic agent.
  • the article of manufacture of 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 comprising a pharmaceutically acceptable buffer. It may further comprise other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • the sequences encoding human BCMA with human IgG1-Fc tag and cynomolgus BCMA extracellular region were inserted into the phr vector to construct expression plasmids, which were then transfected into HEK293.
  • the sequence encoding the human BCMA extracellular region with His tag was inserted into the phr vector to construct an expression plasmid, which was then transfected into HEK293.
  • the specific transfection steps are as follows: HEK293E cells were inoculated in freestyle expression medium (containing 1% FBS, Gibco, 12338-026) at 1 ⁇ 10 6 /mL the day before, and placed in a 37-degree constant temperature shaker (120 rpm) for 24 hours. Hour.
  • the transfection plasmid and transfection reagent PEI were sterilized with a 0.22 ⁇ m filter, and then the transfection plasmid was adjusted to 100 ⁇ g/100 mL cells, and the mass ratio of PEI (1 mg/mL) and plasmid was 3:1, which was taken as Mix 10 mL of Opti-MEM with 200 ⁇ g of plasmid and let it stand for 5 minutes; take another 10 mL of Opti-MEM and 400 ⁇ g of PEI, mix it and let it stand for 5 minutes. The plasmid and PEI were mixed and allowed to stand for 15 minutes.
  • the plasmid and PEI mixture was slowly added to 200 mL of HEK293E cells, and cultured in a shaker with 8% CO 2 , 120 rpm, and 37°C. On day 3 of transfection, 10% volume of feed medium (20 mM glucose + 2 mM L-glutamic acid) was supplemented. On the 6th day after transfection, samples were taken and centrifuged at 4500 rpm for 10 minutes to collect the cell supernatant, and purified according to the method in Test Example 2. The purified protein can be used in the experiments of the following examples or test examples.
  • Human BCMA with human Fc tag human BCMA-ECD-Fc (SEQ ID NO: 1)
  • the underline is the signal peptide sequence; the italic part is the human Fc-linker-tag
  • the underline is the signal peptide sequence; the italic part is His6-linker-tag
  • Cynomolgus monkey BCMA with human Fc tag cynoBCMA-ECD-Fc (SEQ ID NO: 4)
  • the underline is the signal peptide sequence; the italic part is the human Fc-tag
  • Example 2 Protein A affinity chromatography purification of recombinant protein with Fc tag and nickel column purification of recombinant protein with His tag
  • the BCMA supernatant sample expressed by cells was centrifuged at high speed to remove impurities, and then purified by Protein A column. Rinse the column with PBS until the A280 reading drops to baseline.
  • the target protein was eluted with 100mM acetic acid, pH3.5, and neutralized with 1M Tris-HCl, pH8.0. After the eluted sample was properly concentrated, the medium was changed to PBS, and the obtained protein was identified as correct by electrophoresis, peptide map, and LC-MS, and then allocated for use.
  • Example 3 Construction and identification of cell lines expressing recombinant human CD38, human BCMA and cynomolgus BCMA
  • This disclosure constructs a cell line expressing human CHO-K1/hCD38.
  • the full-length human CD38 gene 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-hCD38 ( CRL-11268) to package the virus, after 48 hours of transfection, the virus was collected to infect CHO-K1 cells ( CCL-61). CHOK1 monoclonal cells highly expressing hCD38 were obtained by flow sorting 72 hours after infection.
  • the present disclosure constructs a K562-BCMA cell line expressing human BCMA.
  • the full-length human BCMA gene 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-human BCMA ( CRL-11268) to package the virus, after 48 hours of transfection, the virus was collected to infect K562 cells ( CCL-243).
  • K562 monoclonal cells highly expressing human BCMA were obtained by flow sorting 72 hours after infection.
  • This disclosure also constructs the CHO-K1-cynoBCMA cell line expressing cynomolgus monkey, clones the full-length gene of cynomolgus monkey BCMA into the mammalian cell expression vector pCDH, uses pVSV-G, pCMV-dR8.91 and pCDH-cynoBCMA The three plasmids co-transfected HEK293T cells ( CRL-11268) to package the virus, and after 48 hours of transfection, the virus was collected to infect CHOK1 cells ( CCL-61). CHOK1 monoclonal cells highly expressing cynoBCMA were obtained by flow sorting 72 hours after infection.
  • the present disclosure prepares monoclonal antibodies against human BCMA through hybridoma technology.
  • the obtained antibody specifically binds to human BCMA with high affinity and can cross-react with cynomolgus monkey BCMA; the obtained antibody has better binding activity to human BCMA and cynomolgus monkey BCMA on the cell surface, and the binding activity is affected by the solubility BCMA interference is minimal.
  • Human BCMA-ECD-his and cynoBCMA-ECD-his were used as cross-immunization reagents, Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No.77161) was used as an adjuvant to cross-immunize mice. After the initial immunization and 7 booster immunizations, the mouse 10-6# (titer 625K) with high antibody titer in serum was selected for splenocyte fusion. After fusion, according to the hybridoma cell growth density, the hybridoma culture supernatant is detected, and the antibody that specifically binds to the cell surface BCMA is screened.
  • Monoclonal hybridoma cell lines 4E3, 33H4 and 27 with good activity were screened.
  • Hybridoma cells in the logarithmic growth phase were collected separately, RNA was extracted with NucleoZol (MN) (according to the kit instructions), and reverse transcription was performed (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A).
  • MN NucleoZol
  • the cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and then sequenced.
  • the amino acid sequences of the CDRs and variable regions of 4E3, 33H4 and 27 are as follows:
  • the underlined area is the CDR area obtained according to the Kabat numbering rule
  • the humanization of the mouse monoclonal antibody was carried out according to the methods published in many documents in this field.
  • the homologous sequences of the light chain variable region (VL) and the heavy chain variable region (VH) were searched from the human germline database, The CDR region of the murine antibody was grafted onto the human template, and some residues of VL and VH were mutated, and the constant region of the murine antibody was replaced with a human constant region to obtain the final humanized molecule.
  • Graft represents mouse antibody CDR implanted into human germline FR region sequence.
  • R71A means that R at position 71 is mutated back to A according to the Kabat numbering system. The same below.
  • the specific sequence of the antibody variable region obtained by humanizing the murine antibody 4E3 is as follows (the underline indicates CDR, the same below):
  • the specific sequence of the antibody variable region obtained by humanizing the mouse antibody 33H4 is as follows:
  • the specific sequence of the antibody variable region obtained by humanizing murine antibody 27 is as follows:
  • CD38 binding molecules of the present disclosure may be derived from any suitable antibody. Particularly suitable antibodies are described, for example, in Patent Publication No. WO2020052546 (herein incorporated by reference in its entirety).
  • the CDR and variable region sequences of the anti-CD38 arms in the antibodies of the present disclosure are as follows:
  • variable region The specific sequence of the variable region is as follows:
  • CD3 binding molecules of the present disclosure may be derived from any suitable antibody. Particularly suitable antibodies are described eg in International Application No. WO2020114478 (herein incorporated by reference in its entirety).
  • the CDR and variable region sequences of the anti-CD3 arm in the bispecific antibody of the present disclosure are as follows:
  • variable region The specific sequence of the variable region is as follows:
  • Anti-CD38 antibody variable regions, anti-BCMA antibody variable regions, anti-CD3 antibody variable regions, and IgG 1 mutant IgG 1 (AA) (L234A/L235A) were combined.
  • Format5 is an asymmetric molecular structure, and the complete molecule has three chains, all of which are different, and its structure is shown in Figure 1A.
  • Chain 1 VH(anti-BCMA)-CH1-IgG 1 Fc(Knob,AA)-Linker 1-VH(1149)-Linker 2-VL(1149);
  • Chain 2 VL(anti-BCMA)-CL;
  • the single underlined region is the CDR region of CD38 and CD3 binding domain obtained according to the Kabat numbering rule, and the italicized region is the constant region.
  • Format5-6164 is an asymmetric molecular structure, and the complete molecule has three chains, all of which are different, and its structure is shown in Figure 1A.
  • Chain 1 VH(anti-BCMA)-CH1-IgG 1 Fc(Knob,AA)-Linker 1-VH(1149)-Linker 2-VL(1149);
  • Chain 2 VL(anti-BCMA)-CL;
  • Chain 3 (Hole-5-6164): VH(6164)-Linker 1-VL(6164)-Linker 2-IgG 1 Fc(Hole, AA)-Linker 3-VH(1149)-Linker 4 -VL(1149).
  • the single underlined region is the CDR region of the binding domain of CD38 and CD3 obtained according to the Kabat numbering rule, and the italicized region is the constant region.
  • Format12 is a molecule with a symmetrical structure, including two identical heavy chains (chain 1) and two identical light chains (chain 2), and its structure is shown in Figure 1B.
  • Chain 1 VH(anti-BCMA)-CH1-VH(S107E)-Linker 1-VL(S107E)-Linker 2-IgG 1 Fc(AA)-Linker 3-VH(1149)-Linker 4- VL(1149);
  • Chain 2 VL(anti-BCMA)-CL.
  • Format12-6164 is a molecule with a symmetrical structure, including two identical heavy chains (chain 1) and two identical light chains (chain 2), and its structure is shown in Figure 1B.
  • Chain 1 VH(anti-BCMA)-CH1-VH(6164)-Linker 1-VL(6164)-Linker 2-IgG 1 Fc(AA)-Linker 3-VH(1149)-Linker 4- VL(1149);
  • Chain 2 VL(anti-BCMA)-CL.
  • Format20 is an asymmetric molecular structure, and the complete molecule has three chains, all of which are different, and its structure is shown in Figure 1C.
  • Chain 1 VH(anti-BCMA)-CH1-IgG 1 Fc(Hole,AA);
  • Chain 2 VL(anti-BCMA)-CL;
  • Chain 3 (Knob-20): VH(1149)-Linker 1-VL(1149)-Linker 2-VH(S107E)-Linker 3-VL(S107E)-Linker 4-IgG 1 Fc(Knob, AA).
  • the single underlined region is the CDR region of CD38 and CD3 binding domain obtained according to the Kabat numbering rule, and the italicized region is the constant region.
  • Format20-6164 is an asymmetric molecular structure, and the complete molecule has three chains, all of which are different, and its structure is shown in Figure 1C.
  • Chain 1 VH(anti-BCMA)-CH1-IgG 1 Fc(Hole,AA);
  • Chain 2 VL(anti-BCMA)-CL;
  • Chain 3 (Knob-20-6164): VH(1149)-Linker 1-VL(1149)-Linker 2-VH(6164)-Linker 3-VL(6164)-Linker 4-IgG 1 Fc( Knob, AA).
  • the single underlined region is the CDR region of CD38 and CD3 binding domain obtained according to the Kabat numbering rule, and the italicized region is the constant region.
  • Format21 is a molecule with a symmetrical structure, including two identical heavy chains (chain 1) and two identical light chains (chain 2), and its structure is shown in Figure 1D.
  • Chain 1 VH(1149)-CH1-VH(S107E)-Linker 1-VL(S107E)-Linker 2-IgG 1 Fc(AA)-Linker 3-VL(anti-BCMA)-Linker 4- VH (anti-BCMA);
  • Chain 2 VL(1149)-CL.
  • Format21-6164 is a molecule with a symmetrical structure, including two identical heavy chains (chain 1) and two identical light chains (chain 2), and its structure is shown in Figure 1D.
  • Chain 1 VH(1149)-CH1-VH(6164)-Linker 1-VL(6164)-Linker 2-IgG 1 Fc(AA)-Linker 3-VL(anti-BCMA)-Linker 4- VH (anti-BCMA);
  • Chain 2 VL(1149)-CL, the structure of which is shown in Figure 1D.
  • the single underlined region is the CDR region of the CD38 binding domain obtained according to the Kabat numbering rule, and the italicized region is the constant region.
  • Format24 is an asymmetric molecule with four chains in total, and its structure is shown in Figure 1E.
  • Chain 1 VL(anti-BCMA)-Linker 1-VH(anti-BCMA)-Linker 2-VH(1149)-CH1-IgG 1 Fc(Hole,AA);
  • Chain 2 (two): VL(1149)-CL;
  • Chain 3 (Knob-24): VH(1149)-CH1-IgG1Fc(Knob, AA)-Linker 1 -VL(107E)-Linker 2-VH(107E).
  • the single underlined region is the CDR region of CD38 and CD3 binding domain obtained according to the Kabat numbering rule, and the italicized region is the constant region.
  • Antigen-binding molecules were constructed according to Table 10.
  • T5 means that hu4E3L1 and hu4E3H1 are used as variable regions that bind BCMA
  • T3 means that hu33H4L2 and hu33H4H1 are used as variable regions that bind BCMA
  • T1 means that hu27L1 and hu27H3 are used as variable regions that bind BCMA. Variable area.
  • -5 in T5-5 means adopting Format5 structure
  • -5-6164 in T5-5-6164 means adopting Format5-6164 structure
  • -12 in T5-12 means adopting Format12 structure, and so on .
  • the single underlined region is the CDR region obtained according to the Kabat numbering rule, and the italicized region is the constant region.
  • the positive control molecules GBR-1342 and AMGEN701 used in this disclosure include SEQ ID NO: 178, SEQ ID NO: 179 and SEQ ID NO: 128 in WO2016071355.
  • the amino acid sequence of AMGEN701 includes SEQ ID NO: 74 in WO2017134134.
  • Test example 1 FACS determination of the expression levels of hBCMA and hCD38 on the surface of MM cells
  • the expression levels of MM-related cell lines were detected by FACS.
  • the measurement of CD38 expression is as follows: take the cells with a viability >90% and add the cells to a 96-well plate (3590#, Corning), centrifuge at 300g for 5 minutes, and wash once with 1% BSA . 100 ⁇ L of 10 ⁇ g/mL CD38 antibody prepared in 1% BSA was added respectively, and incubated at 4° C. for 1 hour. Wash twice with 1% BSA. Add 50 ⁇ L of PE-human F(ab') 2 (1:400), and incubate at 4°C for 0.5 hours. Wash twice with 1% BSA.
  • BCMA expression is as follows: take the cells with a viability >90% and add the cells to a 96-well plate (3590#, Corning), centrifuge at 300g for 5 minutes, and wash once with 1% BSA . Add the BCMA antibody (BL2-A:BCMA-APC-A) diluted according to the instructions, and incubate at 4°C for 1 hour. Wash twice with 1% BSA. Add 100 ⁇ L of 1% BSA to resuspend, then detect the fluorescence values of PE and APC on the cell surface with a FACS instrument, and use the geometric mean value for parallel comparison.
  • Test example 2 Affinity of the antibody of the present disclosure to CD38/BCMA/CD3d&CD3e protein
  • this test example uses the instrument Biacore T200 to detect the antibody and hCD3d&CD3e (31.2Kda, Cat.#CT038-H2508H, S.B), hCD38 (30.7Kda, HR) and hBCMA (7.8 Kda, Cat. #BCA-H52y, Acro) binding capacity.
  • the specific method is as follows: use a CM5 biosensing chip coupled with an anti-human antibody to affinity capture the antibody, then flow a solution containing 100nM BCMA-His on the surface of the chip for 180 seconds to saturate the BCMA binding site of the antibody, and then add hCD3d&CD3e Or hCD38, real-time detection with Biacore T200 instrument.
  • the data fitting model adopts 1:1Model. Affinity data for binding of the antibodies of the present disclosure to the corresponding antigens is shown in the table below.
  • the stable transgenic cell line K562/hBCMA overexpressing human BCMA, the stable transgenic cell line CHO K1/hCD38 overexpressing human CD38, and the MM tumor cell RPMI-8226 co-expressing CD38 and BCMA were used as target cells to detect the bispecific
  • the target-specific cytotoxic activity of the specific antibody was tested, and the non-specific cytotoxic activity of the bispecific antibody of the present disclosure was detected by using CHOK1 that does not express CD38 and BCMA as the target cell.
  • Fresh PBMC (purchased from Xuanfeng Biological Company) were centrifuged at 300g for 10 minutes, resuspended with 1640+10% FBS (solution A) containing 200U IL2, and cultured overnight in a T75 culture bottle (density 2 ⁇ 10 6 /mL) ; After collection and centrifugation, resuspend and count with IMDM+10% FBS, DMEM/F-12 1:1+10% FBS and 1640+10% FBS culture medium, adjust the cell number to 1.5 ⁇ 10 6 cells/mL, Add 50 ⁇ L to each well.
  • Collect target cells centrifuge at 1000rpm for 3min, resuspend and count, adjust the cell number to 3 ⁇ 10 5 cells/mL, add 25 ⁇ L to each well, and E:T Ratio is 10:1.
  • the antibody was diluted with the culture medium corresponding to the cells respectively, the initial concentration was 400nM (4 ⁇ final concentration), 10-fold diluted 9 gradients, and 25 ⁇ L was added to each well. Then the cells were cultured in an incubator at 37°C with 5% CO 2 for 48h.
  • Fluorescent signal detection Take out the culture plate, centrifuge at 1000rpm for 3min, pipette 50 ⁇ L of the supernatant into a new 96-well plate, store it in a -20°C refrigerator, and add 50 ⁇ L of one- Glo, incubate at room temperature for 5 minutes, detect the value of luminescence (the luminescence value of cells in the no antibody group is defined as 0 killing), and calculate the killing percentage of different antibodies at different concentrations.
  • Test Example 5 Effect of Soluble BCMA/CD38/APRIL on the In Vitro Cytotoxic Activity of the Antibody of the Disclosure
  • soluble BCMA (average 30ng/mL) in the blood of multiple myeloma patients will interfere with the specific binding of BCMA-CD3 bispecific antibody to the membrane surface of BCMA.
