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WO2022135468A1 - 抗bcma×cd3双特异性抗体及其用途 - Google Patents

抗bcma×cd3双特异性抗体及其用途 Download PDF

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Publication number
WO2022135468A1
WO2022135468A1 PCT/CN2021/140450 CN2021140450W WO2022135468A1 WO 2022135468 A1 WO2022135468 A1 WO 2022135468A1 CN 2021140450 W CN2021140450 W CN 2021140450W WO 2022135468 A1 WO2022135468 A1 WO 2022135468A1
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Prior art keywords
seq
peptide
peptide chain
chain cdrs
chain
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PCT/CN2021/140450
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English (en)
French (fr)
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濮瀑
陈炳良
李莉
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信达生物制药(苏州)有限公司
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Publication of WO2022135468A1 publication Critical patent/WO2022135468A1/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
    • 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
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention generally relates to the fields of immunology and antibody engineering.
  • the present invention relates to novel bispecific antibodies that specifically bind BCMA and CD3.
  • the present invention relates to nucleic acids encoding said anti-BCMAxCD3 bispecific antibodies, vectors comprising said nucleic acids, host cells comprising said nucleic acids or vectors, and pharmaceutical compositions comprising said antibodies or antigen-binding fragments thereof.
  • the present invention relates to the application of these anti-BCMA ⁇ CD3 bispecific antibodies, pharmaceutical compositions and the like in the immunotherapy, prevention and/or diagnosis of diseases.
  • BCMA B cell maturation antigen
  • BAFF B cell activating factor receptor
  • APRIL B cell proliferation-inducing ligand
  • BCMA is predominantly expressed in plasma cells and mature B cell subsets. In 60-70% of multiple myeloma (MM) patients, BCMA is also expressed on the surface of cancerous plasma cells. Serum BCMA levels are elevated in patients with multiple myeloma, and elevated levels correlate with disease status, treatment response, and overall survival. Mice deficient in the BCMA gene had normal B cell levels, but their plasma cell lifespan was significantly shortened. Therefore, BCMA is an ideal target for immunotherapy in multiple myeloma.
  • the present invention provides a bispecific antibody that binds BCMA and CD3 with high target specificity and high affinity, in particular by binding to BCMA expressed on the surface of tumor cells, allowing T cells to be recruited around tumor cells. Antibodies meet this need.
  • the present invention discloses a novel bispecific antibody simultaneously targeting BCMA and CD3, a polynucleotide encoding the bispecific antibody, a vector comprising the polynucleotide, and a polynucleotide comprising the polynucleotide or the vector Host cells, and the use of the bispecific antibodies in the treatment, prevention and/or diagnosis of diseases associated with BCMA activity in an individual.
  • the invention provides a bispecific antibody that specifically binds BCMA and binds CD3 (anti-BCMA x CD3 bispecific antibody) comprising (i) an anti-BCMA antibody or fragment thereof, and (ii) an anti-BCMA CD3 antibody or fragment thereof.
  • the present invention provides an anti-BCMA ⁇ CD3 bispecific antibody in a 1+1 format, as shown in FIG. 1 , which consists of three left-right asymmetrical polypeptide chains, wherein the left half is composed of peptide chains 1 and 1.
  • Peptide chain 2 is composed of peptide chain 3
  • the right half is composed of peptide chain 3
  • peptide chain 1 and peptide chain 2 are the heavy and light chains of antibodies targeting BCMA or CD3, respectively
  • peptide chain 3 is composed of scFv and Fc regions targeting CD3 or BCMA composition.
  • the present invention provides an anti-BCMA ⁇ CD3 bispecific antibody in a 2+1 format, as shown in FIG. 2 , which consists of four left-right asymmetrical polypeptide chains, wherein peptide chain 1 and peptide chain 2 are the antibody heavy and light chains targeting BCMA or CD3, respectively, and peptide 3 consists of an antibody heavy chain targeting BCMA or CD3 and an scFv targeting CD3 or BCMA.
  • the invention provides an anti-BCMAxCD3 bispecific antibody in a 1+1 format, wherein peptide 1 and 2 specifically bind BCMA and peptide 3 specifically binds CD3.
  • the anti-BCMA ⁇ CD3 bispecific antibody provided by the present invention comprises peptide chain 1, peptide chain 2 and peptide chain 3, wherein peptide chain 1 comprises a peptide selected from the group consisting of SEQ ID NO: 1, 3 or 5
  • peptide chain 2 includes 3 light chain CDRs selected from the group consisting of SEQ ID NO: 2, 4 or 6, and the peptide chain 3 includes 3 selected from the group consisting of SEQ ID NO: 7 or 9.
  • the anti-BCMA ⁇ CD3 bispecific antibody provided by the present invention comprises peptide chain 1, peptide chain 2 and peptide chain 3, wherein:
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 1
  • peptide chain 2 comprises 3 light chain CDRs from SEQ ID NO: 2
  • peptide chain 3 comprises 3 light chain CDRs from SEQ ID NO: 7
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 3
  • peptide chain 2 comprises 3 light chain CDRs from SEQ ID NO: 4
  • peptide chain 3 comprises 3 light chain CDRs from SEQ ID NO: 7
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 5
  • peptide chain 2 comprises 3 light chain CDRs from SEQ ID NO: 6
  • peptide chain 3 comprises 3 light chain CDRs from SEQ ID NO: 7
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 1
  • peptide chain 2 comprises 3 light chain CDRs from SEQ ID NO: 2
  • peptide chain 3 comprises 3 light chain CDRs from SEQ ID NO: 9
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 3
  • peptide chain 2 comprises 3 light chain CDRs from SEQ ID NO: 4
  • peptide chain 3 comprises 3 light chain CDRs from SEQ ID NO: 9.