  • the content of APRIL in the serum of MM patients is also the same as that of healthy people 5.9 times that of MM, and soluble CD38 also increased in certain proportion in MM patients.
  • the cell line RPMI-8226 co-expressing CD38 and BCMA was used as the target cell. Soluble BCMA, CD38 and APRIL were added.
  • Fresh PBMC (purchased from Xuanfeng Biological Company) were centrifuged at 300g for 10min, resuspended with 1640+10% FBS (solution A) containing 200U IL2, and cultured overnight in a T75 culture bottle (density 2E6/mL); collected after centrifugation Resuspend and count with 1640+10% FBS medium, adjust the cell number to 1.5 ⁇ 10 6 cells/mL, and add 50 ⁇ L to each well.
  • Collect target cells centrifuge at 1000rpm for 3 minutes, resuspend and count, adjust the cell number to 3 ⁇ 10 5 cells/mL, add 25 ⁇ L to each well, and E:T Ratio is 10:1.
  • the antibody was diluted with the culture medium corresponding to the cells respectively, the initial concentration was 400nM (4 ⁇ final concentration), 10-fold diluted 9 gradients, and 25 ⁇ L was added to each well. Then the cells were cultured in an incubator at 37°C with 5% CO 2 for 48h. Fluorescent signal detection: Take out the culture plate, centrifuge at 1000rpm for 3min, pipette 50 ⁇ L of the supernatant into a new 96-well plate, store it in a -20°C refrigerator, and add 50 ⁇ L of one- Glo, incubate at room temperature for 5 minutes, detect the value of luminescence (the luminescence value of cells in the non-antibody group is defined as 0 killing), and calculate the killing percentage of different antibodies at different concentrations.
  • Relative IC 50 means the ratio of IC50 with soluble protein to IC50 without soluble protein.
  • CD3 T cell engager molecules cause a cytokine storm. Therefore, when developing CD3 T cell engagement molecules, it is necessary to keep cytokines, especially the factor IL-6 that has nothing to do with drug efficacy but can cause side effects, at a low level.
  • Centrifuge CHO-K1 cells at 1000 rpm for 1 minute collect 50 ⁇ L of cell supernatant and dilute 6 times with sample universal diluent, and also dilute IL-6 or IL-2 standard 6 times, add diluted samples or different concentrations to the detection plate
  • reaction wells wash the plate 5 times after incubating at 37°C for 60 minutes, add enzyme conjugate working solution (100 ⁇ L/well), seal the reaction wells with new sealing tape, incubate at 37°C for 30 minutes, wash the plate 5 times, add Color substrate (TMB) 100 ⁇ L/well, incubate at 37°C for 8 minutes in the dark, add reaction stop solution 100 ⁇ L/well, measure OD450 value immediately after mixing (within 3 minutes).
  • TMB
  • Test Example 7 Drug efficacy of antibody in Molp-8 orthotopic tumor model
  • Molp-8 As a typical MM cell line, Molp-8 has higher expression of CD38 and lower expression of BCMA.
  • the bioluminescence signal value is Total Flux (unit, p/s), and the average value is calculated in avg;
  • T/C(%) (T-T0)/(C-C0) ⁇ 100%, where T and C are the bioluminescent signal values of the treatment group and the control group at the end of the experiment; T 0 and C 0 are at the beginning of the experiment bioluminescent signal value.
  • T3-12 and T5-12-6164 showed significant efficacy.
  • Test Example 8 Drug efficacy of antibody in RPMI-8226 orthotopic tumor model
  • RPMI-8226 as a typical MM cell line, has moderate expression of CD38 and BCMA.
  • each antibody was started 4 days after grouping, and this day was defined as Day 0 of the experiment, administered twice a week for a total of 4 times. Photographs and images were taken twice a week, body weight was weighed, and data was recorded. All data were plotted and statistically analyzed using Excel and GraphPad Prism 8 software.
  • the bioluminescence signal value is Total Flux (unit, p/s), and the average value is calculated in avg;
  • T/C(%) (T-T0)/(C-C0) ⁇ 100%, where T and C are the bioluminescent signal values of the treatment group and the control group at the end of the experiment; T 0 and C 0 are at the beginning of the experiment bioluminescent signal value.

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Abstract

本披露涉及特异性结合CD38、BCMA和CD3的抗原结合分子及其医药用途。

Description

特异性结合CD38、BCMA和CD3的抗原结合分子及其医药用途 技术领域
本披露属于生物技术领域，更具体地，本披露涉及抗原结合分子及其应用。
背景技术
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。
多发性骨髓瘤(Multiple myeloma，MM)是全球第二大常见血液癌症，其以骨髓中浆细胞的失控增殖为主要特征，癌变的浆细胞迅速增殖扩散，进而导致单克隆免疫球蛋白的大量产生，进而引起免疫抑制、骨质溶解和末端器官损伤。在全球范围内，有超过13.85万名新确诊MM患者。过去的数十年，由于蛋白酶体抑制剂、免疫调节剂和CD38抗体等新的治疗手段出现，使MM患者的临床治疗效果有很大改善，将患者的预期寿命从3至4年提高到7至8年。但大多数患者仍会因耐药而复发，即便MRD(minimal residual disease)阴性的病人仍会复发，因此，临床迫切需要更加有效的新治疗手段。在靶向MM细胞的同时重塑免疫细胞的抗肿瘤活性的免疫疗法将是非常优异的治疗MM的疗法。
CD38是一类二型跨膜糖蛋白，既能通过与配体CD31相互作用，传导下游信号调节淋巴细胞的激活、B细胞的分化等过程，又能通过外切酶的作用发挥功能。CD38在浆细胞以及多发性骨髓瘤细胞中广泛表达，并且CD38的单抗药物达雷妥尤单抗(Daratumumab)以及Isatuximab在临床上都有较好的治疗响应率以及响应深度，充分证实CD38是针对多发性骨髓瘤的较好的靶点。
BCMA(Tumor necrosis factor receptor superfamily member 17)是一类无信号肽的三型跨膜蛋白，属于TNFR蛋白超家族成员之一。BCMA通过与配体APRIL和BAFF结合，激活下游ERK1/2、NFkB等信号通路，促进B细胞的生长与增殖。在正常的组织细胞中，BCMA主要在B细胞的分化后期，即较成熟的B细胞(Plasmablast、PC)中表达，在发育早期的B细胞以及其他正常组织中无表达。BCMA高选择性地在多发性骨髓瘤细胞表面高表达，且表达量远高于正常B细胞中的表达量。并且，BCMA的近膜端会被γ-secretase剪切，形成可溶性的BCMA(sBCMA)，血浆中的sBCMA在多发性骨髓瘤以及复发型骨髓瘤的病人中明显远高于健康人体，已被作为多发性骨髓瘤疾病进展的重要标记物。
靶向CD3&TAA的双特异性抗体是一种新型的免疫疗法，可以同时结合T细胞和肿瘤细胞，模拟MHC和TCR的相互作用，使T细胞形成裂解性synapse后，释放穿孔素和颗粒酶特异性杀伤肿瘤细胞。活化后的T细胞可以释放细胞因子，启动其他免疫细胞并扩大针对肿瘤的免疫反应，最终导致T细胞的增殖和杀伤肿瘤细胞的级联反应。
发明内容
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BCMA的抗原结合模块、至少一个特异性结合CD38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。这些抗原结合分子能够提供相比BCMA/CD3抗体或CD38/CD3抗体更好的治疗活性，更广地覆盖多发性骨髓瘤病人群体。
在一些实施方案中，如前所述的抗原结合分子，其中所述抗原结合模块是指包含重链可变区和轻链可变区的抗体片段，所述重链可变区和轻链可变区共同构成结合目标抗原(BCMA，CD38或CD3)的结构域。在一些实施方案中，所述抗原结合模块是Fab或scFv。
在一个方面，本披露提供了一种抗原结合分子，所述抗原结合分子包含两个特异性结合BCMA的抗原结合模块、两个特异性结合CD38的抗原结合模块、两个特异性结合CD3的抗原结合模块和Fc区。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块是BCMA-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合CD38的抗原结合模块是CD38-scFv。本披露的BCMA-Fab指该抗原结合模块具有Fab结构，CD3-scFv和CD38-scFv指该抗原结合模块具有scFv结构。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合CD38的抗原结合模块是CD38-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合BCMA的抗原结合模块是BCMA-scFv。本披露的CD38-Fab指该抗原结合模块具有Fab结构，CD3-scFv和BCMA-scFv指该抗原结合模块具有scFv结构。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块是BCMA-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合CD38的抗原结合模块是CD38-scFv；并且一个BCMA-Fab的重链、一个CD3-scFv、Fc区的一个亚基和一个CD38-scFv按N端至C端的顺序分别直接地或通过连接子连接。在一些实施方案中，另一个BCMA-Fab的重链、另一个CD3-scFv、Fc区的另一个亚基和另一个CD38-scFv也按N端至C端的顺序分别直接地或通过连接子连接。在具体的实施方案中，所述抗原结合分子具有如图1B所示的结构。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合CD38的抗原结合模块是CD38-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合BCMA的抗原结合模块是BCMA-scFv；并且一个CD38-Fab的重链、一个CD3-scFv、Fc区的一个亚基和一个BCMA-scFv按N端至C端的顺序分别直接地或通过连接子连接。在一些实施方案中，另一个CD38-Fab的重链、另一个CD3-scFv、Fc区的另一个亚基和另一个BCMA-scFv也按N端至C端的顺序 分别直接地或通过连接子连接。在具体的实施方案中，所述抗原结合分子具有如图1D所示的结构。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-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，和所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL；其中：
所述抗原结合分子包含两条具有式(a)所示结构的第一链和两条具有式(b)所示结构的第二链，
(a)[BCMA-VH]-[CH1]-[CD3-VH]-[连接子1]-[CD3-VL]-[连接子2]-[Fc区的一个亚基]-[连接子3]-[CD38-VH]-[连接子4]-[CD38-VL]，
(b)[BCMA-VL]-[CL]，
式(a)中的连接子1、连接子2、连接子3和连接子4是相同或不同的肽连接子。在一些实施方式中，式(a)中的连接子1、连接子2、连接子3和连接子4的长度为3-15个氨基酸残基。在一些实施方式中，式(a)中的连接子1、连接子3和连接子4的氨基酸序列如SEQ ID NO：99所示，连接子2的氨基酸序列如SEQ ID NO：96所示。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-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，和所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL；其中：
所述抗原结合分子包含两条具有式(c)所示结构的第一链和两条具有式(d)所示结构的第二链，
(c)[CD38-VH]-[CH1]-[CD3-VH]-[连接子1]-[CD3-VL]-[连接子2]-[Fc区的一个亚基]-[连接子3]-[BCMA-VH]-[连接子4]-[BCMA-VL]，
(d)[CD38-VL]-[CL]，
式(c)中的连接子1、连接子2、连接子3和连接子4是相同或不同的肽连接子。在一些实施方式中，式(c)中的连接子1、连接子2、连接子3和连接子4的长度为3-15个氨基酸残基。在一些实施方式中，式(c)中的连接子1、连接子3和连接子4的氨基酸序列如SEQ ID NO：99所示，连接子2的氨基酸序列如SEQ ID NO：96所示。
在一个方面，本披露提供了一种抗原结合分子，所述抗原结合分子包含一个特异性结合BCMA的抗原结合模块、两个特异性结合CD38的抗原结合模块、一个特异性结合CD3的抗原结合模块和Fc区。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合BCMA 的抗原结合模块是BCMA-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合CD38的抗原结合模块是CD38-scFv。在具体的实施方案中，所述抗原结合分子具有如图1A所示的结构。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合CD38的抗原结合模块是CD38-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合BCMA的抗原结合模块是BCMA-scFv。在具体的实施方案中，所述抗原结合分子具有如图1E所示的结构。
在一些实施方案中，如前任一项所述的抗原结合分子，所述Fc区包含能够缔合的第一亚基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；并且，所述特异性结合BCMA的抗原结合模块是BCMA-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合CD38的抗原结合模块是CD38-scFv；并且BCMA-Fab的重链、Fc1和一个CD38-scFv按N端至C端的顺序分别直接地或通过连接子连接，和CD3-scFv、Fc2和另一个CD38-scFv按N端至C端的顺序分别直接地或通过连接子连接。
在一些实施方案中，如前任一项所述的抗原结合分子，所述Fc区包含能够缔合的第一亚基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；并且，所述特异性结合CD38的抗原结合模块是CD38-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合BCMA的抗原结合模块是BCMA-scFv；并且一个CD38-Fab的重链、Fc1和CD3-scFv按N端至C端的顺序分别直接地或通过连接子连接，和BCMA-scFv、另一个CD38-Fab的重链和Fc2按N端至C端的顺序分别直接地或通过连接子连接。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-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，和所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL；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(b)所示结构的第二链和一条具有式(f)所示结构的第三链，
(e)[BCMA-VH]-[CH1]-[Fc1]-[连接子1]-[CD38-VH]-[连接子2]-[CD38-VL]，
(b)[BCMA-VL]-[CL]，
(f)[CD3-VH]-[连接子1]-[CD3-VL]-[连接子2]-[Fc2]-[连接子3]-[CD38-VH]-[连接子4]-[CD38-VL]，
其中，式(e)中的连接子1和连接子2是相同或不同的肽连接子，和式(f)中的连接子1、连接子2、连接子3和连接子4是相同或不同的肽连接子。在一些实施方案中，所述肽连接子是柔性肽连接子。在一些实施方案中，式(e)中的连接子1、 连接子2，以及式(f)中的连接子1、连接子2、连接子3和连接子4的长度为3-15个氨基酸残基。在一些实施方案中，式(e)中的连接子1和连接子2的氨基酸序列如SEQ ID NO：99所示，式(f)中的连接子1、连接子3和连接子4的氨基酸序列如SEQ ID NO：99所示，和式(f)中的连接子2的氨基酸序列如SEQ ID NO：96所示。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-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，和所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL；其中：
所述抗原结合分子包含一条具有式(g)所示结构的第一链、一条具有式(d)所示结构的第二链和一条具有式(h)所示结构的第三链，
(g)[BCMA-VL]-[连接子1]-[BCMA-VH]-[连接子2]-[CD38-VH]-[CH1]-[Fc2]，
(d)[CD38-VL]-[CL]，
(h)[CD38-VH]-[CH1]-[Fc1]-[连接子1]-[CD3-VL]-[连接子2]-[CD3-VH]，