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 5
  • peptide chain 2 comprises 3 light chain CDRs from SEQ ID NO: 6
  • peptide chain 3 comprises 3 light chain CDRs from SEQ ID NO: 9
  • the anti-BCMA ⁇ CD3 bispecific antibody provided by the present invention comprises peptide chain 1, peptide chain 2 and peptide chain 3, wherein:
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 47-49
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 50-52
  • peptide chain 3 includes SEQ ID NOs: 3 heavy chain CDRs shown in 65-67 and 3 light chain CDRs shown in SEQ ID NOs: 68-70;
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 53-55
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 56-58
  • peptide chain 3 includes SEQ ID NOs: 3 heavy chain CDRs shown in 65-67 and 3 light chain CDRs shown in SEQ ID NOs: 68-70;
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 59-61
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 62-64
  • peptide chain 3 includes SEQ ID NOs: 3 heavy chain CDRs shown in 65-67 and 3 light chain CDRs shown in SEQ ID NOs: 68-70;
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 47-49
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 50-52
  • peptide chain 3 includes SEQ ID NOs: 3 heavy chain CDRs shown in 71-73 and 3 light chain CDRs shown in SEQ ID NOs: 74-76;
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 53-55
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 56-58
  • peptide chain 3 includes SEQ ID NOs: 3 heavy chain CDRs shown in 71-73 and 3 light chain CDRs shown in SEQ ID NOs: 74-76; or
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 59-61
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 62-64
  • peptide chain 3 includes SEQ ID NOs: 3 heavy chain CDRs shown in 71-73 and 3 light chain CDRs shown in SEQ ID NOs: 74-76.
  • the anti-BCMA ⁇ CD3 bispecific antibody provided by the present invention comprises peptide chain 1, peptide chain 2 and peptide chain 3, wherein peptide chain 1 comprises a peptide selected from the group consisting of SEQ ID NO: 1, 3 or 5 A VH comprising 3 heavy chain CDRs and having at least 90% identity to SEQ ID NO: 1, 3 or 5, and peptide chain 2 comprising 3 light chain CDRs selected from the group consisting of SEQ ID NO: 2, 4 or 6 and VL having at least 90% identity with SEQ ID NO: 2, 4 or 6, peptide chain 3 comprising 3 heavy chain CDRs selected from the group consisting of SEQ ID NO: 7 or 9 and with SEQ ID NO: 7 or 9 A VH having at least 90% identity and a VL selected from the three light chain CDRs contained in SEQ ID NO: 8 or 10 and having at least 90% identity with SEQ ID NO: 8 or 10.
  • the anti-BCMA ⁇ CD3 bispecific antibody provided by the present invention comprises peptide chain 1, peptide chain 2 and peptide chain 3, wherein:
  • Peptide 1 comprises a VH from the 3 heavy chain CDRs contained in SEQ ID NO: 1 and is at least 90% identical to SEQ ID NO: 1
  • peptide 2 comprises 3 from SEQ ID NO: 2 VL of light chain CDRs and at least 90% identical to SEQ ID NO:2,
  • peptide chain 3 comprising 3 heavy chain CDRs from SEQ ID NO:7 and at least 90% identical to SEQ ID NO:7 a sexual VH and a VL from the three light chain CDRs contained in SEQ ID NO:8 and having at least 90% identity to SEQ ID NO:8;
  • Peptide chain 1 comprises a VH from the 3 heavy chain CDRs contained in SEQ ID NO: 3 and has at least 90% identity with SEQ ID NO: 3
  • peptide chain 2 comprises 3 from SEQ ID NO: 4 VL of light chain CDRs and at least 90% identical to SEQ ID NO:4,
  • peptide chain 3 comprising 3 heavy chain CDRs from SEQ ID NO:7 and at least 90% identical to SEQ ID NO:7 a sexual VH and a VL from the three light chain CDRs contained in SEQ ID NO:8 and having at least 90% identity to SEQ ID NO:8;
  • Peptide chain 1 comprises a VH from the 3 heavy chain CDRs contained in SEQ ID NO: 5 and is at least 90% identical to SEQ ID NO: 5
  • peptide chain 2 comprises 3 from SEQ ID NO: 6 VL of light chain CDRs and at least 90% identical to SEQ ID NO:6,
  • peptide chain 3 comprising 3 heavy chain CDRs from SEQ ID NO:7 and at least 90% identical to SEQ ID NO:7 a sexual VH and a VL from the three light chain CDRs contained in SEQ ID NO:8 and having at least 90% identity to SEQ ID NO:8;
  • Peptide chain 1 comprises a VH from the 3 heavy chain CDRs contained in SEQ ID NO: 1 and has at least 90% identity with SEQ ID NO: 1
  • peptide chain 2 comprises 3 from SEQ ID NO: 2 VL of light chain CDRs and at least 90% identical to SEQ ID NO:2,
  • peptide chain 3 comprising 3 heavy chain CDRs from SEQ ID NO:9 and at least 90% identical to SEQ ID NO:9 a sexual VH and a VL from the three light chain CDRs contained in SEQ ID NO: 10 and having at least 90% identity to SEQ ID NO: 10;
  • Peptide chain 1 comprises a VH from 3 heavy chain CDRs contained in SEQ ID NO:3 and has at least 90% identity with SEQ ID NO:3, and peptide chain 2 comprises 3 from SEQ ID NO:4 VL of light chain CDRs and at least 90% identical to SEQ ID NO:4, peptide chain 3 comprising 3 heavy chain CDRs from SEQ ID NO:9 and at least 90% identical to SEQ ID NO:9 A sexual VH and a VL from the three light chain CDRs contained in SEQ ID NO: 10 and having at least 90% identity to SEQ ID NO: 10; or
  • Peptide chain 1 comprises a VH from the 3 heavy chain CDRs contained in SEQ ID NO: 5 and has at least 90% identity with SEQ ID NO: 5
  • peptide chain 2 comprises 3 from SEQ ID NO: 6 VL of light chain CDRs and at least 90% identical to SEQ ID NO:6,
  • peptide chain 3 comprising 3 heavy chain CDRs from SEQ ID NO:9 and at least 90% identical to SEQ ID NO:9
  • the anti-BCMA ⁇ CD3 bispecific antibody provided by the present invention comprises peptide chain 1, peptide chain 2 and peptide chain 3, wherein peptide chain 1 comprises a compound selected from the group consisting of SEQ ID NO: 1, 3 or 5
  • peptide chain 1 comprises a compound selected from the group consisting of SEQ ID NO: 1, 3 or 5
  • the VH, peptide chain 2 comprises a VL shown in SEQ ID NO: 2, 4 or 6
  • peptide chain 3 comprises a VH selected from SEQ ID NO: 7 or 9 and is selected from SEQ ID NO: 8 or 10 VL shown.