其中，式(g)中的连接子1和连接子2是相同或不同的肽连接子，和式(h)中的连接子1和连接子2是相同或不同的肽连接子。在一些实施方案中，所述肽连接子是柔性肽连接子。在一些实施方案中，式(g)中的连接子1、连接子2，以及式(h)中的连接子1和连接子2的长度为3-15个氨基酸残基。在一些实施方案中，式(g)中的连接子1和连接子2的氨基酸序列如SEQ ID NO：99所示，式(h)中的连接子1的氨基酸序列如SEQ ID NO：97所示，和式(h)中连接子2的氨基酸序列如SEQ ID NO：99所示。
在一个方面，本披露提供了一种抗原结合分子，所述抗原结合分子包含一个特异性结合BCMA的抗原结合模块、一个特异性结合CD38的抗原结合模块、一个特异性结合CD3的抗原结合模块和Fc区。在具体的实施方案中，所述抗原结合分子具有如图1C所示的结构。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块是BCMA-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合CD38的抗原结合模块是CD38-scFv。
在一些实施方案中，如前任一项所述的抗原结合分子，所述Fc区包含能够缔合的第一亚基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；并且CD38-scFv、CD3-scFv和Fc1按N端至C端的顺序分别直接地或通过连接子连接，和BCMA-Fab的重链和Fc2按N端至C端的顺序分别直接地或通过连接子连接。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-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，和所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL；其中：
所述抗原结合分子包含一条具有式(i)所示结构的第一链、一条具有式(b)所示结构的第二链和一条具有式(j)所示结构的第三链，
(i)[BCMA-VH]-[CH1]-[Fc2]，
(b)[BCMA-VL]-[CL]，
(j)[CD38-VH]-[连接子1]-[CD38-VL]-[连接子2]-[CD3-VH]-[连接子3]-[CD3-VL]-[连接子4]-[Fc1]，
其中，式(j)中的连接子1、连接子2、连接子3和连接子4是相同或不同的肽连接子。在一些实施方案中，所述肽连接子是柔性肽连接子。在一些实施方案中，式(j)中的连接子1、连接子2、连接子3和连接子4的长度为3-15个氨基酸残基。在一些实施方案中，式(j)中的连接子1和连接子3的氨基酸序列如SEQ ID NO：99所示，式(j)中的连接子2的氨基酸序列如SEQ ID NO：97所示，和式(j)中的连接子4的氨基酸序列如SEQ ID NO：96所示。
在一些实施方案中，如前任一项所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-VL，其中：
(i)所述BCMA-VH包含SEQ ID NO：23中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：24中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的氨基酸序列；或
(ii)所述BCMA-VH包含SEQ ID NO：25中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：26中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的氨基酸序列；或
(iii)所述BCMA-VH包含SEQ ID NO：27中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：28中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的氨基酸序列。
在一些实施方案中，如前所述的抗原结合分子，所述BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号规则定义的。
在一些实施方案中，如前任一项所述的抗原结合分子，
(i)所述BCMA-VH具有：包含SEQ ID NO：5的氨基酸序列的BCMA-HCDR1、包含SEQ ID NO：6的氨基酸序列的BCMA-HCDR2和包含SEQ ID NO：7的氨基酸序列的BCMA-HCDR3；并且所述BCMA-VL具有：包含SEQ ID NO：8的氨基酸序列的BCMA-LCDR1、包含SEQ ID NO：9的氨基酸序列的BCMA-LCDR2和包含SEQ ID NO：10的氨基酸序列的BCMA-LCDR3，或
(ii)所述BCMA-VH具有：包含SEQ ID NO：11的氨基酸序列的 BCMA-HCDR1、包含SEQ ID NO：12的氨基酸序列的BCMA-HCDR2和包含SEQ ID NO：13的氨基酸序列的BCMA-HCDR3；并且所述BCMA-VL具有：包含SEQ ID NO：14的氨基酸序列的BCMA-LCDR1、包含SEQ ID NO：15的氨基酸序列的BCMA-LCDR2和包含SEQ ID NO：16的氨基酸序列的BCMA-LCDR3，或
(iii)所述BCMA-VH具有：包含SEQ ID NO：17的氨基酸序列的BCMA-HCDR1、包含SEQ ID NO：18的氨基酸序列的BCMA-HCDR2和包含SEQ ID NO：19的氨基酸序列的BCMA-HCDR3；并且所述BCMA-VL具有：包含SEQ ID NO：20的氨基酸序列的BCMA-LCDR1、包含SEQ ID NO：21的氨基酸序列的BCMA-LCDR2和包含SEQ ID NO：22的氨基酸序列的BCMA-LCDR3。
在一些实施方案中，如前所述的抗原结合分子，所述BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3是根据Kabat编号规则定义的。
在一些实施方案中，如前所述的抗原结合分子，其中：
(i)所述BCMA-VH具有：包含SEQ ID NO：5的氨基酸序列的BCMA-HCDR1，包含SEQ ID NO：6的氨基酸序列的BCMA-HCDR2，和包含SEQ ID NO：7的氨基酸序列的BCMA-HCDR3；并且所述BCMA-VL具有：包含SEQ ID NO：8的氨基酸序列的BCMA-LCDR1，包含SEQ ID NO：9的氨基酸序列的BCMA-LCDR2，和包含SEQ ID NO：10的氨基酸序列的BCMA-LCDR3。在一些实施方案中，所述BCMA-VH和/或所述BCMA-VL是鼠源的或人源化的。在一些实施方案中，所述BCMA-VH和/或所述BCMA-VL是人源化的。在一些实施方案中，所述BCMA-VH具有来源于IGHV1-46*01的重链框架区，并且其是未被取代的或具有选自48I、67A、71A、73K、76T和93V组成的组中的一个或多个氨基酸取代；和/或所述BCMA-VL具有来源于IGKV1-39*01的轻链框架区，并且其是未被取代的或具有选自43S、45Q、48V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，上述可变区和CDR是根据Kabat编号规则定义的。
在一些实施方案中，如前所述的抗原结合分子，其中：
(i)所述BCMA-VH包含与SEQ ID NO：23具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，和所述BCMA-VL包含与SEQ ID NO：24具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列。在一些实施方案中，所述BCMA-VH包含SEQ ID NO：23的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：24的氨基酸序列。
在一些实施方案中，如前所述的抗原结合分子，其中：
(i)所述BCMA-VH包含与选自由SEQ ID NO：29、SEQ ID NO：30和SEQ ID NO：31组成的组的氨基酸序列分别具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，和所述BCMA-VL包含与选自由SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34和SEQ ID NO：35组成的组的氨基酸序列分别 具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列。在一些实施方案中，所述BCMA-VH包含选自由SEQ ID NO：29、SEQ ID NO：30和SEQ ID NO：31组成的组的氨基酸序列，和所述BCMA-VL包含选自由SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34和SEQ ID NO：35组成的组的氨基酸序列。在一些实施方案中，所述特异性结合BCMA的抗原结合模块与包含SEQ ID NO：29和SEQ ID NO：32的抗原结合模块是功能等价的。在一些实施方案中，所述BCMA-VH包含SEQ ID NO：29的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：32的氨基酸序列。
在一些实施方案中，如前所述的抗原结合分子，其中：
(ii)所述BCMA-VH具有：包含SEQ ID NO：11的氨基酸序列的BCMA-HCDR1、包含SEQ ID NO：12的氨基酸序列的BCMA-HCDR2和包含SEQ ID NO：13的氨基酸序列的BCMA-HCDR3；并且所述BCMA-VL具有：包含SEQ ID NO：14的氨基酸序列的BCMA-LCDR1、包含SEQ ID NO：15的氨基酸序列的BCMA-LCDR2和包含SEQ ID NO：16的氨基酸序列的BCMA-LCDR3。在一些实施方案中，所述BCMA-VH和/或所述BCMA-VL是鼠源的或人源化的。在一些实施方案中，所述BCMA-VH和/或所述BCMA-VL是人源化的。在一些实施方案中，所述BCMA-VH具有来源于IGHV7-4-1*02的重链框架区，并且其是未被取代的或具有选自2I、44V、45F、46K、75A、76N和93L组成的组中的一个或多个氨基酸取代；和/或所述BCMA-VL具有来源于IGKV1-27*01的轻链框架区，并且其是未被取代的或具有选自43S和66D组成的组中的一个或多个氨基酸取代。在一些实施方案中，上述可变区和CDR是根据Kabat编号规则定义的。
在一些实施方案中，如前所述的抗原结合分子，其中：
(ii)所述BCMA-VH包含与SEQ ID NO：25具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，和所述BCMA-VL包含与SEQ ID NO：26具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列。在一些实施方案中，所述BCMA-VH包含SEQ ID NO：25的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：26的氨基酸序列。
在一些实施方案中，如前所述的抗原结合分子，其中：
(ii)所述BCMA-VH包含与选自由由SEQ ID NO：36、SEQ ID NO：37和SEQ ID NO：38组成的组的氨基酸序列分别具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，和所述BCMA-VL包含与SEQ ID NO：39或SEQ ID NO：40的氨基酸序列分别具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列。在一些实施方案中，所述BCMA-VH包含选自由SEQ ID NO：36、SEQ ID NO：37和SEQ ID NO：38组成的组的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：39或SEQ ID NO：40的氨基酸序列。在一些实施方案中，所述特异性结合BCMA的抗原结合模块与包含SEQ ID NO：36和SEQ ID NO： 40的抗原结合模块是功能等价的。在一些实施方案中，所述BCMA-VH包含SEQ ID NO：36的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：40的氨基酸序列。
在一些实施方案中，如前所述的抗原结合分子，其中：
(iii)所述BCMA-VH具有：包含SEQ ID NO：17的氨基酸序列的BCMA-HCDR1、包含SEQ ID NO：18的氨基酸序列的BCMA-HCDR2和包含SEQ ID NO：19的氨基酸序列的BCMA-HCDR3；并且所述BCMA-VL具有：包含SEQ ID NO：20的氨基酸序列的BCMA-LCDR1、包含SEQ ID NO：21的氨基酸序列的BCMA-LCDR2和包含SEQ ID NO：22的氨基酸序列的BCMA-LCDR3。在一些实施方案中，所述BCMA-VH和/或所述BCMA-VL是鼠源的或人源化的。在一些实施方案中，所述BCMA-VH和/或所述BCMA-VL是人源化的。在一些实施方案中，所述BCMA-VH具有来源于IGHV1-46*01的重链框架区，并且其是未被取代的或具有选自38K、48I、66T、67A、71S、73K和78A组成的组中的一个或多个氨基酸取代；和/或所述BCMA-VL具有来源于IGKV2-28*01的轻链框架区，并且其是未被取代的或具有选自2V、45K和87F组成的组中的一个或多个氨基酸取代。在一些实施方案中，上述可变区和CDR是根据Kabat编号规则定义的。
在一些实施方案中，如前所述的抗原结合分子，其中：
(iii)所述BCMA-VH包含与SEQ ID NO：27具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，和所述BCMA-VL包含与SEQ ID NO：28具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列。在一些实施方案中，所述BCMA-VH包含SEQ ID NO：27的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：28的氨基酸序列。
在一些实施方案中，如前所述的抗原结合分子，其中：
(iii)所述BCMA-VH包含与选自由由SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43和SEQ ID NO：44组成的组的氨基酸序列分别具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，和所述BCMA-VL包含与选自由SEQ ID NO：45或SEQ ID NO：46组成的组的氨基酸序列分别具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列。在一些实施方案中，所述BCMA-VH包含选自由SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43和SEQ ID NO：44组成的组的氨基酸序列，和所述BCMA-VL包含选自由SEQ ID NO：45或SEQ ID NO：46组成的组的氨基酸序列。在一些实施方案中，所述特异性结合BCMA的抗原结合模块与包含SEQ ID NO：43和SEQ ID NO：45的抗原结合模块是功能等价的。在一些实施方案中，所述BCMA-VH包含SEQ ID NO：43的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：45的氨基酸序列。
在一些实施方案中，如前所述的抗原结合分子，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：
(i)所述CD3-HCDR1、CD3-HCDR2和CD3-HCDR3分别包含SEQ ID NO： 63中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3的氨基酸序列；和所述CD3-LCDR1、CD3-LCDR2和CD3-LCDR3分别包含SEQ ID NO：64中的CD3-LCDR1、CD3-LCDR2和CD3-LCDR3的氨基酸序列，或
(ii)所述CD3-HCDR1、CD3-HCDR2和CD3-HCDR3分别包含SEQ ID NO：65中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3的氨基酸序列；和所述CD3-LCDR1、CD3-LCDR2和CD3-LCDR3分别包含SEQ ID NO：64中的CD3-LCDR1、CD3-LCDR2和CD3-LCDR3的氨基酸序列。
在一些实施方案中，如前所述的抗原结合分子，所述CD3-HCDR1、CD3-HCDR2、CD3-HCDR3、CD3-LCDR1、CD3-LCDR2和CD3-LCDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号规则定义的。
在一些实施方案中，如前所述的抗原结合分子，其中：
(i)所述CD3-VH具有：包含SEQ ID NO：55的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：56的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：57的氨基酸序列的CD3-HCDR3；并且所述CD3-VL具有：包含SEQ ID NO：58的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：59的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：60的氨基酸序列的CD3-LCDR3，或
(ii)所述CD3-VH具有：包含SEQ ID NO：55的氨基酸序列的CD3-HCDR1，包含SEQ ID NO：61的氨基酸序列的CD3-HCDR2，和包含SEQ ID NO：62的氨基酸序列的CD3-HCDR3；并且所述CD3-VL具有：包含SEQ ID NO：58的氨基酸序列的CD3-LCDR1，包含SEQ ID NO：59的氨基酸序列的CD3-LCDR2，和包含SEQ ID NO：60的氨基酸序列的CD3-LCDR3。
在一些实施方案中，如前所述的抗原结合分子，所述CD3-HCDR1、CD3-HCDR2、CD3-HCDR3、CD3-LCDR1、CD3-LCDR2和CD3-LCDR3是根据Kabat编号规则定义的。
在一些实施方案中，如前所述的抗原结合分子，其中：
(i)所述CD3-VH包含与SEQ ID NO：63具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，和所述CD3-VL包含与SEQ ID NO：64具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列。在一些实施方案中，所述特异性结合CD3的抗原结合模块与包含SEQ ID NO：63和SEQ ID NO：64的抗原结合模块是功能等价的。在一些实施方案中，所述CD3-VH包含SEQ ID NO：63的氨基酸序列，和所述CD3-VL包含SEQ ID NO：64的氨基酸序列。
在一些实施方案中，如前所述的抗原结合分子，其中：
(ii)所述CD3-VH包含与SEQ ID NO：65具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，和所述CD3-VL包含与SEQ ID NO：64具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列。在一 些实施方案中，所述特异性结合CD3的抗原结合模块与包含SEQ ID NO：65和SEQ ID NO：64的抗原结合模块是功能等价的。在一些实施方案中，所述CD3-VH包含SEQ ID NO：65的氨基酸序列，和所述CD3-VL包含SEQ ID NO：64的氨基酸序列。
在一些实施方案中，如前所述的抗原结合分子，所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL，其中所述CD38-VH中的CD38-HCDR1、CD38-HCDR2和CD38-HCDR3分别包含SEQ ID NO：53中的CD38-HCDR1、CD38-HCDR2和CD38-HCDR3的氨基酸序列，并且所述CD38-VL中的CD38-LCDR1、CD38-LCDR2和CD38-LCDR3分别包含SEQ ID NO：54中的CD38-LCDR1、CD38-LCDR2和CD38-LCDR3的氨基酸序列。在一些实施方案中，所述CD38-HCDR1、CD38-HCDR2、CD38-HCDR3、CD38-LCDR1、CD38-LCDR2和CD38-LCDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号规则定义的。
在一些实施方案中，如前所述的抗原结合分子，所述CD38-VH具有：包含SEQ ID NO：47的氨基酸序列的CD38-HCDR1，包含SEQ ID NO：48的氨基酸序列的CD38-HCDR2，和包含SEQ ID NO：49的氨基酸序列的CD38-HCDR3；并且所述CD38-VL具有：包含SEQ ID NO：50的氨基酸序列的CD38-LCDR1，包含SEQ ID NO：51的氨基酸序列的CD38-LCDR2，和包含SEQ ID NO：52的氨基酸序列的CD38-LCDR3。在一些实施方案中，所述CD38-HCDR1、CD38-HCDR2、CD38-HCDR3、CD38-LCDR1、CD38-LCDR2和CD38-LCDR3是根据Kabat编号规则定义的。
在一些实施方案中，如前所述的抗原结合分子，其中：
所述CD38-VH包含与SEQ ID NO：53具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列，和所述CD38-VL包含与SEQ ID NO：54具有至少90％、95％、96％、97％、98％或99％的序列同一性的氨基酸序列。在一些实施方案中，所述特异性结合CD38的抗原结合模块与包含SEQ ID NO：53和SEQ ID NO：54的抗原结合模块是功能等价的。在一些实施方案中，所述CD38-VH包含SEQ ID NO：53的氨基酸序列，和所述CD38-VL包含SEQ ID NO：54的氨基酸序列。
在一些实施方案中，如前任一项所述的抗原结合分子，其连接子是肽连接子。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如L 1-(GGGGS) n-L 2所示的氨基酸序列，其中L 1是键、A、GS、GGS或GGGS(SEQ ID NO：102)，n是0、1、2、3、4、5、6、7、8、9或10，L 2是键、G、GG、GGG或GGGG(SEQ ID NO：103)，且所述连接子不是键。在一些实施方案中，所述的肽连接子序列如SEQ ID NO：96至SEQ ID NO：99所示。
GGG(SEQ ID NO：96)
GGGGS(SEQ ID NO：97)
GGGGSGGGGS(SEQ ID NO：98)
GGGGSGGGGSGGGGS(SEQ ID NO：99)
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Fc区。在一些实施方案中，所述Fc区是IgG Fc区，优选为IgG 1Fc区。在一些实施方案中，所述Fc区包含一个或多个氨基酸取代，所述的氨基酸取代能够减少Fc区与Fc受体的结合。在一些实施方案中，所述的氨基酸取代能够减少其与Fcγ受体的结合。在一些实施方案中，所述Fc区是人IgG 1Fc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。在一些实施方案中，所述Fc1具有SEQ ID NO：68的氨基酸序列。
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Fc区，所述Fc区包含能够相互缔合的第一亚基Fc1和第二亚基Fc2，所述Fc1和Fc2各自独立地具有一个或多个减少Fc区同源二聚化的氨基酸取代。在一些实施方案中，所述Fc1和Fc2各自独立地具有一个或多个根据杵臼技术的的氨基酸取代。在一些实施方案中，所述Fc1具有根据杵臼技术的凸起结构，和所述Fc2具有根据杵臼技术的孔结构。在一些实施方案中，所述Fc1在366位置的氨基酸残基为W；和所述Fc2在366位置的氨基酸残基为S、在368位置的氨基酸残基为A、和在407位置的氨基酸残基为V，编号依据为EU索引。在一些实施方案中，所述Fc1具有SEQ ID NO：100的氨基酸序列，和所述Fc2具有SEQ ID NO：101的氨基酸序列。
在一些实施方案中，如前任一项所述的抗原结合分子，
所述抗原结合分子具有：两条包含SEQ ID NO：76的氨基酸序列的第一链和两条包含SEQ ID NO：74的氨基酸序列的第二链；或
所述抗原结合分子具有：两条包含SEQ ID NO：75的氨基酸序列的第一链和两条包含SEQ ID NO：74的氨基酸序列的第二链；或
所述抗原结合分子具有：两条包含SEQ ID NO：82的氨基酸序列的第一链和两条包含SEQ ID NO：81的氨基酸序列的第二链；或
所述抗原结合分子具有：两条包含SEQ ID NO：83的氨基酸序列的第一链和两条包含SEQ ID NO：81的氨基酸序列的第二链；或
所述抗原结合分子具有：两条包含SEQ ID NO：90的氨基酸序列的第一链和两条包含SEQ ID NO：89的氨基酸序列的第二链；或
所述抗原结合分子具有：两条包含SEQ ID NO：91的氨基酸序列的第一链和两条包含SEQ ID NO：89的氨基酸序列的第二链；或
所述抗原结合分子具有：两条包含SEQ ID NO：77的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链；或
所述抗原结合分子具有：两条包含SEQ ID NO：78的氨基酸序列的第一链、 两条包含SEQ ID NO：71的氨基酸序列的第二链；或
所述抗原结合分子具有：两条包含SEQ ID NO：85的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链；或
所述抗原结合分子具有：两条包含SEQ ID NO：86的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链；或
所述抗原结合分子具有：两条包含SEQ ID NO：93的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链；或
所述抗原结合分子具有：两条包含SEQ ID NO：94的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链；或
所述抗原结合分子具有：一条包含SEQ ID NO：73的氨基酸序列的第一链、一条包含SEQ ID NO：74的氨基酸序列的第二链和一条包含SEQ ID NO：66的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：73的氨基酸序列的第一链、一条包含SEQ ID NO：74的氨基酸序列的第二链和一条包含SEQ ID NO：67的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：80的氨基酸序列的第一链、一条包含SEQ ID NO：81的氨基酸序列的第二链和一条包含SEQ ID NO：66的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：80的氨基酸序列的第一链、一条包含SEQ ID NO：81的氨基酸序列的第二链和一条包含SEQ ID NO：67的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：88的氨基酸序列的第一链、一条包含SEQ ID NO：89的氨基酸序列的第二链和一条包含SEQ ID NO：66的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：88的氨基酸序列的第一链、一条包含SEQ ID NO：89的氨基酸序列的第二链和一条包含SEQ ID NO：67的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：84的氨基酸序列的第一链、一条包含SEQ ID NO：81的氨基酸序列的第二链和一条包含SEQ ID NO：69的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：84的氨基酸序列的第一链、一条包含SEQ ID NO：81的氨基酸序列的第二链和一条包含SEQ ID NO：70的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：92的氨基酸序列的第一链、一条包含SEQ ID NO：89的氨基酸序列的第二链和一条包含SEQ ID NO：69的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：92的氨基酸序列的第一链、一条包含SEQ ID NO：89的氨基酸序列的第二链和一条包含SEQ ID NO：70的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：79的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链和一条包含SEQ ID NO：72的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：87的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链和一条包含SEQ ID NO：72的氨基酸序列的第三链；或
所述抗原结合分子具有：一条包含SEQ ID NO：95的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链和一条包含SEQ ID NO：72的氨基酸序列的第三链。
在一些实施方案中，如前任一项所述的抗原结合分子，其具有：两条包含SEQ ID NO：76的氨基酸序列的第一链和两条包含SEQ ID NO：74的氨基酸序列的第二链。
在一些实施方案中，如前任一项所述的抗原结合分子，其具有：两条包含SEQ ID NO：82的氨基酸序列的第一链和两条包含SEQ ID NO：81的氨基酸序列的第二链。
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，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。
在另一个方面，本披露还提供一种宿主细胞，其包含前述的分离的核酸。
在另一个方面，本披露还提供一种治疗疾病的方法，所述方法包括向受试者施用治疗有效量的前述任一项所述的抗原结合分子或组合物。
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组合物在制备治疗或预防疾病的药物中的用途。
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组合物。在一些实施方式中，所述药物用于治疗疾病。
在一些实施方式中，所述疾病是淋巴谱系或骨髓谱系的造血系统肿瘤或自身免疫性疾病。在一些实施方式中，所述淋巴谱系或骨髓谱系的造血系统肿瘤选自：多发性骨髓瘤、浆细胞瘤、浆细胞白血病、巨球蛋白血症、淀粉样变性、华氏巨球蛋白血症、孤立性骨浆细胞瘤、髓外浆细胞瘤、骨硬化性骨髓瘤、重链病、意义不明确的单克隆丙种球蛋白病以及郁积型多发性骨髓瘤、B-细胞淋巴瘤、伯基 特淋巴瘤、何杰金淋巴瘤和多毛细胞淋巴瘤；所述自身免疫性疾病选自：类风湿性关节炎、系统性红斑性狼疮、哮喘、炎症性肠道疾病、多发性硬化、克罗恩氏病、胃炎、桥本氏甲状腺炎、强直性脊柱炎和移植物对抗宿主疾病。
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。
附图说明
图1A：Format5的结构示意图；图1B：Format12的结构示意图；图1C：Format20的结构示意图；图1D：Format21的结构示意图；图1E：Format24的结构示意图。
图2：抗体结合RPMI-8226细胞的能力。
图3：抗体对CHO K1细胞的细胞毒活性。
具体实施方式
术语
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。
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：5的氨基酸序列的BCMA-HCDR1，其明确的包含氨基酸序列如SEQ ID NO：5所示的BCMA-HCDR1。
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。
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。
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。
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，如化学修饰也可能是可用的。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。
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。示例性的，本文中的抗原结合分子是三特异性抗原结合分子(例如：三特异性抗体)。术语“三特异性抗原结合分子”指能够特异性结合三个不同抗原的抗原结合分子。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。“天然抗体”指天然存在的免疫球蛋白分子。例如，天然IgG抗体是约150,000道尔顿的异四聚糖蛋白，由二硫键结合的两条相同轻链和两条相同重链构成。从N至C端，每条重链具有一个可变区(VH)，又称作可变重域、重链可变区，接着是三个恒定域(CH1、CH2和CH3)。类似地，从N至C端，每条轻链具有一个可变区(VL)，又称作可变轻域，或轻链可变域，接着是一个恒定轻域(轻链恒定区、CL)。
术语“可变区”或“可变域”指抗原结合分子中结合抗原的域。本文中，特异性结合BCMA的抗原结合模块中的重链可变区标示为BCMA-VH，轻链可变区标示为BCMA-VL；特异性结合CD38的抗原结合模块中的重链可变区标示为CD38-VH，轻链可变区标示为CD38-VL；特异性结合CD3的抗原结合模块中的重链可变区标示为CD3-VH，轻链可变区标示为CD3-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，BCMA-VH中的3个CDR区分别标示为BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3；BCMA-VL中的3个CDR区分别标示为BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3； CD38-VH中的3个CDR区分别标示为CD38-HCDR1、CD38-HCDR2和CD38-HCDR3；CD38-VL中的3个CDR区分别标示为CD38-LCDR1、CD38-LCDR2和CD38-LCDR3；CD3-VH中的3个CDR区分别标示为CD3-HCDR1、CD3-HCDR2和CD3-HCDR3；CD3-VL中的3个CDR区分别标示为CD3-LCDR1、CD3-LCDR2和CD3-LCDR3。每个VH和VL由从氨基末端排到羧基末端按以下顺序排列的三个CDR和四个FR构成：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。
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。
表1.CDR编号系统之间的关系
CDR IMGT Kabat AbM Chothia Contact
HCDR1 27-38 31-35 26-35 26-32 30-35
HCDR2 56-65 50-65 50-58 52-56 47-58
HCDR3 105-117 95-102 95-102 95-102 93-101
LCDR1 27-38 24-34 24-34 24-34 30-36
LCDR2 56-65 50-56 50-56 50-56 46-55
LCDR3 105-117 89-97 89-97 89-97 89-96
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。
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，所述部分与完整抗体所结合的抗原相结合。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。Fab指由免疫球蛋白的VH和CH1(Fab重链)与VL和CL(Fab轻链)组成的蛋白质。本披露中，BCMA-Fab表示具有Fab结构的特异性结合BCMA的抗原结合模块，BCMA-scFv表示具有scFv结构的特异性结合BCMA的抗原结合模块，以此类推。
术语“scFv”是指单链抗体(single chain antibody fragment，scFv)，其由抗体重链可变区(VH)和轻链可变区(VL)通过15～20个氨基酸的短肽接头连接而成的抗体。
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。
术语“嵌合”抗体指抗体中的重和/或轻链的一部分自特定的来源或物种衍生，而重和/或轻链的剩余部分自不同来源或物种衍生的抗体。
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。
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：用于测定抗体KD的方法包括使用生物传感系统例如系统测量表面等离子体共振，或通过溶液平衡滴定法(SET)测量溶液中的亲和力。
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该抗原内的表位。通常，抗体以约1×10 -6M或更小(例如约1×10 -7M，约1×10 -8M或更小)的平衡解离常数(KD)结合抗原或抗原内的表位。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
Figure PCTCN2022105578-appb-000001
表面等离子共振测定法所测量的。然而，特异性结合至抗原或抗原内的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。
术语“抗原结合模块”指特异性结合目标抗原的多肽分子。抗原结合模块包括如本文中定义的抗体及其片段。具体的抗原结合模块包括抗体的抗原结合域，其包含抗体重链可变区和抗体轻链可变区。术语“特异性结合BCMA的抗原结合模块”是指能够以足够的亲和力结合BCMA的模块。在某些实施例中，特异性结 合BCMA的抗原结合模块具有以下的平衡解离常数(KD)：<约1μM、<约100nM或<约10nM，其是通过FACS、表面等离子共振等方法测量的。在某些实施例中，特异性结合BCMA的抗原结合模块结合来自不同物种的BCMA中的保守表位。术语“特异性结合CD38的抗原结合模块”是指能够以足够的亲和力结合CD38的模块。在某些实施例中，特异性结合CD38的抗原结合模块具有以下的平衡解离常数(KD)：<约1μM、<约100nM或<约10nM，其是通过FACS、表面等离子共振等方法测量的。在某些实施例中，特异性结合CD38的抗原结合模块结合来自不同物种的CD38中的保守表位。术语“特异性结合CD3的抗原结合模块”是指能够以足够的亲和力结合CD3的模块。在某些实施例中，特异性结合CD3的抗原结合模块具有以下的平衡解离常数(KD)：<约1μM、<约200nM、<约100nM、和/或>50nM，其是通过FACS、表面等离子共振等方法测量的。在某些实施例中，抗CD3抗体结合来自不同物种的CD3中的保守表位。抗原结合模块包括如本文定义的抗体片段，例如Fab或scFv。
术语“效应子功能”指那些可归于抗体Fc区(天然序列Fc区或氨基酸序列变体Fc区)且随抗体同种型而变化的生物学活性。抗体效应子功能的例子包括：C1q结合和补体依赖性细胞毒性；Fc受体结合；抗体依赖性细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体(例如B细胞受体)下调；和B细胞活化。
术语“单克隆抗体”指基本上均质的抗体的群，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。
术语“抗原”是指能够由诸如抗原结合蛋白(例如抗体)的选择性结合剂结合，且另外能够用于动物中以产生能够结合该抗原的抗体的分子或分子部分。抗原可具有一个或多个能够与不同的抗原结合蛋白(例如抗体)相互作用的表位。
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。当术语“键”出现在结构单元，其表示该单元没有氨基酸，单元两侧的单元直接连接。
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。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。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)密码子的第三位被混合碱基和/或脱氧肌苷残基取代。
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相应天然存在的氨基酸的人工化学模拟物，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。
术语序列“同一性”指，当对两条序列进行最佳比对时，必要时引入间隙，以获取最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，两条序列的氨基酸/核酸在等价位置相同的程度(百分比)。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。
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。
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。
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。本文中包括具有与在初始转化细胞中筛选或选择的相同功能或生物学活性的突变体后代。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)。
如在本申请中所使用的，表述“细胞”、“细胞系”和“细胞培养物”可以互换使用，并且所有这样的名称均包括子代。因而，词语“转化体”和“转化的细胞”包括原代受试者细胞和来源于其的培养物，而与传代的次数无关。还应理解的是，由于有意或无意的突变，使得并非所有子代均具有完全相同的DNA内容物。包括与筛选出其的原始转化细胞具有相同功能或生物活性的突变子代。
“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。
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。
术语“药学上可接受的载体”指药学配制剂中与活性成分不同的,且对受试者无毒的成分。药学可接受载剂包括但不限于缓冲剂、赋形剂、稳定剂或防腐剂。
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。除非指出时，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。
“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。
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。
“治疗(treatment或treat)”和“处理”(及其语法变型)指试图改变所治疗个体的天然过程的临床干预，并且可以为了预防或者在临床病理学的过程期间实施。治疗的期望效果包括但不限于预防疾病的发生或再发生，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，使用本披露的抗体来延迟疾病的形成或减缓疾病的进展。
“有效量”一般是足以降低症状的严重程度及/或频率、消除这些症状及/或潜在病因、预防症状及/或其潜在病因出现及/或改良或改善由疾病状态引起或与其相关的损伤(例如肺病)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。
本披露的抗原结合分子
本披露提供了抗原结合分子，其具有诸多有利的特性，例如亲和力、对细胞表面抗原的特异性、在抗原存在下特异性激活T细胞的活性、治疗活性、安全性(如更低的细胞因子释放)、药物代谢动力学特性和成药性(如产率、纯度和稳定性等)。
示例性的抗原结合分子
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BCMA的抗原结合模块、至少一个特异性结合CD38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。特别的，本披露的抗原结合分子具有以下至少一种功能活性：