  • the anti-BCMA ⁇ CD3 bispecific antibody provided by the present invention comprises peptide chain 1, peptide chain 2 and peptide chain 3, wherein:
  • Peptide chain 1 includes VH shown in SEQ ID NO: 1
  • peptide chain 2 includes VL shown in SEQ ID NO: 2
  • peptide chain 3 includes VH shown in SEQ ID NO: 7 and VL shown in SEQ ID NO: 8;
  • Peptide chain 1 includes VH shown in SEQ ID NO:3
  • peptide chain 2 includes VL shown in SEQ ID NO:4
  • peptide chain 3 includes VH shown in SEQ ID NO:7 and VL shown in SEQ ID NO:8;
  • Peptide chain 1 includes VH shown in SEQ ID NO:5
  • peptide chain 2 includes VL shown in SEQ ID NO:6
  • peptide chain 3 includes VH shown in SEQ ID NO:7 and VL shown in SEQ ID NO:8;
  • Peptide chain 1 includes VH shown in SEQ ID NO: 1
  • peptide chain 2 includes VL shown in SEQ ID NO: 2
  • peptide chain 3 includes VH shown in SEQ ID NO: 9 and VL shown in SEQ ID NO: 10;
  • Peptide chain 1 includes VH shown in SEQ ID NO:3
  • peptide chain 2 includes VL shown in SEQ ID NO:4
  • peptide chain 3 includes VH shown in SEQ ID NO:9 and VL shown in SEQ ID NO:10; or
  • Peptide chain 1 includes VH shown in SEQ ID NO: 5
  • peptide chain 2 includes VL shown in SEQ ID NO: 6
  • peptide chain 3 includes VH shown in SEQ ID NO: 9 and VL shown in SEQ ID NO: 10.
  • the anti-BCMAxCD3 bispecific antibody provided by the present invention comprises peptide chain 1, peptide chain 2 and peptide chain 3, wherein peptide chain 1 and peptide chain 3 comprise the same or different Fc regions.
  • the Fc regions contained in peptide chain 1 and peptide chain 3 have "knob” and "hole” structures, respectively, which interact to stabilize the steric structure of the bispecific antibody.
  • variable regions contained in peptide chain 1 and peptide chain 3 are homologous or heterologous to the Fc region.
  • the variable regions contained in Peptide 1 and Peptide 3 are linked to the Fc region directly or via a linker.
  • the linker is a flexible linker commonly used in the art.
  • variable region of the heavy chain and the variable region of the light chain are formed by a linker to form an scFv.
  • the linker connecting the variable region of the heavy chain and the variable region of the light chain is a flexible linker commonly used in the art.
  • the present invention provides an anti-BCMAxCD3 bispecific antibody comprising Peptide 1, Peptide 2 and Peptide 3, wherein:
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11 sequence, or consists of SEQ ID NO: 11;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO: 12;
  • peptide chain 3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO: 13;
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14 sequence, or consisting of SEQ ID NO: 14;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO: 15;
  • peptide chain 3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO: 16;
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 17 sequence, or consists of SEQ ID NO: 17;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO: 18;
  • peptide chain 3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO: 19;
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:20 sequence, or consists of SEQ ID NO:20;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO: 21;
  • peptide chain 3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO: 22;
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:23 sequence, or consists of SEQ ID NO: 23;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO: 24; A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO: 25; or
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:26 sequence, or consists of SEQ ID NO:26;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO:27;
  • peptide chain 3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO:28.
  • the present invention provides an anti-BCMA x CD3 bispecific antibody in a 2+1 format, which consists of peptide chain 1, peptide chain 3 and two peptide chains 2, comprising two antigen recognition sites recognizing BCMA and an antigen recognition site that recognizes CD3, as shown in Figure 2.
  • the present invention provides an anti-BCMA ⁇ CD3 bispecific antibody in a 2+1 format, wherein the scFv domain that specifically binds to CD3 is attached to the C-terminus of peptide chain 1 directly or through a linker, thereby constituting the peptide chain 3.
  • the scFv is formed by joining the heavy chain variable region and light chain variable region that specifically binds CD3 by a suitable linker known in the art.
  • the heavy chain variable domain of peptide 1 and the light chain variable domain of peptide 2 are paired to form the first antigen recognition site of BCMA
  • the heavy chain variable knot of peptide 3 Domain pairing with the light chain variable domain of peptide 2 forms the second antigen recognition site of BCMA
  • the scFv of peptide 3 forms the antigen recognition site of CD3.
  • the respective Fc domains of peptide chain 1 and peptide chain 3 interact to form an Fc region.
  • the respective Fc domains of Peptide 1 and Peptide 3 contain a stabilizing interaction mutation, eg, a "knob-in-hole" mutation.
  • the anti-BCMA ⁇ CD3 bispecific antibody in 2+1 format provided by the present invention comprises peptide chain 1, 2 peptide chains 2 and peptide chain 3, wherein:
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 1
  • peptide chain 2 comprises 3 light chain CDRs from SEQ ID NO: 2
  • the N-terminus of peptide chain 3 comprises from SEQ ID NO: 2.
  • NO: 1 contains 3 heavy chain CDRs and the C-terminus contains 3 heavy chain CDRs from SEQ ID NO: 7 and 3 light chain CDRs from SEQ ID NO: 8;
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 3
  • peptide chain 2 comprises 3 light chain CDRs from SEQ ID NO: 4
  • the N-terminus of peptide chain 3 comprises from SEQ ID NO: 4.
  • NO:3 contains 3 heavy chain CDRs and the C-terminus contains 3 heavy chain CDRs from SEQ ID NO:7 and 3 light chain CDRs from SEQ ID NO:8;
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 5
  • peptide chain 2 comprises 3 light chain CDRs from SEQ ID NO: 6, and the N-terminus of peptide chain 3 comprises from SEQ ID NO: 6.
  • NO:5 contains 3 heavy chain CDRs and the C-terminus contains 3 heavy chain CDRs from SEQ ID NO:7 and 3 light chain CDRs from SEQ ID NO:8;
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 1
  • peptide chain 2 comprises 3 light chain CDRs from SEQ ID NO: 2
  • the N-terminus of peptide chain 3 comprises from SEQ ID NO: 2.
  • NO:1 contains 3 heavy chain CDRs and the C-terminus contains 3 heavy chain CDRs from SEQ ID NO:9 and 3 light chain CDRs from SEQ ID NO:10;
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 3
  • peptide chain 2 comprises 3 light chain CDRs from SEQ ID NO: 4
  • the N-terminus of peptide chain 3 comprises from SEQ ID NO: 4 : the 3 heavy chain CDRs contained in 3 and the C-terminus comprises the 3 heavy chain CDRs from SEQ ID NO:9 and the 3 light chain CDRs from SEQ ID NO:10; or
  • Peptide chain 1 comprises 3 heavy chain CDRs from SEQ ID NO: 5
  • peptide 2 comprises 3 light chain CDRs from SEQ ID NO: 6
  • the N-terminus of peptide chain 3 comprises from SEQ ID NO: 6.
  • NO:5 contains the 3 heavy chain CDRs and the C-terminus contains the 3 heavy chain CDRs from SEQ ID NO:9 and the 3 light chain CDRs from SEQ ID NO:10.
  • the anti-BCMA ⁇ CD3 bispecific antibody in 2+1 format provided by the present invention comprises peptide chain 1, 2 peptide chains 2 and peptide chain 3, wherein:
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 47-49
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 50-52
  • the N-terminus of peptide chain 3 includes SEQ ID NO: 50-52.
  • the 3 heavy chain CDRs shown in ID NOs: 47-49 and the C-terminus comprises the 3 heavy chain CDRs shown in SEQ ID NOs: 65-67 and the 3 light chain CDRs shown in SEQ ID NOs: 68-70;
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 53-55
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 56-58
  • the N-terminus of peptide chain 3 includes SEQ ID NO: 56-58.
  • the 3 heavy chain CDRs shown in ID NOs: 53-55 and the C-terminus comprises the 3 heavy chain CDRs shown in SEQ ID NOs: 65-67 and the 3 light chain CDRs shown in SEQ ID NOs: 68-70;
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 59-61
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 62-64
  • the N-terminus of peptide chain 3 includes SEQ ID NO: 62-64.
  • the 3 heavy chain CDRs shown in ID NOs: 59-61 and the C-terminus comprises the 3 heavy chain CDRs shown in SEQ ID NOs: 65-67 and the 3 light chain CDRs shown in SEQ ID NOs: 68-70;
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 47-49
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 50-52
  • the N-terminus of peptide chain 3 includes SEQ ID NO: 50-52.
  • the 3 heavy chain CDRs shown in ID NOs: 47-49 and the C-terminus comprises the 3 heavy chain CDRs shown in SEQ ID NOs: 71-73 and the 3 light chain CDRs shown in SEQ ID NOs: 74-76;
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 53-55
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 56-58
  • the N-terminus of peptide chain 3 includes SEQ ID NO: 56-58.
  • the 3 heavy chain CDRs shown in ID NOs: 53-55 and the C-terminus comprises the 3 heavy chain CDRs shown in SEQ ID NOs: 71-73 and the 3 light chain CDRs shown in SEQ ID NOs: 74-76; or
  • Peptide chain 1 includes 3 heavy chain CDRs shown in SEQ ID NOs: 59-61
  • peptide chain 2 includes 3 light chain CDRs shown in SEQ ID NOs: 62-64
  • the N-terminus of peptide chain 3 includes SEQ ID NO: 62-64.
  • the 3 heavy chain CDRs shown in ID NOs: 59-61 and the C-terminus comprise the 3 heavy chain CDRs shown in SEQ ID NOs: 71-73 and the 3 light chain CDRs shown in SEQ ID NOs: 74-76.
  • the anti-BCMA ⁇ CD3 bispecific antibody in 2+1 format provided by the present invention comprises peptide chain 1, 2 peptide chains 2 and peptide chain 3, wherein:
  • Peptide chain 1 comprises VH shown in SEQ ID NO: 1
  • peptide chain 2 comprises VL shown in SEQ ID NO: 2
  • N-terminal of peptide chain 3 comprises VH shown in SEQ ID NO: 1
  • C-terminal comprises SEQ ID NO: 1 : VH shown in 7 and VL shown in SEQ ID NO: 8;
  • Peptide chain 1 comprises VH shown in SEQ ID NO: 3
  • peptide chain 2 comprises VL shown in SEQ ID NO: 4
  • N-terminal of peptide chain 3 comprises VH shown in SEQ ID NO: 3
  • C-terminal comprises SEQ ID NO: 3 : VH shown in 7 and VL shown in SEQ ID NO: 8;
  • Peptide chain 1 comprises VH shown in SEQ ID NO: 5
  • peptide chain 2 comprises VL shown in SEQ ID NO: 6
  • N-terminal of peptide chain 3 comprises VH shown in SEQ ID NO: 5
  • C-terminal comprises SEQ ID NO: 5 : VH shown in 7 and VL shown in SEQ ID NO: 8;
  • Peptide chain 1 comprises VH shown in SEQ ID NO: 1
  • peptide chain 2 comprises VL shown in SEQ ID NO: 2
  • N-terminal of peptide chain 3 comprises VH shown in SEQ ID NO: 1
  • C-terminal comprises SEQ ID NO: 1 VH shown in: 9 and VL shown in SEQ ID NO: 10;
  • Peptide chain 1 comprises VH shown in SEQ ID NO: 3
  • peptide chain 2 comprises VL shown in SEQ ID NO: 4
  • N-terminal of peptide chain 3 comprises VH shown in SEQ ID NO: 3
  • C-terminal comprises SEQ ID NO: 3 : VH shown in 9 and VL shown in SEQ ID NO: 10; or
  • Peptide chain 1 comprises VH shown in SEQ ID NO: 5
  • peptide chain 2 comprises VL shown in SEQ ID NO: 6
  • N-terminal of peptide chain 3 comprises VH shown in SEQ ID NO: 5
  • C-terminal comprises SEQ ID NO: 5 : VH shown in 9 and VL shown in SEQ ID NO: 10.