a.针对CD38/BCMA/CD3d&CD3e的高亲和力。在一些实施方式中，所述的抗原结合分子以小于200nM的KD与CD38/BCMA/CD3d&CD3e结合。在一些实施方式中，所述的抗原结合分子以小于10nM的KD与CD38或BCMA结合，并且以小于200nM的KD与CD3d&CD3e结合。所述KD是通过表面等离子共振确定的。
b.针对细胞表面抗原的高亲和力。具体测试方法见测试例2。
c.针对表达BCMA细胞的体外特异性杀伤活性。在一些实施方式中，所述抗原结合分子可特异性杀伤表达BCMA和/或CD38的细胞，但不能杀伤不表达BCMA和CD38的细胞。具体测试方法见测试例4。
d.体外特异性杀伤活性不受可溶性BCMA/CD38/APRIL的影响。具体测试方法见测试例5。
e.诱导低水平的细胞因子(IL-6和IL-2)释放。具体测试方法见测试例6。
f.更强的体内治疗活性。具体测试方法见测试例7、8。
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BCMA的抗原结合模块、至少一个特异性结合CD38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BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-VL，其中：
(i)所述BCMA-VH具有：氨基酸序列如SEQ ID NO：5所示的BCMA-HCDR1、氨基酸序列如SEQ ID NO：6所示的BCMA-HCDR2和氨基酸序列如SEQ ID NO：7所示的BCMA-HCDR3；并且所述BCMA-VL具有：氨基酸序列如SEQ ID NO：8所示的BCMA-LCDR1、氨基酸序列如SEQ ID NO：9所示的BCMA-LCDR2和氨基酸序列如SEQ ID NO：10所示的BCMA-LCDR3；或
(ii)所述BCMA-VH具有：氨基酸序列如SEQ ID NO：11所示的 BCMA-HCDR1、氨基酸序列如SEQ ID NO：12所示的BCMA-HCDR2和氨基酸序列如SEQ ID NO：13所示的BCMA-HCDR3；并且所述BCMA-VL具有：氨基酸序列如SEQ ID NO：14所示的BCMA-LCDR1、氨基酸序列如SEQ ID NO：15所示的BCMA-LCDR2和氨基酸序列如SEQ ID NO：16所示的BCMA-LCDR3；或
(iii)所述BCMA-VH具有：氨基酸序列如SEQ ID NO：17所示的BCMA-HCDR1、氨基酸序列如SEQ ID NO：18所示的BCMA-HCDR2和氨基酸序列如SEQ ID NO：19所示的BCMA-HCDR3；并且所述BCMA-VL具有：氨基酸序列如SEQ ID NO：20所示的BCMA-LCDR1、氨基酸序列如SEQ ID NO：21所示的BCMA-LCDR2和氨基酸序列如SEQ ID NO：22所示的BCMA-LCDR3；
所述BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3是根据Kabat编号规则定义的。
在一些实施方案中，如前所述的抗原结合分子，其中：
所述重链可变区BCMA-VH的氨基酸序列如SEQ ID NO：29所示，和所述轻链可变区BCMA-VL的氨基酸序列如SEQ ID NO：32所示，或
所述重链可变区BCMA-VH的氨基酸序列如SEQ ID NO：36所示，和所述轻链可变区BCMA-VL的氨基酸序列如SEQ ID NO：40所示，或
所述重链可变区BCMA-VH的氨基酸序列如SEQ ID NO：43所示，和所述轻链可变区BCMA-VL的氨基酸序列如SEQ ID NO：45所示，
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：
(i)所述CD3-VH具有：氨基酸序列如SEQ ID NO：55所示的CD3-HCDR1、氨基酸序列如SEQ ID NO：56所示的CD3-HCDR2和氨基酸序列如SEQ ID NO：57所示的CD3-HCDR3；并且所述CD3-VL具有：氨基酸序列如SEQ ID NO：58所示的CD3-LCDR1、氨基酸序列如SEQ ID NO：59所示的CD3-LCDR2和氨基酸序列如SEQ ID NO：60所示的CD3-LCDR3；或
(ii)所述CD3-VH具有：氨基酸序列如SEQ ID NO：55所示的CD3-HCDR1、氨基酸序列如SEQ ID NO：61所示的CD3-HCDR2和氨基酸序列如SEQ ID NO：62所示的CD3-HCDR3；并且所述CD3-VL具有：氨基酸序列如SEQ ID NO：58所示的CD3-LCDR1、氨基酸序列如SEQ ID NO：59所示的CD3-LCDR2和氨基酸序列如SEQ ID NO：60所示的CD3-LCDR3；
所述CD3-HCDR1、CD3-HCDR2、CD3-HCDR3、CD3-LCDR1、CD3-LCDR2和CD3-LCDR3是根据Kabat编号规则定义的。
在一些实施方案中，如前任一项所述的抗原结合分子，所述重链可变区CD3-VH的氨基酸序列如SEQ ID NO：63所示，和所述轻链可变区CD3-VL的氨基酸序列如SEQ ID NO：64所示；或所述重链可变区CD3-VH的氨基酸序列如SEQ  ID NO：65所示，和所述轻链可变区CD3-VL的氨基酸序列如SEQ ID NO：64所示。
在一些实施方案中，如前任一项所述的抗原结合分子，其中所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL，其中：
所述CD38-VH具有：氨基酸序列如SEQ ID NO：47所示的CD38-HCDR1、氨基酸序列如SEQ ID NO：48所示的CD38-HCDR2和氨基酸序列如SEQ ID NO：49所示的CD38-HCDR3；并且所述CD38-VL具有：氨基酸序列如SEQ ID NO：50所示的CD38-LCDR1、氨基酸序列如SEQ ID NO：51所示的CD38-LCDR2和氨基酸序列如SEQ ID NO：52所示的CD38-LCDR3；
所述CD38-HCDR1、CD38-HCDR2、CD38-HCDR3、CD38-LCDR1、CD38-LCDR2和CD38-LCDR3是根据Kabat编号规则定义的。
在一些实施方案中，如前任一项所述的抗原结合分子，所述重链可变区CD38-VH的氨基酸序列如SEQ ID NO：53所示，和所述轻链可变区CD38-VL的氨基酸序列如SEQ ID NO：54所示。
在一些实施方案中，如前任一项所述的抗原结合分子，其中
(i)所述BCMA-VH具有：氨基酸序列如SEQ ID NO：5所示的BCMA-HCDR1、氨基酸序列如SEQ ID NO：6所示的BCMA-HCDR2和氨基酸序列如SEQ ID NO：7所示的BCMA-HCDR3；并且所述BCMA-VL具有：氨基酸序列如SEQ ID NO：8所示的BCMA-LCDR1、氨基酸序列如SEQ ID NO：9所示的BCMA-LCDR2和氨基酸序列如SEQ ID NO：10所示的BCMA-LCDR3，和
所述CD3-VH具有：氨基酸序列如SEQ ID NO：55所示的CD3-HCDR1、氨基酸序列如SEQ ID NO：61所示的CD3-HCDR2和氨基酸序列如SEQ ID NO：62所示的CD3-HCDR3；并且所述CD3-VL具有：氨基酸序列如SEQ ID NO：58所示的CD3-LCDR1、氨基酸序列如SEQ ID NO：59所示的CD3-LCDR2和氨基酸序列如SEQ ID NO：60所示的CD3-LCDR3，和
所述CD38-VH具有：氨基酸序列如SEQ ID NO：47所示的CD38-HCDR1、氨基酸序列如SEQ ID NO：48所示的CD38-HCDR2和氨基酸序列如SEQ ID NO：49所示的CD38-HCDR3；并且所述CD38-VL具有：氨基酸序列如SEQ ID NO：50所示的CD38-LCDR1、氨基酸序列如SEQ ID NO：51所示的CD38-LCDR2和氨基酸序列如SEQ ID NO：52所示的CD38-LCDR3；或
(ii)所述BCMA-VH具有：氨基酸序列如SEQ ID NO：11所示的BCMA-HCDR1、氨基酸序列如SEQ ID NO：12所示的BCMA-HCDR2和氨基酸序列如SEQ ID NO：13所示的BCMA-HCDR3；并且所述BCMA-VL具有：氨基酸序列如SEQ ID NO：14所示的BCMA-LCDR1、氨基酸序列如SEQ ID NO：15所示的BCMA-LCDR2和氨基酸序列如SEQ ID NO：16所示的BCMA-LCDR3，和
所述CD3-VH具有：氨基酸序列如SEQ ID NO：55所示的CD3-HCDR1、氨 基酸序列如SEQ ID NO：56所示的CD3-HCDR2和氨基酸序列如SEQ ID NO：57所示的CD3-HCDR3；并且所述CD3-VL具有：氨基酸序列如SEQ ID NO：58所示的CD3-LCDR1、氨基酸序列如SEQ ID NO：59所示的CD3-LCDR2和氨基酸序列如SEQ ID NO：60所示的CD3-LCDR3，和
所述CD38-VH具有：氨基酸序列如SEQ ID NO：47所示的CD38-HCDR1、氨基酸序列如SEQ ID NO：48所示的CD38-HCDR2和氨基酸序列如SEQ ID NO：49所示的CD38-HCDR3；并且所述CD38-VL具有：氨基酸序列如SEQ ID NO：50所示的CD38-LCDR1、氨基酸序列如SEQ ID NO：51所示的CD38-LCDR2和氨基酸序列如SEQ ID NO：52所示的CD38-LCDR3；
所述BCMA-HCDR1、BCMA-HCDR2、BCMA-HCDR3、BCMA-LCDR1、BCMA-LCDR2、BCMA-LCDR3、CD3-HCDR1、CD3-HCDR2、CD3-HCDR3、CD3-LCDR1、CD3-LCDR2、CD3-LCDR3、CD38-HCDR1、CD38-HCDR2、CD38-HCDR3、CD38-LCDR1、CD38-LCDR2和CD38-LCDR3是根据Kabat编号规则定义的。
在一些实施方案中，如前任一项所述的抗原结合分子，所述重链可变区BCMA-VH的氨基酸序列如SEQ ID NO：29所示，所述轻链可变区BCMA-VL的氨基酸序列如SEQ ID NO：32所示，所述重链可变区CD3-VH的氨基酸序列如SEQ ID NO：65所示，所述轻链可变区CD3-VL的氨基酸序列如SEQ ID NO：64所示，所述重链可变区CD38-VH的氨基酸序列如SEQ ID NO：53所示，和所述轻链可变区CD38-VL的氨基酸序列如SEQ ID NO：54所示。
在一些实施方案中，如前任一项所述的抗原结合分子，所述重链可变区BCMA-VH的氨基酸序列如SEQ ID NO：36所示，所述轻链可变区BCMA-VL的氨基酸序列如SEQ ID NO：40所示，所述重链可变区CD3-VH的氨基酸序列如SEQ ID NO：63所示，所述轻链可变区CD3-VL的氨基酸序列如SEQ ID NO：64所示，所述重链可变区CD38-VH的氨基酸序列如SEQ ID NO：53所示，和所述轻链可变区CD38-VL的氨基酸序列如SEQ ID NO：54所示。
在一些实施方案中，如前任一项所述的抗原结合分子，其中所述抗原结合分子还包含Fc区，所述Fc区包含能够缔合的两个亚基。在一些实施方案中，所述两个亚基的氨基酸序列如SEQ ID NO：68所示。在一些实施方案中，所述两个亚基的氨基酸序列分别如SEQ ID NO：100和SEQ ID NO：101所示。
抗原结合分子的结构
本披露提供一种三特异性的抗原结合分子，其可以是三价、四价、五价、六价及更多价的。所述抗原结合分子包含抗原结合模块，该抗原结合模块可以是任意的能够与目标抗原结合的多肽分子，例如抗体片段。在一些实施方案中，所述抗原结合模块是指包含重链可变区和轻链可变区的抗体片段，所述重链可变区和 轻链可变区共同构成结合目标抗原的结构域。在一些实施方案中，所述抗原结合模块是Fab或scFv。本披露的抗原结合分子中的抗原结合模块可以以任意的，但不影响结合活性的方式连接。示例性的，抗原结合分子的结构示意图如图1A、图1B、图1C、图1D或图1E所示。
抗原结合分子的变体
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。
取代、插入、和删除变体
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。取代诱变感兴趣的位点包括CDR和FR。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“例示性取代”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。
表2.氨基酸的取代
原始残基 示例性取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp,Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸 Leu
依照常见的侧链特性，氨基酸可以如下分组:
(1)疏水性的：正亮氨酸，Met，Ala，Val，Leu，Ile；
(2)中性，亲水性的：Cys，Ser，Thr，Asn，Gln；
(3)酸性的：Asp，Glu；
(4)碱性的：His，Lys，Arg；
(5)影响链取向的残基：Gly，Pro；
(6)芳香族的：Trp，Tyr，Phe。
非保守取代会需要用这些类别之一的成员替换另一个类别的成员。
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。特别地，经常靶向HCDR3和LCDR3。
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。此类变化可以例如在CDR中的抗原接触残基外部。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。
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，鉴定一个残基或靶残基组(例如带电荷的残基，诸如Arg、Asp、His、Lys和Glu)，并且用中性或带负电荷的氨基酸(例如，Ala或聚丙氨酸)替换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。
氨基酸序列插入包括长度范围为1个残基至含有100或更多个残基的多肽的氨基和/或羧基端融合，和单个或多个氨基酸残基的序列内插入。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抗体的N或C端与酶或延长抗体的血清半衰期的多肽的融合物。
Fc区的改造
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IgG 1Fc区或IgG 4Fc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IgG 1Fc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IgG 1Fc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IgG 4Fc区，在228等位置的氨基酸残基取代可降低的效应子功能。
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。基于不同来源，所述Fc区的第356位氨基酸残基可以是E或D，第358位氨基酸残基可以是M或L。在一些实施例方式中，所述Fc区的第356位氨基酸残基可以是E，第358位氨基酸残基是M。在一些实施例方式中，所述Fc区的第356位氨基酸残基可以是D，第358位氨基酸残基是L。
抗原结合分子可包含与Fc区的两个亚基融合的不同抗原结合模块，因此可能 导致不期望的同源二聚化。为了提高产率和纯度，因此在本发明的抗原结合分子的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：
表3.KIH突变组合
Figure PCTCN2022105578-appb-000002
在一些实施方式中，具体的Fc区的氨基酸序列如下：
>IgG 1Fc(Knob)(SEQ ID NO：100)
Figure PCTCN2022105578-appb-000003
>IgG 1Fc(Hole)(SEQ ID NO：101)
Figure PCTCN2022105578-appb-000004
除了杵臼技术外，用于修饰多特异性抗体的重链的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WO96/27011、WO98/050431、EP1870459、WO2007/110205、WO 007/147901、WO2009/089004、WO2010/129304、WO2011/90754、WO2011/143545、WO2012/058768、WO2013/157954和WO013/096291。
Fc区的C末端可以是以氨基酸残基PGK结束的完整C末端；也可以是缩短的C末端，例如在所述缩短的C末端中已经去除了一个或两个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。
重组方法
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。
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原结合分子的情况下，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。
在具有异二聚体重链的双特异性抗原结合分子的情况下，需要四个核酸，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。
在一个实施方案中，本披露提供了编码如前所述的抗原结合分子的分离的核酸。此类核酸可以给自独立的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，在适合抗原结合分子表达的条件下，培养包含编码所述抗原结合分子的核酸的宿主细胞，如上文所提供的，和任选地从宿主细胞(或宿主细胞培养基)回收所述抗原结合分子。
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原结合分子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。
用于克隆或表达编码抗原结合分子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，抗原结合分子可在细菌中产生，特别是当抗原结合分子不需要糖基化和Fc效应子功能时。在表达后，抗原结合分子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。
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原结合分子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原结合分子。适于表达(糖基化)抗原结合分子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、US6040498、US6420548、US7125978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。关于适合产生抗原结合分子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。
免疫缀合物
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。
诊断与治疗组合物
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目标抗原的存在。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。
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目标抗原的存在的方法。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BCMA、CD38或CD3是用于选择患者的生物标志物。
可使用本披露的抗原结合分子来诊断的例示性病症，例如B细胞障碍或自身免疫性疾病，所述B细胞障碍选自：多发性骨髓瘤、浆细胞瘤、浆细胞白血病、巨球蛋白血症、淀粉样变性、华氏巨球蛋白血症、孤立性骨浆细胞瘤、髓外浆细胞瘤、骨硬化性骨髓瘤、重链病、意义不明确的单克隆丙种球蛋白病以及郁积型多发性骨髓瘤；所述自身免疫性疾病是全身性红斑狼疮。
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)。
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。
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。用于体内施用的配制剂一般是无菌的。无菌性可容易地实现，例如通过穿过无菌滤膜过滤。
治疗方法与施用途径
本文提供的任何抗原结合分子可用于治疗方法。
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B细胞障碍或自身免疫性疾病，所述B细胞障碍选自：多发性骨髓瘤、浆细胞瘤、浆细胞白血病、巨球蛋白血症、淀粉样变性、华氏巨球蛋白血症、孤立性骨浆细胞瘤、髓外浆细胞瘤、骨硬化性骨髓瘤、重链病、意义不明确的单克隆丙种球蛋白病以及郁积型多发性骨髓瘤；所述自身免疫性疾病是全身性红斑狼疮。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。
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。
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。
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。
本披露的抗原结合分子将以符合良好医疗实践的方式配制、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。
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。
制品
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。
实施例与测试例