  • the present invention provides an anti-BCMAxCD3 bispecific antibody in a 2+1 format comprising Peptide 1, 2 Peptide 2 and Peptide 3, wherein:
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:29 sequence, or consists of SEQ ID NO:29;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO:30;
  • peptide chain 3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO:31;
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:32 sequence, or consists of SEQ ID NO:32;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO:33;
  • peptide chain 3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO:34;
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:35 sequence, or consists of SEQ ID NO:35;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO:36;
  • peptide chain 3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO:37;
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:38 sequence, or consists of SEQ ID NO:38;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO:39;
  • peptide chain 3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO:40;
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:41 sequence, or consists of SEQ ID NO:41;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO:42;
  • peptide chain 3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO: 43; or
  • Peptide chain 1 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:44 sequence, or consists of SEQ ID NO:44;
  • peptide chain 2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A sequence of 99% or more sequence identity, or consisting of SEQ ID NO:45;
  • peptide chain 3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, A sequence of 96%, 97%, 98%, 99% or more sequence identity, or consisting of SEQ ID NO:46.
  • the present invention also provides a polynucleotide (nucleic acid) encoding the anti-BCMAxCD3 bispecific antibody of the present invention, a vector, preferably an expression vector, comprising said polynucleotide.
  • the present invention provides host cells comprising the polynucleotides or vectors of the present invention.
  • the present invention also provides a method for producing an anti-BCMA ⁇ CD3 bispecific antibody of the present invention, comprising step (i) culturing a host of the present invention under conditions suitable for expressing the anti-BCMA ⁇ CD3 bispecific antibody of the present invention cells, and (ii) recovery of the anti-BCMAxCD3 bispecific antibody of the invention.
  • the present invention provides a diagnostic kit and pharmaceutical composition comprising the anti-BCMAxCD3 bispecific antibody of the present invention. Further, use of the anti-BCMA ⁇ CD3 bispecific antibody, diagnostic kit or pharmaceutical composition of the present invention is also provided for treating, preventing and/or diagnosing diseases related to BCMA activity, especially for treating, Prevention and/or diagnosis of multiple myeloma.
  • the present invention provides a method of treating a disease associated with BCMA activity comprising administering to a patient in need thereof a therapeutically effective amount of an anti-BCMA x CD3 bispecific antibody of the present invention, or a pharmaceutical composition of the present invention .
  • the disease is a cancer overexpressing BCMA, more preferably the disease is multiple myeloma.
  • Figure 1 Schematic representation of the 1+1 format bispecific antibody structure, wherein Figure 1B shows the structure of the individual chains that make up the 1+1 format bispecific antibody structure.
  • FIG. 1 Schematic diagram of the 2+1 format structure.
  • Example antibody-mediated killing of PBMCs on L363 cells 6A.
  • FIG. 8 Example antibody-mediated killing of multiple myeloma cell lines by PBMCs, 8A.
  • Figure 9 Flow cytometry detection of cytokine release accompanying the killing of NCl-H929 cells by exemplary antibody-induced PBMCs.
  • FIG. 12 Example antibody-mediated activation of T cells in PBMCs, 12A. Percentage of CD25+/CD69+ double positive cells in CD8+ T cells during example antibody-mediated killing of NCl-H929 cells by PBMCs (reactive CD8+ T cells 12B. Percentage of CD25+/CD69+ double-positive cells in CD4+ T cells during PBMC mediated killing of NCl-H929 cells by an example antibody (reflecting the degree of activation of CD4+ T cells); 12C.
  • Example antibody-mediated killing of NCl-H929 cells Percentage of CD25+/CD69+ double positive cells in CD8+ T cells during PBMC-mediated killing of L363 cells (reflecting the degree of activation of CD8+ T cells); 12D.
  • Example CD4+ T cells during antibody-mediated killing of L363 cells by PBMC Percentage of CD25+/CD69+ double positive cells in cells (response to degree of activation of CD4+ T cells); 12E. Percentage of CD25+/CD69+ double positive cells among CD8+ T cells during example antibody-mediated killing of RPPMI8226 cells by PBMC (response Degree of activation of CD8+ T cells); 12F. Percentage of CD25+/CD69+ double positive cells in CD4+ T cells during example antibody-mediated killing of RPPMI8226 cells by PBMC (reflecting the degree of activation of CD4+ T cells).
  • Example antibody promotes CD8+ T cell proliferation, 13A.
  • Example antibody dose-dependently promotes CD8+ T cell proliferation in the presence of L363 cells, 13B.
  • Example antibody is non-specific for CD8 in the presence of BCMA-negative NUGC4 cells +T cell proliferation.
  • FIG. 14 Exemplary antibody promotes CD4+ T cell proliferation, 14A. Exemplary antibody dose-dependently promotes CD4+ T cell proliferation in the presence of L363 cells, 14B. Exemplary antibody does not non-specific CD4 in the presence of BCMA-negative NUGC4 cells +T cell proliferation.
  • Figure 15 Tumor inhibitory effect of exemplary antibodies in the NCl-H929 tumor-bearing humanized mouse model.
  • antibody is used herein in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (eg, , bispecific antibodies), single chain antibodies, whole antibodies or antibody fragments thereof exhibiting the desired antigen-binding activity.
  • An intact antibody will generally contain at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, eg, antibodies naturally occurring in camels may contain only heavy chains.
  • antigen-binding fragment refers to a molecule other than an intact antibody that comprises a portion of the intact antibody and binds the antigen to which the intact antibody binds.
  • antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies (dAbs); linear antibodies; single chain antibodies (eg, scFv); single domains Antibodies (single domain antibodies); antigen-binding fragments of bivalent or bispecific antibodies; camelid antibodies; and other fragments that exhibit the desired ability to bind antigen (eg, BCMA and/or CD3).
  • binding and “specific binding” mean that the binding of the antibody is selective for the antigen and can be distinguished from unwanted or nonspecific interactions.