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。
实施例1.人BCMA和食蟹猴BCMA的表达
编码带人IgG1-Fc标签的人BCMA和食蟹猴BCMA胞外区序列插入phr载体中，构建成表达质粒，然后转染HEK293。编码带His标签的人BCMA胞外区序列插入phr载体中，构建成表达质粒，然后转染HEK293。具体转染步骤为：前一天将HEK293E细胞以1×10 6/mL接种于freestyle表达培养基(含有1％FBS，Gibco，12338-026)，放置于37度恒温摇床(120rpm)继续培养24小时。24小时后，将转染质粒和转染试剂PEI用0.22μm的滤器除菌，然后将转染质粒调整为100μg/100mL细胞，PEI(1mg/mL)和质粒的质量比为3:1，取10mL的Opti-MEM和200μg质粒混匀，静置5min；另取10mL的Opti-MEM和400μg PEI混匀，静置5min。将质粒和PEI进行混匀，静置15min。将质粒和PEI混合物缓慢加入200mL HEK293E的细胞中，放入8％CO 2、120rpm、37℃的摇床中培养。转染第3天，补充10％体积的补料培养基(20mM葡萄糖+2mM L-谷氨酸)。待转染第6天，取样4500rpm离心10min收集细胞上清，参照测试例2的方法纯化。纯化后的蛋白可用于下述各实施例或测试例实验中。
其中人BCMA-ECD-Fc、人BCMA-ECD-his、cynoBCMA-ECD-his和cynoBCMA-ECD-Fc的氨基酸序列如下所示。cynoBCMA-ECD-his购自ACRO公司。
(1)带人Fc标签的人BCMA：人BCMA-ECD-Fc(SEQ ID NO：1)
Figure PCTCN2022105578-appb-000005
注：下划线为信号肽序列；斜体部分为人Fc-连接子-tag
(2)带His标签的人BCMA的ECD：人BCMA-ECD-His(SEQ ID NO：2)
Figure PCTCN2022105578-appb-000006
注：下划线为信号肽序列；斜体部分为His6-连接子-tag
(3)带His标签的食蟹猴BCMA的ECD：cynoBCMA-ECD-His(SEQ ID NO：3)
Figure PCTCN2022105578-appb-000007
注：斜体部分为连接子-10His-tag
(4)带人Fc标签的食蟹猴BCMA：cynoBCMA-ECD-Fc(SEQ ID NO：4)
Figure PCTCN2022105578-appb-000008
注：下划线为信号肽序列；斜体部分为人Fc-tag
实施例2.Protein A亲和层析纯化带Fc标签的重组蛋白和镍柱纯化带His标签的重组蛋白
带人Fc标签的蛋白的纯化：将细胞表达的BCMA上清样品高速离心去除杂质，通过Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用100mM乙酸pH3.5洗脱目的蛋白，用1M Tris-HCl，pH8.0中和。洗脱样品适当浓缩后换液到PBS中，将得到的蛋白经电泳，肽图，LC-MS鉴定为正确后分装备用。
带人His标签的蛋白的纯化：将细胞表达的BCMA上清样品高速离心去除杂质。用含有PBS缓冲液平衡镍柱，冲洗2-5倍柱体积，将上清样品以一定流速上Ni Sepharose excel柱。用PBS缓冲液冲洗柱子，至A280读数降至基线，再后用PBS+10mM咪唑冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液，最后用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰，浓缩换液，将得到的蛋白经电泳，肽图，LC-MS鉴定为正确后分装备用。
实施例3.表达重组人CD38，人BCMA和食蟹猴BCMA细胞系的构建和鉴定
本披露构建了表达人CHO-K1/hCD38的细胞株。将人CD38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-hCD38三种质粒共同转染HEK293T细胞(
Figure PCTCN2022105578-appb-000009
CRL-11268)来包装病毒，转染48小时后，收集病毒感染CHO-K1细胞(
Figure PCTCN2022105578-appb-000010
CCL-61)。感染72小时后通过流式分选获取高表达hCD38的CHOK1单克隆细胞。
为筛选能和细胞表面BCMA有较好结合的抗体，本披露构建了表达人BCMA的K562-BCMA细胞株。将人BCMA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-人BCMA三种质粒共同转染HEK293T细胞(
Figure PCTCN2022105578-appb-000011
CRL-11268)来包装病毒，转染48小时后，收集病毒感染K562细胞(
Figure PCTCN2022105578-appb-000012
CCL-243)。感染72小时后通过流式分选获取高表达人BCMA的K562单克隆细胞。
本披露还构建了表达食蟹猴的CHO-K1-cynoBCMA细胞株，将食蟹猴BCMA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-cynoBCMA三种质粒共同转染HEK293T细胞(
Figure PCTCN2022105578-appb-000013
CRL-11268)来包装病毒，转染48小时后，收集病毒感染CHOK1细胞(
Figure PCTCN2022105578-appb-000014
CCL-61)。感染72小时后通过流式分选获取高表达cynoBCMA的CHOK1单克隆细胞。
实施例4.抗人BCMA杂交瘤抗体的筛选和鉴定
本披露通过杂交瘤技术制备了针对人BCMA的单克隆抗体。所得抗体与人BCMA以较高的亲和力特异性结合，并且可以与食蟹猴BCMA发生交叉反应；所得抗体与细胞表面的人BCMA和食蟹猴BCMA有较好的结合活性，且该结合活性受可溶性BCMA干扰极小。
将人BCMA-ECD-his和cynoBCMA-ECD-his作为交叉免疫试剂，
Figure PCTCN2022105578-appb-000015
Gold Adjuvant(Sigma Cat No.T2684)与Thermo
Figure PCTCN2022105578-appb-000016
Alum(Thermo Cat No.77161)作为佐剂交叉免疫小鼠。经过初次免疫和7次加强免疫后选择血清中抗体滴度高小鼠10-6#(滴度625K)进行脾细胞融合。融合后根据杂交瘤细胞生长密度，对杂交瘤培养上清进行检测，并筛选特异性结合细胞表面BCMA的抗体。
筛选得到活性好的单克隆杂交瘤细胞株4E3、33H4和27。分别收集对数生长期杂交瘤细胞，用NucleoZol(MN)提取RNA(按照试剂盒说明书步骤)，并进行反转录(PrimeScript TM Reverse 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。4E3、33H4和27的CDR和可变区的氨基酸序列如下：
表4.BCMA抗体CDR
Figure PCTCN2022105578-appb-000017
>4E3鼠源重链可变区(SEQ ID NO：23)
Figure PCTCN2022105578-appb-000018
Figure PCTCN2022105578-appb-000019
>4E3鼠源轻链可变区(SEQ ID NO：24)
Figure PCTCN2022105578-appb-000020
>33H4鼠源重链可变区(SEQ ID NO：25)
Figure PCTCN2022105578-appb-000021
>33H4鼠源轻链可变区(SEQ ID NO：26)
Figure PCTCN2022105578-appb-000022
>27鼠源重链可变区(SEQ ID NO：27)
Figure PCTCN2022105578-appb-000023
>27鼠源轻链可变区(SEQ ID NO：28)
Figure PCTCN2022105578-appb-000024
注：下划线标记区为根据Kabat编号规则获得的CDR区
实施例5.抗人BCMA单克隆抗体的人源化设计
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，将鼠源抗体的CDR区移植到人源模板上，并对VL和VH的部分残基进行突变，将鼠源抗体的恒定区替换为人恒定区，得到最终的人源化分子。
1. 4E3的人源FR区的选择和回复突变
表5. 4E3的人源FR区选择和回复突变
Figure PCTCN2022105578-appb-000025
注：Graft代表鼠抗体CDR植入人种系FR区序列。示例性的，R71A表示依照Kabat编号系统，将71位R突变回A。下同。
鼠源抗体4E3人源化得到的抗体可变区具体序列如下(下划线表示CDR，下同)：
>hu4E3H1(SEQ ID NO：29)
Figure PCTCN2022105578-appb-000026
>hu4E3H2(SEQ ID NO：30)
Figure PCTCN2022105578-appb-000027
>hu4E3H3(SEQ ID NO：31)
Figure PCTCN2022105578-appb-000028
>hu4E3L1(SEQ ID NO：32)
Figure PCTCN2022105578-appb-000029
>hu4E3L2(SEQ ID NO：33)
Figure PCTCN2022105578-appb-000030
>hu4E3L3(SEQ ID NO：34)
Figure PCTCN2022105578-appb-000031
>hu4E3L4(SEQ ID NO：35)
Figure PCTCN2022105578-appb-000032
2. 33H4的人源FR区的选择和回复突变
表6. 33H4的人源FR区选择和回复突变
Figure PCTCN2022105578-appb-000033
鼠源抗体33H4人源化得到的抗体可变区具体序列如下：
>hu33H4H1(SEQ ID NO：36)
Figure PCTCN2022105578-appb-000034
>hu33H4H2(SEQ ID NO：37)
Figure PCTCN2022105578-appb-000035
>hu33H4H3(SEQ ID NO：38)
Figure PCTCN2022105578-appb-000036
>hu33H4L1(SEQ ID NO：39)
Figure PCTCN2022105578-appb-000037
>hu33H4L2(SEQ ID NO：40)
Figure PCTCN2022105578-appb-000038
表7. 27的人源FR区选择和回复突变
Figure PCTCN2022105578-appb-000039
鼠源抗体27人源化得到的抗体可变区具体序列如下：
>hu27H1(SEQ ID NO：41)
Figure PCTCN2022105578-appb-000040
>hu27H2(SEQ ID NO：42)
Figure PCTCN2022105578-appb-000041
>hu27H3(SEQ ID NO：43)
Figure PCTCN2022105578-appb-000042
Figure PCTCN2022105578-appb-000043
>hu27H4(SEQ ID NO：44)
Figure PCTCN2022105578-appb-000044
>hu27L1(SEQ ID NO：45)
Figure PCTCN2022105578-appb-000045
>hu27L2(SEQ ID NO：46)
Figure PCTCN2022105578-appb-000046
实施例6.本披露抗体的制备及鉴定
本披露CD38结合分子可以来源于任意适宜的抗体。特别适宜的抗体描述于例如专利公开号WO2020052546(通过援引完整收入本文)。
本披露抗体中的抗CD38臂的CDR和可变区序列如下所示：
表8.CD38臂的CDR
Figure PCTCN2022105578-appb-000047
可变区具体序列如下：
>1149-VH(SEQ ID NO：53)
Figure PCTCN2022105578-appb-000048
>1149-VL(SEQ ID NO：54)
Figure PCTCN2022105578-appb-000049
本披露CD3结合分子可以来源于任意适宜的抗体。特别适宜的抗体描述于例如国际申请号WO2020114478(通过援引完整收入本文)。
本披露双特异性抗体中的抗CD3臂的CDR和可变区序列如下所示：
表9.CD3臂的CDR
Figure PCTCN2022105578-appb-000050
可变区具体序列如下：
>S107E-VH(SEQ ID NO：63)
Figure PCTCN2022105578-appb-000051
>S107E-VL(SEQ ID NO：64)
Figure PCTCN2022105578-appb-000052
>6164-VH(SEQ ID NO：65)
Figure PCTCN2022105578-appb-000053
>6164-VL(SEQ ID NO：64)
将抗CD38抗体可变区、抗BCMA抗体可变区、抗CD3抗体可变区以及IgG 1突变体IgG 1(AA)(L234A/L235A)组合。各形成不同形式的抗原结合分子，分别为Format5，Format5-6164，Format12，Format12-6164，Format20，Format20-6164，Format21，Format21-6164，Format24。
Format5为非对称结构分子，完整分子共三条链，三条链均不相同，其结构如图1A所示。
链1：VH(anti-BCMA)-CH1-IgG 1Fc(Knob,AA)-连接子1-VH(1149)-连接子2-VL(1149)；
链2：VL(anti-BCMA)-CL；
链3(Hole-5)：VH(S107E)-连接子1-VL(S107E)-连接子2-IgG 1Fc(Hole,AA)-连接子3-VH(1149)-连接子4-VL(1149)。
>Hole-5(SEQ ID NO：66)
Figure PCTCN2022105578-appb-000054
注：单下划线标记区为根据Kabat编号规则获得的CD38、CD3结合结构域的CDR区，斜体为恒定区。
Format5-6164为非对称结构分子，完整分子共三条链，三条链均不相同，其结构如图1A所示。
链1：VH(anti-BCMA)-CH1-IgG 1Fc(Knob,AA)-连接子1-VH(1149)-连接子2-VL(1149)；
链2：VL(anti-BCMA)-CL；
链3：(Hole-5-6164)：VH(6164)-连接子1-VL(6164)-连接子2-IgG 1Fc(Hole,AA)-连接子3-VH(1149)-连接子4-VL(1149)。
>Hole-5-6164(SEQ ID NO：67)
Figure PCTCN2022105578-appb-000055
注：单下划线标记区为根据Kabat编号规则获得的CD38、CD3结合结构域的 CDR区，斜体为恒定区。
Format12为对称结构分子，包含两条相同的重链(链1)和两条相同的轻链(链2)，其结构如图1B所示。
链1：VH(anti-BCMA)-CH1-VH(S107E)-连接子1-VL(S107E)-连接子2-IgG 1Fc(AA)-连接子3-VH(1149)-连接子4-VL(1149)；
链2：VL(anti-BCMA)-CL。
Format12-6164为对称结构分子，包含两条相同的重链(链1)和两条相同的轻链(链2)，其结构如图1B所示。
链1：VH(anti-BCMA)-CH1-VH(6164)-连接子1-VL(6164)-连接子2-IgG 1Fc(AA)-连接子3-VH(1149)-连接子4-VL(1149)；
链2：VL(anti-BCMA)-CL。
>IgG 1Fc(AA)(SEQ ID NO：68)
Figure PCTCN2022105578-appb-000056
Format20为非对称结构分子，完整分子共三条链，三条链均不相同，其结构如图1C所示。
链1：VH(anti-BCMA)-CH1-IgG 1Fc(Hole,AA)；
链2：VL(anti-BCMA)-CL；
链3(Knob-20)：VH(1149)-连接子1-VL(1149)–连接子2-VH(S107E)-连接子3-VL(S107E)-连接子4-IgG 1Fc(Knob,AA)。
>Knob-20(SEQ ID NO：69)
Figure PCTCN2022105578-appb-000057
注：单下划线标记区为根据Kabat编号规则获得的CD38、CD3结合结构域的CDR区，斜体为恒定区。
Format20-6164为非对称结构分子，完整分子共三条链，三条链均不相同，其结构如图1C所示。
链1：VH(anti-BCMA)-CH1-IgG 1Fc(Hole,AA)；
链2：VL(anti-BCMA)-CL；
链3(Knob-20-6164)：VH(1149)-连接子1-VL(1149)-连接子2-VH(6164)-连接子3-VL(6164)-连接子4-IgG 1Fc(Knob,AA)。
>Knob-20-6164(SEQ ID NO：70)
Figure PCTCN2022105578-appb-000058
注：单下划线标记区为根据Kabat编号规则获得的CD38、CD3结合结构域的CDR区，斜体为恒定区。
Format21为对称结构分子，包含两条相同的重链(链1)和两条相同的轻链(链2)，其结构如图1D所示。
链1：VH(1149)-CH1-VH(S107E)-连接子1-VL(S107E)-连接子2-IgG 1Fc(AA)-连接子3-VL(anti-BCMA)-连接子4-VH(anti-BCMA)；
链2：VL(1149)-CL。
Format21-6164为对称结构分子，包含两条相同的重链(链1)和两条相同的轻链(链2)，其结构如图1D所示。
链1：VH(1149)-CH1-VH(6164)-连接子1-VL(6164)-连接子2-IgG 1Fc(AA)-连接子3-VL(anti-BCMA)-连接子4-VH(anti-BCMA)；
链2：VL(1149)-CL，其结构如图1D所示。
>VL(1149)-CL(SEQ ID NO：71)
Figure PCTCN2022105578-appb-000059
Figure PCTCN2022105578-appb-000060
注：单下划线标记区为根据Kabat编号规则获得的CD38结合结构域的CDR区，斜体为恒定区。
Format24为非对称结构分子，完整分子共四条链，其结构如图1E所示。
链1：VL(anti-BCMA)-连接子1-VH(anti-BCMA)-连接子2-VH(1149)-CH1-IgG 1Fc(Hole,AA)；
链2(两条)：VL(1149)-CL；
链3(Knob-24)：VH(1149)-CH1-IgG 1Fc(Knob,AA)-连接子1-VL(107E)-连接子2-VH(107E)。
>Knob-24(SEQ ID NO：72)
Figure PCTCN2022105578-appb-000061
注：单下划线标记区为根据Kabat编号规则获得的CD38、CD3结合结构域的CDR区，斜体为恒定区。
根据表10构建抗原结合分子，编号中，T5表示采用hu4E3L1、hu4E3H1作为结合BCMA的可变区，T3表示采用hu33H4L2、hu33H4H1作为结合BCMA的可变区，T1表示采用hu27L1、hu27H3作为结合BCMA的可变区。
表10.本披露的三特异性抗体
编号 链1 链2 链3
T5-5 SEQ ID NO：73 SEQ ID NO：74 SEQ ID NO：66
T5-5-6164 SEQ ID NO：73 SEQ ID NO：74 SEQ ID NO：67
T5-12 SEQ ID NO：75 SEQ ID NO：74  
T5-12-6164 SEQ ID NO：76 SEQ ID NO：74  
T5-21 SEQ ID NO：77 SEQ ID NO：71  
T5-21-6164 SEQ ID NO：78 SEQ ID NO：71  
T5-24 SEQ ID NO：79 SEQ ID NO：71 SEQ ID NO：72
T3-5 SEQ ID NO：80 SEQ ID NO：81 SEQ ID NO：66
T3-5-6164 SEQ ID NO：80 SEQ ID NO：81 SEQ ID NO：67
T3-12 SEQ ID NO：82 SEQ ID NO：81  
T3-12-6164 SEQ ID NO：83 SEQ ID NO：81  
T3-20 SEQ ID NO：84 SEQ ID NO：81 SEQ ID NO：69
T3-20-6164 SEQ ID NO：84 SEQ ID NO：81 SEQ ID NO：70
T3-21 SEQ ID NO：85 SEQ ID NO：71  
T3-21-6164 SEQ ID NO：86 SEQ ID NO：71  
T3-24 SEQ ID NO：87 SEQ ID NO：71 SEQ ID NO：72
T1-5 SEQ ID NO：88 SEQ ID NO：89 SEQ ID NO：66
T1-5-6164 SEQ ID NO：88 SEQ ID NO：89 SEQ ID NO：67
T1-12 SEQ ID NO：90 SEQ ID NO：89  
T1-12-6164 SEQ ID NO：91 SEQ ID NO：89  
T1-20 SEQ ID NO：92 SEQ ID NO：89 SEQ ID NO：69
T1-20-6164 SEQ ID NO：92 SEQ ID NO：89 SEQ ID NO：70
T1-21 SEQ ID NO：93 SEQ ID NO：71  
T1-21-6164 SEQ ID NO：94 SEQ ID NO：71  
T1-24 SEQ ID NO：95 SEQ ID NO：71 SEQ ID NO：72
注：T5-5中的-5表示采用Format5的结构；T5-5-6164中的-5-6164表示采用Format5-6164的结构；T5-12中的-12表示采用Format12的结构，以此类推。
>SEQ ID NO：73
Figure PCTCN2022105578-appb-000062
>SEQ ID NO：74
Figure PCTCN2022105578-appb-000063
>SEQ ID NO：75
Figure PCTCN2022105578-appb-000064
Figure PCTCN2022105578-appb-000065
>SEQ ID NO：76
Figure PCTCN2022105578-appb-000066