  • the ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) or optical interferometry of biofilm layers (ForteBio) or other conventional binding assays known in the art.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • FormeBio optical interferometry of biofilm layers
  • antibodies with a KD of about 1x10-7 or less, a KD of about 1x10-8 or less, a KD of about 1x10-9 or less, a KD of about 1x10-10 or A lower KD, about 1 x 10-11 or lower KD binds to BCMA or CD3, then the antibody is an antibody that "specifically binds to BCMA or CD3."
  • antibodies that specifically bind human BCMA or CD3 may be cross-reactive with BCMA or CD3 proteins from other species.
  • antibodies specific for human BCMA or CD3, in some embodiments, can cross-react with cynomolgus BCMA or CD3.
  • Methods for determining cross-reactivity include those described in the Examples as well as standard assays known in the art, eg, by using bio-light interference, or flow cytometry techniques.
  • single-chain variable fragment or "scFv” is a small-molecule genetically engineered antibody that uses genetic engineering methods at the DNA level to combine the variable heavy (VH) and light chain variable regions of a native antibody. (VL) linked (usually via a synthetic linking peptide (or “linker”)) small-molecule recombinant antibody.
  • scFv single-chain antibodies Compared with intact antibody molecules, scFv single-chain antibodies have the following advantages: they contain a complete antibody variable region and retain the antigen specificity and binding activity of the original antibody; they do not contain the Fc region of the antibody molecule, so they have weak immunogenicity and are used for The human body is not easy to generate an immune response; it is easy to operate and suitable for use as a genetic engineering component to prepare other antigen-specific binding molecules with new properties, such as full-length antibodies, scFv-Fc, etc.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region generally extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed . Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • knock-in-hole refers to the creation of a "knob” structure on one Fc chain of a bispecific antibody molecule described herein, and a "hole” structure on the other chain, such that the hole is identical to the knob or a similar size, suitably placed so that when the two Fcs interact, the knob of one Fc can be positioned in the corresponding hole of the other Fc, thereby stabilizing the structure of the heteromultimer (see, e.g., US Pat. No. 5,731,168 ).
  • knobs can be constructed by replacing small amino acid side chains with larger side chains, according to the state of the art.
  • sockets can be constructed by replacing large amino acid side chains with smaller side chains.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in antibody binding to an antigen.
  • the variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (see, e.g., Kindt et al. Kuby Immunology, 6th ed ., WH Freeman and Co. p. 91 (2007)).
  • FRs conserved framework regions
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • CDR regions are antibody variable domains that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and /or regions containing antigen contact residues ("antigen contact points").
  • the CDRs are mainly responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are numbered sequentially from the N-terminus and are commonly referred to as CDR1, CDR2 and CDR3.
  • the CDRs located within the variable domains of antibody heavy chains are also referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable domains of antibody light chains are referred to as LCDR1, LCDR2 and LCDR3.
  • its CDR sequence can be determined using various schemes well known in the art, for example: Chothia (Chothia) based on the three-dimensional structure of the antibody and topology of the CDR loops. Chothia et al.
  • CDRs can also be determined based on having the same Kabat numbering positions as the reference CDR sequence.
  • a residue position in an antibody variable region refers to the numbering system according to the Kabat ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed . Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the CDR boundaries of the variable regions of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different.
  • the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined by the present invention.
  • Antibodies with different specificities have different CDRs.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding.
  • the minimal binding unit can be a sub-portion of a CDR.
  • the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • cytotoxic agent refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction.
  • chemotherapeutic agent includes chemical compounds useful in the treatment of cancer.
  • small molecule drug refers to low molecular weight organic compounds capable of modulating biological processes.
  • Small molecule is defined as a molecule with a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD.
  • Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As therapeutic agents, small molecules can be more cell permeable, less susceptible to degradation, and less susceptible to eliciting an immune response than macromolecules.
  • the term "functional Fc region” refers to an Fc region that possesses the "effector functions" of a native sequence Fc region.
  • exemplary “effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; Such effector functions generally require the association of an Fc region with a binding domain (eg, an antibody variable domain), and can be assessed using a variety of assays, such as those disclosed herein.
  • therapeutic agent encompasses any substance that is effective in preventing or treating tumors (eg, cancer), including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulatory agents .
  • immunomodulator refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response.
  • the immune response can be a humoral response or a cellular response.
  • an “effective amount” refers to an amount or dose of an antibody or fragment or conjugate or composition of the invention which, after administration to the patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.
  • an “effective amount” can be distinguished as a “therapeutically effective amount” and a “prophylactically effective amount”.
  • An effective amount can be readily determined by the attending physician, who is skilled in the art, by taking into account a variety of factors such as the species, size, age and general health of the mammal, the specific disease involved, the degree or severity of the disease, the individual patient response, the particular antibody administered, the mode of administration, the bioavailability characteristics of the administered formulation, the chosen dosing regimen, and the use of any concomitant therapy.
  • an effective amount of a bispecific antibody of the invention preferably inhibits a measurable parameter (eg, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, or even in comparison to a control More preferably at least about 50%, 60% or 70% and still more preferably at least about 80% or 90%.
  • a measurable parameter eg, tumor growth rate, tumor volume, etc.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the original primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity in the originally transformed cell.
  • multispecific antibody refers to an antibody having at least two different antigen-binding sites, each of which is associated with a different epitope of the same antigen or Binds to different epitopes of different antigens.
  • Multispecific antibodies are antibodies that have binding specificities for at least two different antigenic epitopes.
  • bispecific antibodies having binding specificities for a first antigen or target (BCMA) and a second antigen target (CD3). Given that the antibody construct according to the invention is (at least) bispecific, it is not naturally occurring and it is distinct from the naturally occurring product.
  • a "bispecific" antibody or immunoglobulin is an artificial hybrid antibody or immunoglobulin having at least two different binding sides with different specificities.
  • target means BCMA or CD3.
  • first target and second target means BCMA as the first target and CD3 as the second target or vice versa.
  • cytokine is a generic term for proteins released by one cell population to act as intercellular mediators on another cell.
  • cytokines are lymphokines, monokines, interleukins (IL) such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factors, such as TNF- ⁇ or TNF- ⁇ ; and other polypeptide factors, including LIF and kit ligand (KL) and Gamma-interferon.
  • cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequence cytokines, including synthetically produced small molecule entities, and pharmaceutically acceptable derivatives and salts.
  • immunoconjugate is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.
  • mammals include, but are not limited to, domestic animals (eg, cattle, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, , mice and rats).
  • domestic animals eg, cattle, sheep, cats, dogs, and horses
  • primates eg, humans and non-human primates such as monkeys
  • rabbits eg, mice and rats
  • rodents eg, mice and rats.
  • the individual or subject is a human.
  • isolated antibody is an antibody that has been separated from components of its natural environment.
  • the antibody is purified to greater than 95% or 99% purity, such as by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed phase) HPLC) determined.
  • electrophoresis eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography eg, ion exchange or reversed phase
  • the sequences are aligned for optimal comparison purposes (e.g., between the first and second amino acid sequences or nucleic acid sequences for optimal alignment. Gaps are introduced in one or both or non-homologous sequences can be discarded for comparison purposes).
  • the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (at http://www.gcg.com) is used that has been integrated into the GAP program of the GCG software package available), using the Blossum 62 matrix or the PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6, to determine the distance between two amino acid sequences percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences.
  • a particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • pharmaceutical adjuvant refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., with which the active substance is administered.
  • composition refers to a composition that is in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients.
  • combination therapy refers to the administration of two or more therapeutic agents to treat a cancer or infection as described in this disclosure.
  • administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, eg, in a single capsule having a fixed ratio of active ingredients.
  • administration includes co-administration of the individual active ingredients in multiple or separate containers such as tablets, capsules, powders and liquids. Powders and/or liquids can be reconstituted or diluted to the desired dose prior to administration.
  • such administration also includes the sequential use of each type of therapeutic agent at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the drug combination in the treatment of the disorders or conditions described herein.
  • treating refers to slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the occurrence or progression of a disease or disorder or symptoms of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for preventive regimens.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • BCMA, target BCMA, human BCMA refers to the human B cell maturation target, also known as BCMA, TR17_human, TNFRSF17 (UniProt Q02223).
  • the extracellular domain of BCMA consists of amino acids 1-54 (or 5-51) according to UniProt.
  • Antibody against BCMA refers to an antibody that specifically binds to BCMA.
  • the anti-BCMA antibody binds to an unrelated non-BCMA protein to an extent about 10-fold, preferably >100-fold, less than the antibody binds to BCMA, as measured, eg, by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the antibody that binds to BCMA has a dissociation constant (Kd) of 10-7 M or less, preferably 10-8 M to 10-13 M, preferably 10-9 M to 10-13 M.
  • the anti-BCMA antibody binds to an epitope of BCMA that is conserved in BCMA from different species, preferably in humans and cynomolgus monkeys.
  • the "anti-BCMA antibody” referred to herein comprises a heavy chain variable region comprising a CDR from SEQ ID NO: 1, 3 or 5 and a light chain variable region, the light chain may be The variable regions comprise CDRs from SEQ ID NO: 2, 4 or 6.
  • T cells or T lymphocytes are a class of lymphocytes that play a central role in cell-mediated immunity.
  • the specificity of T cell responses is mediated by the recognition of antigens (displayed in the context of the major histocompatibility complex MHC) by TCRs.
  • the CD3 receptor complex is a protein complex that includes a CD3 ⁇ (gamma) chain, a CD3 ⁇ (delta) chain, and two CD3 ⁇ (epsilon) chains present on the cell surface, involved in the activation of cytotoxic T cells ( CD8+ naive T cells) and T helper cells (CD4+ naive T cells).
  • Clustering of CD3 on T cells such as by immobilized anti-CD3 antibodies, results in T cell activation, which is analogous to T cell receptor engagement, but independent of its clone-specific specificity.
  • CD3 was found to be membrane bound to all mature T cells, and this high specificity, coupled with its presence at all stages of T cell development, makes it a useful immunohistochemical marker for T cells in tissue sections. It is envisaged that the antibody constructs according to the present invention generally and advantageously show less non-specific T cell activation, which is not required in specific immunotherapy. This means a reduced risk of side effects.
  • an “anti-CD3 antibody” as referred to herein refers to an antibody that binds CD3.
  • an “anti-CD3 antibody” referred to herein comprises a heavy chain variable region comprising a CDR from SEQ ID NO: 7 or 9 and a light chain variable region, the light chain variable region comprising CDRs from SEQ ID NO: 8 or 10.
  • anti-BCMA and CD3 bispecific antibody refers to a bispecific antibody capable of binding the targets BCMA and CD3 with sufficient affinity to recruit T cells for redirected lysis of target cells.
  • Engaged T cells are capable of continuous target cell lysis and are not affected by immune evasion mechanisms that interfere with peptide antigen processing and presentation or clone T cell differentiation.
  • the "anti-BCMA and CD3 bispecific antibody” referred to herein comprises a heavy chain variable region and a light chain variable region targeting BCMA, the heavy chain variable region comprising a variable region derived from SEQ ID NO: 1 , 3 or 5 CDRs, the light chain variable region comprises the CDRs from SEQ ID NO: 2, 4 or 6, and the heavy chain variable region and light chain variable region targeting CD3, the heavy chain variable region comprising from The CDRs of SEQ ID NO: 7 or 9, the light chain variable region comprises the CDRs from SEQ ID NO: 8 or 10.
  • the antibodies can be used as diagnostic and/or therapeutic agents targeting BCMA-expressing cancers.
  • the anti-BCMA ⁇ CD3 bispecific antibody provided by the present invention has the following advantages:
  • the anti-BCMA ⁇ CD3 bispecific antibodies of the present invention are formed by assembling different anti-BCMA antibodies with anti-CD3 antibodies with different affinities, and the anti-BCMA ⁇ CD3 bispecific antibody-induced PBMC pairs The detection of T cell activation and the release level of various cytokines is introduced into the tumor cell killing experiment, which is conducive to the comprehensive evaluation of the efficacy and safety of BCMA/CD3 double antibody in the early stage (in vitro screening stage). Screening for differentiated molecules with similar maximal killing and lower levels of cytokine release in the in vitro screening stage for in vivo and toxicological experiments will help reduce the risk of cytokine storm during the clinical application of such dual antibodies.