>SEQ ID NO：77
Figure PCTCN2022105578-appb-000067
Figure PCTCN2022105578-appb-000068
>SEQ ID NO：78
Figure PCTCN2022105578-appb-000069
>SEQ ID NO：79
Figure PCTCN2022105578-appb-000070
Figure PCTCN2022105578-appb-000071
>SEQ ID NO：80
Figure PCTCN2022105578-appb-000072
>SEQ ID NO：81
Figure PCTCN2022105578-appb-000073
>SEQ ID NO：82
Figure PCTCN2022105578-appb-000074
Figure PCTCN2022105578-appb-000075
>SEQ ID NO：83
Figure PCTCN2022105578-appb-000076
>SEQ ID NO：84
Figure PCTCN2022105578-appb-000077
>SEQ ID NO：85
Figure PCTCN2022105578-appb-000078
Figure PCTCN2022105578-appb-000079
>SEQ ID NO：86
Figure PCTCN2022105578-appb-000080
>SEQ ID NO：87
Figure PCTCN2022105578-appb-000081
Figure PCTCN2022105578-appb-000082
>SEQ ID NO：88
Figure PCTCN2022105578-appb-000083
>SEQ ID NO：89
Figure PCTCN2022105578-appb-000084
>SEQ ID NO：90
Figure PCTCN2022105578-appb-000085
>SEQ ID NO：91
Figure PCTCN2022105578-appb-000086
>SEQ ID NO：92
Figure PCTCN2022105578-appb-000087
>SEQ ID NO：93
Figure PCTCN2022105578-appb-000088
Figure PCTCN2022105578-appb-000089
>SEQ ID NO：94
Figure PCTCN2022105578-appb-000090
>SEQ ID NO：95
Figure PCTCN2022105578-appb-000091
注：单下划线标记区为根据Kabat编号规则获得的CDR区，斜体为恒定区。
>CH1(SEQ ID NO：104)
Figure PCTCN2022105578-appb-000092
>CL(SEQ ID NO：105)
Figure PCTCN2022105578-appb-000093
本披露所用的阳性对照分子GBR-1342和AMGEN701。GBR-1342的氨基酸序列包括WO2016071355中的SEQ ID NO：178、SEQ ID NO：179和SEQ ID NO：128。AMGEN701的氨基酸序列包括WO2017134134中的SEQ ID NO：74。
测试例
测试例1.FACS测定MM细胞表面hBCMA和hCD38的表达量
为测试MM细胞表面BCMA和CD38的表达量，通过FACS对MM相关细胞系进行表达量检测。CD38表达量测定具体如下：取活率>90％的细胞按每孔细胞数1×10 5个将细胞加至96孔板(3590#，Corning)中，300g离心5分钟，1％BSA洗一次。分别取100μL 1％BSA配制好的10μg/mL的CD38抗体加入，4℃孵育1小时。1％BSA洗两次。加入PE-人F(ab’) 2(1:400)50μL，4℃孵育0.5小时。1％BSA洗两次。加入100μL 1％BSA重悬，上机读值。BCMA表达量测定具体如下：取活率>90％的细胞按每孔细胞数1×10 5个将细胞加至96孔板(3590#，Corning)中，300g离心5分钟，1％BSA洗一次。取按说明书稀释好的BCMA抗体(BL2-A:BCMA-APC-A)加入，4℃孵育1小时。1％BSA洗两次。加入100μL1％BSA重悬，然后用FACS仪器检测细胞表面的PE和APC的荧光值，并采用几何平均值进行平行比较。
表11.MM细胞株CD38和BCMA表达量鉴定
Figure PCTCN2022105578-appb-000094
测试例2.本披露的抗体对CD38/BCMA/CD3d&CD3e蛋白的亲和力
为测试本披露的抗体对CD38/BCMA/CD3D&E的亲和力，本测试例用仪器Biacore T200检测了抗体与hCD3d&CD3e(31.2Kda，Cat.#CT038-H2508H，S.B)，hCD38(30.7Kda，HR)和hBCMA(7.8Kda，Cat.#BCA-H52y，Acro)的结合能力。具体方法如下：用偶联有抗人抗体的CM5生物传感芯片亲和捕获抗体，然后于芯片表面流经含100nM BCMA-His的溶液180秒，使抗体的BCMA结合位点饱和，再加入hCD3d&CD3e或者hCD38，用Biacore T200仪器实时检测。数据拟合模型采用1:1Model。本披露的抗体与相应抗原结合的亲和力数据如下表所示。
表12.抗体的亲和力测定
Figure PCTCN2022105578-appb-000095
注：为方便纯化，Format24的Knob-24链的C端含有6His。
结果显示，本披露的抗体有较好的CD38/BCMA结合力，并且能结合CD3d&CD3e。
测试例3.FACS测定抗体对MM细胞表面抗原的亲和力
为测试本披露的抗体与MM细胞表面hCD38和hBCMA的结合能力，本测试例用流式细胞术方法检测了CD38/BCMA-CD3三特异性抗体与CD38和BCMA共表达的MM肿瘤细胞RPMI-8226的结合能力。具体如下：上述细胞培养于含10％FBS的1640的培养基中，放置于37℃，5％CO 2培养箱中，培养2天，按每孔细胞数1×10 5个将细胞加至细胞板中，300g离心分钟，1％BSA洗一次。用Mix-n-Stain CF 647Antibody Labeling Kits(MX647S100-1KT#，Sigma)直标抗体，并将荧光标记后的抗体梯度稀释，按每孔100μL加入细胞板中，4℃孵育1小时，1％BSA洗板三次，每孔加入100μL PBS读板。结果如图2所示，本披露的抗体能非常好的结合到BCMA和CD38共表达的肿瘤细胞表面，其效果优于阳性对照。
测试例4.抗体的体外细胞毒活性
为测试本披露的抗体作为T细胞衔接分子对肿瘤细胞的杀伤活性。用过表达人BCMA的稳转细胞株K562/hBCMA，过表达人CD38的稳转细胞株CHO K1/hCD38和CD38和BCMA共表达的MM肿瘤细胞RPMI-8226作为靶细胞来检测本披露的双特异性抗体的靶点特异性细胞毒活性,并用不表达CD38和BCMA的CHOK1作为靶细胞来检测本披露的双特异性抗体的非特异性细胞毒活性。新鲜 PBMC(购自轩峰生物公司)300g离心10分钟，用含有200U IL2的1640+10％FBS(A液)重悬，置于T75的培养瓶培养过夜(密度为2×10 6/mL)；收集离心后分别用IMDM+10％FBS、DMEM/F-12 1:1+10％FBS和1640+10％FBS的培养基重悬、计数，调整细胞数为1.5×10 6个/mL，每孔加入50μL。收集靶细胞，1000rpm离心3min，重悬、计数，调整细胞数为3×10 5细胞/mL，每孔加入25μL，E:T Ratio为10:1。抗体用细胞分别对应的培养基稀释，起始浓度为400nM(4×终浓度)，10倍稀释9个梯度，每孔加入25μL。然后将细胞放在37℃，5％CO 2的培养箱培养48h。萤光信号检测：取出培养板，1000rpm离心3min，吸取50μL上清于新的96孔板，冻存于-20℃冰箱，在原来含有细胞的板子中，按1:1的比例加入50μL one-Glo，室温孵育5min，检测发光的数值(将未加抗体组的细胞的发光值定义为0杀伤)，计算不同抗体在不同浓度下的杀伤百分比。
表13-1.抗体对K562/hBCMA的细胞毒活性
抗体 IC50(pM) 裂解率％
T1-12 0.034 98.6
T3-12 0.110 98.7
T5-12 0.012 99.5
T5-12-6164 0.148 99.5
T1-21 0.085 98.8
T3-21 0.024 99.4
AMGEN701 0.343 99.5
表13-2.抗体对CHO K1/hCD38的细胞毒活性
Figure PCTCN2022105578-appb-000096
表13-3.抗体对RPMI8266的细胞毒活性
Figure PCTCN2022105578-appb-000097
Figure PCTCN2022105578-appb-000098
注：Format5-6164和Format24的Knob链C端含有6His。
结果见表13-1至13-3和图3，本披露的抗体的细胞毒活性是CD38和BCMA靶点特异性的，其效果优于阳性对照；同时，图3显示在没有CD38以及BCMA表达的CHOK1细胞系上，本披露的抗体表现出更弱的细胞毒活性，具有更好的安全性。
测试例5.可溶性BCMA/CD38/APRIL对本披露抗体的体外细胞毒活性的影响
多发性骨髓瘤病人血液中存在的高浓度可溶性BCMA(平均30ng/mL)会干扰BCMA-CD3双特异性抗体特异性结合到膜表面BCMA，其次，APRIL在MM病人血清中的含量也是健康人中的5.9倍，可溶性CD38在MM病人中也有一定比例的增高。为测试本披露的抗体体外肿瘤细胞杀伤的活性是否受可溶性BCMA/CD38/APRIL的影响，采用共表达CD38和BCMA的细胞系RPMI-8226作为靶细胞，在细胞毒活性测试时，分别添加或者一起添加了可溶性BCMA、CD38和APRIL。新鲜PBMC(购自轩峰生物公司)300g离心10min，用含有200U IL2的1640+10％FBS(A液)重悬，置于T75的培养瓶培养过夜(密度为2E6/mL)；收集离心后分别用1640+10％FBS的培养基重悬、计数，调整细胞数为1.5×10 6细胞/mL，每孔加入50μL。收集靶细胞，1000rpm离心3分钟，重悬、计数，调整细胞数为3×10 5细胞/mL，每孔加入25μL，E:T Ratio为10:1。抗体用细胞分别对应的培养基稀释，起始浓度为400nM(4×终浓度)，10倍稀释9个梯度，每孔加入25μL。然后将细胞放在37℃，5％CO 2的培养箱培养48h。萤光信号检测：取出培养板，1000rpm离心3min，吸取50μL上清于新的96孔板，冻存于-20℃冰箱，在原来含有细胞的板子中，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(将未加抗体组的细胞的发光值定义为0杀伤)，计算不同抗体在不同浓度下的杀伤百分比。
表14.可溶性蛋白对抗体细胞杀伤活性的影响
Figure PCTCN2022105578-appb-000099
Figure PCTCN2022105578-appb-000100
注：“相对IC 50”表示添加可溶性蛋白的IC50与不添加可溶性蛋白的IC50的比值。
结果显示，本披露的抗体的细胞毒活性受可溶性BCMA/CD38/APRIL影响极小。与没有可溶性蛋白存在的条件下进行对比，30ng/mL的可溶性BCMA、5ng/mL的可溶性CD38以及100ng/mL的可溶性APRIL分别存在或者共同存在仅使本披露的抗体的细胞毒作用IC 50变至原来的0.8～2.5倍，而AMGEN701在可溶性蛋白存在时，IC 50却增加到原来的10倍。
测试例6.抗体诱导的细胞因子释放水平检测
CD3T细胞衔接分子会引起细胞因子风暴。因此开发CD3T细胞衔接分子时，需要能将细胞因子，尤其是和药效无关但会引起副作用的因子IL-6，保持在较低水平。
将CHO-K1细胞1000rpm离心分钟，收集50μL的细胞上清用标本通用稀释液稀释6倍，IL-6或IL-2标准品亦稀释6倍，在检测板中加入稀释好的样品或者不同浓度标准品(100μL/孔)，用封板胶纸封住反应孔，37℃孵育90分钟后洗板5次，加入生物素化抗体工作液(100μL/孔)，用新封板胶纸封住反应孔，37℃孵育60分钟后洗板5次，加入酶结合物工作液(100μL/孔)，用新封板胶纸封住反应孔，37℃孵育30分钟后洗板5次，加入显色底物(TMB)100μL/孔，避光37℃孵育8分钟，加入反应终止液100μL/孔，混匀后即刻测量OD450值(3分钟内)。
表15.IL-6与IL-2的释放(CHO K1)
Figure PCTCN2022105578-appb-000101
结果显示，本披露的抗体带来的IL6和IL-2的释放水平均低于GBR-1342，提示本披露的抗体具有更好的安全性。
体内活性生物学评价
测试例7.抗体在Molp-8原位瘤模型中的药效
Molp-8作为典型的MM细胞系，有较高的CD38表达和较低的BCMA表达。
为测试本披露的抗体在Molp-8原位瘤模型中的药效，将Molp-8/lucG细胞1.5×10 6细胞/200μL/小鼠通过尾静脉接种于79只NDG小鼠体内。肿瘤细胞接种后1天，将两名供体新鲜分离的PBMCs以1:1比例混合，以5×10 6/小鼠注射到小鼠腹腔。肿瘤细胞接种后4天，每只小鼠腹腔注射生物发光底物(15mg/mL)，按 照10ml/kg体积注射，通过异氟烷麻醉，注射10分钟后通过小动物成像系统拍照成像。去除体重过小的、生物发光信号值过大和过小的，按生物发光信号将小鼠随机分组，每组7只。分组当天开始腹腔注射各抗体，并将该天定义为该实验Day0，每周2次。每周拍照成像2次，称体重，记录数据。所有数据使用Excel和GraphPad Prism 8软件进行作图及统计分析。
生物发光信号值为Total Flux(单位，p/s)，平均值以avg计算；
抑瘤率(％)＝1-T/C(％)。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生物发光信号值；T 0、C 0为实验开始时的生物发光信号值。实验结果显示，T3-12和T5-12-6164都表现出了明显药效。
表16.抗体在Molp-8原位瘤模型中的药效
Figure PCTCN2022105578-appb-000102
测试例8.抗体在RPMI-8226原位瘤模型的药效
RPMI-8226作为典型的MM细胞系，有中等的CD38表达和BCMA表达。
将RPMI-8226-lucG细胞5×10 6细胞/200μL/小鼠通过尾静脉接种于NDG小鼠体内。肿瘤细胞接种15天，每只小鼠腹腔注射生物发光底物(15mg/mL)，按照10mL/kg体积注射，通过异氟烷麻醉，注射10分钟后通过小动物成像系统拍照成像。去除体重过小的、生物发光信号值过大和过小的，按生物发光信号将小鼠随机分组，每组7只。分组后1天，将两名供体新鲜分离的PBMCs以1:1比例混合，以5×10 6/小鼠注射到小鼠腹腔。分组后4天开始腹腔注射各抗体，并将该天定义为该实验Day0，每周2次，共给药4次。每周拍照成像2次，称体重，记录数据。所有数据使用Excel和GraphPad Prism 8软件进行作图及统计分析。
生物发光信号值为Total Flux(单位，p/s)，平均值以avg计算；
抑瘤率(％)＝1-T/C(％)。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生物发光信号值；T 0、C 0为实验开始时的生物发光信号值。
表17.抗体在RPMI-8226原位瘤模型中的药效
给药组 给药剂量(mg/kg) 抑瘤率％
空白(PBS) / /
T3-12 0.01 14.4
T3-12 0.03 96.9
T3-12 0.1 >100
T5-12-6164 0.03 50.2
GBR-1342 0.015 -263.0
GBR-1342 0.05 -17.8
结果显示，T3-12和T5-12-6164都表现出了明显的药效，均比等摩尔的GBR-1342表现出更好的药效。在给药过程中，荷瘤小鼠对各抗体均能较好耐受，体重没有明显波动。
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。

Claims (19)

  1. 一种特异性结合BCMA、CD38和CD3的抗原结合分子，其包含至少一个特异性结合BCMA的抗原结合模块、至少一个特异性结合CD38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。
  2. 根据权利要求1所述的抗原结合分子，所述抗原结合分子包含两个特异性结合BCMA的抗原结合模块、两个特异性结合CD38的抗原结合模块、两个特异性结合CD3的抗原结合模块和Fc区；
    优选地，
    所述特异性结合BCMA的抗原结合模块是BCMA-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合CD38的抗原结合模块是CD38-scFv；或
    所述特异性结合CD38的抗原结合模块是CD38-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合BCMA的抗原结合模块是BCMA-scFv；
    更优选地，
    所述特异性结合BCMA的抗原结合模块是BCMA-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合CD38的抗原结合模块是CD38-scFv；并且BCMA-Fab的重链、CD3-scFv、Fc区的一个亚基和CD38-scFv按N端至C端的顺序分别直接地或通过连接子连接；或
    所述特异性结合CD38的抗原结合模块是CD38-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合BCMA的抗原结合模块是BCMA-scFv；并且CD38-Fab的重链、CD3-scFv、Fc区的一个亚基和BCMA-scFv按N端至C端的顺序分别直接地或通过连接子连接。
  3. 根据权利要求1或2所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-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，和所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL；其中：
    所述抗原结合分子包含两条具有式(a)所示结构的第一链和两条具有式(b)所示结构的第二链：
    (a)[BCMA-VH]-[CH1]-[CD3-VH]-[连接子1]-[CD3-VL]-[连接子2]-[Fc区的一个亚基]-[连接子3]-[CD38-VH]-[连接子4]-[CD38-VL]，
    (b)[BCMA-VL]-[CL]，
    式(a)中的连接子1、连接子2、连接子3和连接子4是相同或不同的肽连接子；或
    所述抗原结合分子包含两条具有式(c)所示结构的第一链和两条具有式(d)所示结构的第二链：
    (c)[CD38-VH]-[CH1]-[CD3-VH]-[连接子1]-[CD3-VL]-[连接子2]-[Fc区的一个亚基]-[连接子3]-[BCMA-VH]-[连接子4]-[BCMA-VL]，
    (d)[CD38-VL]-[CL]，
    式(c)中的连接子1、连接子2、连接子3和连接子4是相同或不同的肽连接子；
    优选地，
    式(a)中的连接子1、连接子2、连接子3和连接子4的长度为3-15个氨基酸残基；或
    式(c)中的连接子1、连接子2、连接子3和连接子4的长度为3-15个氨基酸残基；
    更优选地，
    式(a)中的连接子1、连接子3和连接子4的氨基酸序列如SEQ ID NO：99所示，连接子2的氨基酸序列如SEQ ID NO：96所示；或
    式(c)中的连接子1、连接子3和连接子4的氨基酸序列如SEQ ID NO：99所示，连接子2的氨基酸序列如SEQ ID NO：96所示。
  4. 根据权利要求1所述的抗原结合分子，所述抗原结合分子包含一个特异性结合BCMA的抗原结合模块、两个特异性结合CD38的抗原结合模块、一个特异性结合CD3的抗原结合模块和Fc区；
    优选地，
    所述特异性结合BCMA的抗原结合模块是BCMA-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合CD38的抗原结合模块是CD38-scFv；或
    所述特异性结合CD38的抗原结合模块是CD38-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合BCMA的抗原结合模块是BCMA-scFv；
    更优选地，所述Fc区包含能够缔合的第一亚基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；并且，
    所述特异性结合BCMA的抗原结合模块是BCMA-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合CD38的抗原结合模块是CD38-scFv；并且BCMA-Fab的重链、Fc1和一个CD38-scFv按N端至C端的顺序分别直接地或通过连接子连接，和CD3-scFv、Fc2和另一个CD38-scFv按N端至C端的顺序分别直接地或通过连接子连接；或
    所述特异性结合CD38的抗原结合模块是CD38-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合BCMA的抗原结合模块是BCMA-scFv；并且一个CD38-Fab的重链、Fc1和CD3-scFv按N端至C端的顺序分别直接地或通过连接子连接，和BCMA-scFv、另一个CD38-Fab的重链和Fc2按N端至C端的顺序分别直接地或通过连接子连接。
  5. 根据权利要求1或4所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-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，和所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL；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(b)所示结构的第二链和一条具有式(f)所示结构的第三链：
    (e)[BCMA-VH]-[CH1]-[Fc1]-[连接子1]-[CD38-VH]-[连接子2]-[CD38-VL]，
    (b)[BCMA-VL]-[CL]，
    (f)[CD3-VH]-[连接子1]-[CD3-VL]-[连接子2]-[Fc2]-[连接子3]-[CD38-VH]-[连接子4]-[CD38-VL]，
    其中，式(e)中的连接子1和连接子2是相同或不同的肽连接子，和式(f)中的连接子1、连接子2、连接子3和连接子4是相同或不同的肽连接子；或
    所述抗原结合分子包含一条具有式(g)所示结构的第一链、两条具有式(d)所示结构的第二链和一条具有式(h)所示结构的第三链：
    (g)[BCMA-VL]-[连接子1]-[BCMA-VH]-[连接子2]-[CD38-VH]-[CH1]-[Fc2]，
    (d)[CD38-VL]-[CL]，
    (h)[CD38-VH]-[CH1]-[Fc1]-[连接子1]-[CD3-VL]-[连接子2]-[CD3-VH]，
    其中，式(g)中的连接子1和连接子2是相同或不同的肽连接子，和式(h)中的连接子1和连接子2是相同或不同的肽连接子；
    优选地，
    式(e)中的连接子1、连接子2，以及式(f)中的连接子1、连接子2、连接子3和连接子4的长度为3-15个氨基酸残基；或
    式(g)中的连接子1、连接子2，以及式(h)中的连接子1和连接子2的长度为3-15个氨基酸残基；
    更优选地，
    式(e)中的连接子1和连接子2的氨基酸序列如SEQ ID NO：99所示，式(f)中的连接子1、连接子3和连接子4的氨基酸序列如SEQ ID NO：99所示，和式(f)中的连接子2的氨基酸序列如SEQ ID NO：96所示；或
    式(g)中的连接子1和连接子2的氨基酸序列如SEQ ID NO：99所示，式(h) 中的连接子1的氨基酸序列如SEQ ID NO：97所示，和式(h)中连接子2的氨基酸序列如SEQ ID NO：99所示。
  6. 根据权利要求1所述的抗原结合分子，所述抗原结合分子包含一个特异性结合BCMA的抗原结合模块、一个特异性结合CD38的抗原结合模块、一个特异性结合CD3的抗原结合模块和Fc区；
    优选地，
    所述特异性结合BCMA的抗原结合模块是BCMA-Fab，所述特异性结合CD3的抗原结合模块是CD3-scFv和所述特异性结合CD38的抗原结合模块是CD38-scFv；
    更优选地，
    所述Fc区包含能够缔合的第一亚基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；并且CD38-scFv、CD3-scFv和Fc1按N端至C端的顺序分别直接地或通过连接子连接，和BCMA-Fab的重链和Fc2按N端至C端的顺序分别直接地或通过连接子连接。
  7. 根据权利要求1或6所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-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，和所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL；其中：
    所述抗原结合分子包含一条具有式(i)所示结构的第一链、一条具有式(b)所示结构的第二链和一条具有式(j)所示结构的第三链：
    (i)[BCMA-VH]-[CH1]-[Fc2]，
    (b)[BCMA-VL]-[CL]，
    (j)[CD38-VH]-[连接子1]-[CD38-VL]-[连接子2]-[CD3-VH]-[连接子3]-[CD3-VL]-[连接子4]-[Fc1]，
    其中，式(j)中的连接子1、连接子2、连接子3和连接子4是相同或不同的肽连接子；
    优选地，
    式(j)中的连接子1、连接子2、连接子3和连接子4的长度为3-15个氨基酸残基；
    更优选地，
    式(j)中的连接子1和连接子3的氨基酸序列如SEQ ID NO：99所示，式(j)中的连接子2的氨基酸序列如SEQ ID NO：97所示，和式(j)中的连接子4的氨基酸序列如SEQ ID NO：96所示。
  8. 根据权利要求1至7任一项所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-VL，其中：
    (i)所述BCMA-VH包含SEQ ID NO：23中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：24中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的氨基酸序列；或
    (ii)所述BCMA-VH包含SEQ ID NO：25中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：26中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的氨基酸序列；或
    (iii)所述BCMA-VH包含SEQ ID NO：27中的BCMA-HCDR1、BCMA-HCDR2和BCMA-HCDR3的氨基酸序列，和所述BCMA-VL包含SEQ ID NO：28中的BCMA-LCDR1、BCMA-LCDR2和BCMA-LCDR3的氨基酸序列；
    优选地，
    (i)所述BCMA-VH具有：SEQ ID NO：5的BCMA-HCDR1、SEQ ID NO：6的BCMA-HCDR2和SEQ ID NO：7的BCMA-HCDR3；并且所述BCMA-VL具有：SEQ ID NO：8的BCMA-LCDR1、SEQ ID NO：9的BCMA-LCDR2和SEQ ID NO：10的BCMA-LCDR3，或
    (ii)所述BCMA-VH具有SEQ ID NO：11的BCMA-HCDR1、SEQ ID NO：12的BCMA-HCDR2和SEQ ID NO：13的BCMA-HCDR3；并且所述BCMA-VL具有SEQ ID NO：14的BCMA-LCDR1、SEQ ID NO：15的BCMA-LCDR2和SEQ ID NO：16的BCMA-LCDR3，或
    (iii)所述BCMA-VH具有SEQ ID NO：17的BCMA-HCDR1、SEQ ID NO：18的BCMA-HCDR2和SEQ ID NO：19的BCMA-HCDR3；并且所述BCMA-VL具有SEQ ID NO：20的BCMA-LCDR1、SEQ ID NO：21的BCMA-LCDR2和SEQ ID NO：22的BCMA-LCDR3。
  9. 根据权利要求8所述的抗原结合分子，其中：
    (i)所述BCMA-VH如SEQ ID NO：23所示，和所述BCMA-VL如SEQ ID NO：24所示，或
    所述BCMA-VH如SEQ ID NO：29、SEQ ID NO：30和SEQ ID NO：31中任一所示，和所述BCMA-VL如SEQ ID NO：32、SEQ ID NO：33、SEQ ID NO：34和SEQ ID NO：35中任一所示，
    优选地，
    所述BCMA-VH如SEQ ID NO：29所示，和所述BCMA-VL如SEQ ID NO：32所示；或
    (ii)所述BCMA-VH如SEQ ID NO：25所示，和所述BCMA-VL如SEQ ID NO： 26所示，或
    所述BCMA-VH如SEQ ID NO：36、SEQ ID NO：37和SEQ ID NO：38中任一所示，和所述BCMA-VL如SEQ ID NO：39或SEQ ID NO：40之一所示，
    优选地，
    所述BCMA-VH如SEQ ID NO：36所示，和所述BCMA-VL如SEQ ID NO：40所示；或
    (iii)所述BCMA-VH如SEQ ID NO：27所示，和所述BCMA-VL如SEQ ID NO：28所示，或
    所述BCMA-VH如SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43和SEQ ID NO：44中任一所示，和所述BCMA-VL如SEQ ID NO：45或SEQ ID NO：46之一所示，
    优选地，
    所述BCMA-VH如SEQ ID NO：43所示，和所述BCMA-VL如SEQ ID NO：45所示。
  10. 根据权利要求1至9任一项所述的抗原结合分子，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：
    (i)所述CD3-VH包含SEQ ID NO：63中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3的氨基酸序列；和所述CD3-VL包含SEQ ID NO：64中的CD3-LCDR1、CD3-LCDR2和CD3-LCDR3的氨基酸序列，或
    (ii)所述CD3-VH包含SEQ ID NO：65中的CD3-HCDR1、CD3-HCDR2和CD3-HCDR3的氨基酸序列；和所述CD3-VL包含SEQ ID NO：64中的CD3-LCDR1、CD3-LCDR2和CD3-LCDR3的氨基酸序列；
    优选地，
    (i)所述CD3-VH具有如SEQ ID NO：55所示的CD3-HCDR1，如SEQ ID NO：56所示的CD3-HCDR2，和如SEQ ID NO：57所示的CD3-HCDR3；并且所述CD3-VL具有如SEQ ID NO：58所示的CD3-LCDR1，如SEQ ID NO：59所示的CD3-LCDR2，和如SEQ ID NO：60所示的CD3-LCDR3，或
    (ii)所述CD3-VH具有如SEQ ID NO：55所示的CD3-HCDR1，如SEQ ID NO：61所示的CD3-HCDR2，和如SEQ ID NO：62所示的CD3-HCDR3；并且所述CD3-VL具有如SEQ ID NO：58所示的CD3-LCDR1，如SEQ ID NO：59所示的CD3-LCDR2，和如SEQ ID NO：60所示的CD3-LCDR3；
    更优选地，
    所述CD3-VH如SEQ ID NO：63所示，和所述CD3-VL如SEQ ID NO：64所示；或
    所述CD3-VH如SEQ ID NO：65所示，和所述CD3-VL如SEQ ID NO：64 所示。
  11. 根据权利要求1至10任一项所述的抗原结合分子，所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL，其中所述CD38-VH包含SEQ ID NO：53中的CD38-HCDR1、CD38-HCDR2和CD38-HCDR3的氨基酸序列，并且所述CD38-VL包含SEQ ID NO：54中的CD38-LCDR1、CD38-LCDR2和CD38-LCDR3的氨基酸序列；
    优选地，
    所述CD38-VH具有如SEQ ID NO：47所示的CD38-HCDR1，如SEQ ID NO：48所示的CD38-HCDR2，和如SEQ ID NO：49所示的CD38-HCDR3；并且所述CD38-VL具有如SEQ ID NO：50所示的CD38-LCDR1，如SEQ ID NO：51所示的CD38-LCDR2，和如SEQ ID NO：52所示的CD38-LCDR3；
    更优选地，
    所述CD38-VH如SEQ ID NO：53所示，和所述CD38-VL如SEQ ID NO：54所示。
  12. 根据权利要求1至11任一项所述的抗原结合分子，所述特异性结合BCMA的抗原结合模块包含重链可变区BCMA-VH和轻链可变区BCMA-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和所述特异性结合CD38的抗原结合模块包含重链可变区CD38-VH和轻链可变区CD38-VL，其中：
    (i)所述BCMA-VH具有：如SEQ ID NO：5所示的BCMA-HCDR1、如SEQ ID NO：6所示的BCMA-HCDR2和如SEQ ID NO：7所示的BCMA-HCDR3；并且所述BCMA-VL具有：如SEQ ID NO：8所示的BCMA-LCDR1、如SEQ ID NO：9所示的BCMA-LCDR2和如SEQ ID NO：10所示的BCMA-LCDR3，和
    所述CD3-VH具有：如SEQ ID NO：55所示的CD3-HCDR1，如SEQ ID NO：61所示的CD3-HCDR2，和如SEQ ID NO：62所示的CD3-HCDR3；并且所述CD3-VL具有：如SEQ ID NO：58所示的CD3-LCDR1，如SEQ ID NO：59所示的CD3-LCDR2，和如SEQ ID NO：60所示的CD3-LCDR3，和
    所述CD38-VH具有：如SEQ ID NO：47所示的CD38-HCDR1，如SEQ ID NO：48所示的CD38-HCDR2，和如SEQ ID NO：49所示的CD38-HCDR3；并且所述CD38-VL具有：如SEQ ID NO：5所示的CD38-LCDR1，如SEQ ID NO：51所示的CD38-LCDR2，和如SEQ ID NO：52所示的CD38-LCDR3；或
    (ii)所述BCMA-VH具有：如SEQ ID NO：11所示的BCMA-HCDR1、如SEQ ID NO：12所示的BCMA-HCDR2和如SEQ ID NO：13所示的BCMA-HCDR3；并且所述BCMA-VL具有：如SEQ ID NO：14所示的BCMA-LCDR1、如SEQ ID  NO：15所示的BCMA-LCDR2和如SEQ ID NO：16所示的BCMA-LCDR3，和
    所述CD3-VH具有：如SEQ ID NO：55所示的CD3-HCDR1，如SEQ ID NO：56所示的CD3-HCDR2，和如SEQ ID NO：57所示的CD3-HCDR3；并且所述CD3-VL具有：如SEQ ID NO：58所示的CD3-LCDR1，如SEQ ID NO：59所示的CD3-LCDR2，和如SEQ ID NO：60所示的CD3-LCDR3，和
    所述CD38-VH具有：如SEQ ID NO：47所示的CD38-HCDR1，如SEQ ID NO：48所示的CD38-HCDR2，和如SEQ ID NO：49所示的CD38-HCDR3；并且所述CD38-VL具有：如SEQ ID NO：50所示的CD38-LCDR1，如SEQ ID NO：51所示的CD38-LCDR2，和如SEQ ID NO：52所示的CD38-LCDR3；
    优选地，
    (i)所述BCMA-VH如SEQ ID NO：29所示，所述BCMA-VL如SEQ ID NO：32所示，所述CD3-VH如SEQ ID NO：65所示，所述CD3-VL如SEQ ID NO：64所示，所述CD38-VH如SEQ ID NO：53所示，和所述CD38-VL如SEQ ID NO：54所示；或
    (ii)所述BCMA-VH如SEQ ID NO：36所示，所述BCMA-VL如SEQ ID NO：40所示，所述CD3-VH如SEQ ID NO：63所示，所述CD3-VL如SEQ ID NO：64所示，所述CD38-VH如SEQ ID NO：53所示，和所述CD38-VL如SEQ ID NO：54所示。
  13. 根据权利要求1至12任一项所述的抗原结合分子，所述抗原结合分子包含Fc区，所述Fc区优选为IgG Fc区，更优选为IgG 1 Fc区；
    优选地，所述Fc区包含一个或多个氨基酸取代，所述的氨基酸取代能够减少Fc区与Fc受体的结合；
    更优选地，所述的氨基酸取代能够减少其与Fcγ受体的结合；
    特别地，所述Fc区是人IgG 1 Fc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。
  14. 根据权利要求4至13任一项所述的抗原结合分子，所述抗原结合分子包含Fc区，所述Fc区包含能够缔合的第一亚基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；
    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；
    更优选地，
    所述Fc1在366位置的氨基酸残基为W；和所述Fc2在366位置的氨基酸残基为S、在368位置的氨基酸残基为A、和在407位置的氨基酸残基为V，编号依 据为EU索引。
  15. 根据权利要求1所述的抗原结合分子，
    所述抗原结合分子具有：两条包含SEQ ID NO：76的氨基酸序列的第一链和两条包含SEQ ID NO：74的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：75的氨基酸序列的第一链和两条包含SEQ ID NO：74的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：82的氨基酸序列的第一链和两条包含SEQ ID NO：81的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：83的氨基酸序列的第一链和两条包含SEQ ID NO：81的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：90的氨基酸序列的第一链和两条包含SEQ ID NO：89的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：91的氨基酸序列的第一链和两条包含SEQ ID NO：89的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：77的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：78的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：85的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：86的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：93的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：94的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：73的氨基酸序列的第一链、一条包含SEQ ID NO：74的氨基酸序列的第二链和一条包含SEQ ID NO：66的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：73的氨基酸序列的第一链、一条包含SEQ ID NO：74的氨基酸序列的第二链和一条包含SEQ ID NO：67的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：80的氨基酸序列的第一链、一条包含SEQ ID NO：81的氨基酸序列的第二链和一条包含SEQ ID NO：66的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：80的氨基酸序列的第一链、一条包含SEQ ID NO：81的氨基酸序列的第二链和一条包含SEQ ID NO：67的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：88的氨基酸序列的第一链、一条包含SEQ ID NO：89的氨基酸序列的第二链和一条包含SEQ ID NO：66的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：88的氨基酸序列的第一链、一条包含SEQ ID NO：89的氨基酸序列的第二链和一条包含SEQ ID NO：67的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：84的氨基酸序列的第一链、一条包含SEQ ID NO：81的氨基酸序列的第二链和一条包含SEQ ID NO：69的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：84的氨基酸序列的第一链、一条包含SEQ ID NO：81的氨基酸序列的第二链和一条包含SEQ ID NO：70的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：92的氨基酸序列的第一链、一条包含SEQ ID NO：89的氨基酸序列的第二链和一条包含SEQ ID NO：69的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：92的氨基酸序列的第一链、一条包含SEQ ID NO：89的氨基酸序列的第二链和一条包含SEQ ID NO：70的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：79的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链和一条包含SEQ ID NO：72的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：87的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链和一条包含SEQ ID NO：72的氨基酸序列的第三链；或
    所述抗原结合分子具有：一条包含SEQ ID NO：95的氨基酸序列的第一链、两条包含SEQ ID NO：71的氨基酸序列的第二链和一条包含SEQ ID NO：72的氨基酸序列的第三链；
    优选地，
    所述抗原结合分子具有：两条包含SEQ ID NO：76的氨基酸序列的第一链和两条包含SEQ ID NO：74的氨基酸序列的第二链；或
    所述抗原结合分子具有：两条包含SEQ ID NO：82的氨基酸序列的第一链和两条包含SEQ ID NO：81的氨基酸序列的第二链。
  16. 一种药物组合物，其含有：
    治疗有效量的权利要求1至15任一项所述的抗原结合分子，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂；
    优选地，所述的药物组合物中还包含至少一种第二治疗剂。
  17. 分离的核酸，其编码权利要求1至15任一项所述的抗原结合分子。
  18. 一种宿主细胞，其包含如权利要求17所述的分离的核酸。
  19. 一种治疗疾病的方法，所述方法包括向受试者施用权利要求1至15任一项所述的抗原结合分子或权利要求16所述的药物组合物的步骤；
    优选地，所述疾病是淋巴谱系或骨髓谱系的造血系统肿瘤或自身免疫性疾病；
    更优选地，所述淋巴谱系或骨髓谱系的造血系统肿瘤选自：多发性骨髓瘤、浆细胞瘤、浆细胞白血病、巨球蛋白血症、淀粉样变性、华氏巨球蛋白血症、孤立性骨浆细胞瘤、髓外浆细胞瘤、骨硬化性骨髓瘤、重链病、意义不明确的单克隆丙种球蛋白病以及郁积型多发性骨髓瘤、B-细胞淋巴瘤、伯基特淋巴瘤、何杰金淋巴瘤和多毛细胞淋巴瘤；所述自身免疫性疾病选自：类风湿性关节炎、系统性红斑性狼疮、哮喘、炎症性肠道疾病、多发性硬化、克罗恩氏病、胃炎、桥本氏甲状腺炎、强直性脊柱炎和移植物对抗宿主疾病。
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