  • the BCMA/CD3 dual antibody of the present invention simultaneously binds BCMA on the surface of multiple myeloma cells and CD3 on the surface of primary T cells, and mediates the killing of BCMA-positive tumor cells by T cells.
  • the bispecific antibodies of the present invention are capable of dose-dependently inducing killing of PBMCs against multiple myeloma cells with different levels of BCMA expression.
  • the anti-BCMAxCD3 antibodies of the invention mediate killing of human multiple myeloma cells and dose-dependently activate CD8+ T cells and CD4+ T cells isolated from PBMCs. In some embodiments, the antibodies of the invention promote the proliferative capacity of human CD8+ T, CD4+ T cells.
  • the antibodies of the present invention can effectively inhibit the growth of tumors, and the tumor inhibition rate can reach 62%, or even 108%, compared to the control.
  • anti-BCMAxCD3 bispecific antibodies with amino acid changes are contemplated herein, wherein the amino acid changes comprise amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes described in the present invention occur in regions outside the variable region of the heavy chain and/or outside the variable region of the light chain.
  • substitutions are conservative substitutions.
  • Conservative substitutions refer to the substitution of one amino acid by another amino acid within the same class, e.g., substitution of an acidic amino acid by another acidic amino acid, substitution of a basic amino acid by another basic amino acid, or substitution of a neutral amino acid by another neutral amino acid replacement. Exemplary permutations are shown in the following table:
  • the substitutions occur in the CDR regions of the antibody.
  • the variant obtained has a modification (eg, improvement) in certain biological properties (eg, increased affinity) relative to the parent antibody and/or will have certain biological properties that are substantially retained of the parent antibody.
  • exemplary substitutional variants are affinity matured antibodies.
  • the antibodies provided herein are altered to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites. When an antibody contains an Fc region, the carbohydrate attached to it can be varied. In some applications, modifications to remove unwanted glycosylation sites may be useful, such as removal of fucose moieties to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al. (2002) ) JBC277:26733). In other applications, galactosylation modifications can be made to modify complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Fc region variants can include human Fc region sequences (eg, human IgGl, IgG2, IgG3, or IgG4 Fc regions) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.
  • human Fc region sequences eg, human IgGl, IgG2, IgG3, or IgG4 Fc regions
  • amino acid modifications eg, substitutions
  • Fc variants see US Pat. No. 7,332,581, US Pat. No. 6,737,056, US Pat. No. 6,737,056; WO 2004/056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001), USA Patent No.
  • cysteine-engineered antibodies eg, "thioMAbs”
  • cysteine residues of the antibody are replaced with cysteine residues.
  • Cysteine engineered antibodies can be generated as described, eg, in US Pat. No. 7,521,541.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available.
  • Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol,
  • the invention provides nucleic acids encoding any of the above anti-BCMA, anti-CD3 and anti-BCMAxCD3 antibodies or antigen-binding fragments thereof.
  • the present invention also encompasses nucleic acids that hybridize to the above-mentioned nucleic acids under stringent conditions, nucleic acids having one or more substitutions (eg conservative substitutions), deletions or insertions compared to the above-mentioned nucleic acids, or at least 80% compared to the above-mentioned nucleic acids , at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical nucleic acid sequences.
  • the present invention provides a vector comprising the above-described nucleic acid.
  • the vector is an expression vector.
  • the present invention provides a host cell comprising the nucleic acid or the vector.
  • Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells) or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
  • the present invention provides compositions comprising any of the anti-BCMAxCD3 antibodies or antigen-binding fragments thereof described herein, preferably the compositions are pharmaceutical compositions.
  • the composition further comprises pharmaceutical excipients.
  • a composition eg, a pharmaceutical composition
  • compositions of the present invention may also contain one or more other active ingredients as required for the particular indication being treated, preferably with those active ingredients that do not adversely affect each other's activity.
  • active ingredients such as chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective active agents, small molecule drugs or immunomodulators, and the like.
  • the active ingredients are suitably combined in amounts effective for the intended application.
  • Anti-BCMA and anti-CD3 bispecific antibody molecules were designed in two different formats: 1+1 format and 2+1 format, where the numbers indicate the number of antigen recognition sites that bind the antigens BCMA and CD3, respectively.
  • 1+1 format indicates that the bispecific antibody has an antigen recognition site that binds the antigen BCMA and an antigen recognition site that binds CD3.
  • FIG. 1A The schematic diagram of the structure of the 1+1 format bispecific antibody is shown in Figure 1A, which is composed of three asymmetrical left and right polypeptide chains, of which the left half is composed of peptide chain 1 and peptide chain 2, and the right half is composed of peptide chain 3. .
  • the structures of peptide chain 1, peptide chain 2, and peptide chain 3 are shown in Figure 1B.
  • Peptide chain 1 includes a heavy chain variable domain, an immunoglobulin CH1 domain, and an Fc domain in sequence from the N-terminus to the C-terminus.
  • Chain 2 contains the light chain variable domain and immunoglobulin CL domain from the N-terminus to the C-terminus in turn, and the peptide chain 3 from the N-terminus to the C-terminus contains the antibody heavy chain variable region and the light chain variable region by artificial A single chain antibody (single chain Fv, scFv) and an Fc domain linked by a synthetic linker.
  • the heavy chain variable domains of peptide 1 and the light chain variable domains of peptide 2 are paired to form the antigen recognition site of BCMA, and the scFv of peptide 3 forms the antigen recognition site of CD3.
  • the respective Fc domains of peptide chain 1 and peptide chain 3 interact to form an Fc region.
  • the respective Fc domains of Peptide 1 and Peptide 3 contain a stabilizing interaction mutation, eg, a "knob-in-hole" mutation.
  • FIG. 2A The structure of the 2+1 format bispecific antibody is shown in Figure 2A, which consists of four polypeptide chains that are left and right asymmetrical, of which the left half is composed of peptide chain 1 and peptide chain 2, and the right half is composed of peptide chain 2 and peptide chain.
  • Chain 3 composition The structures of peptide chain 1, peptide chain 2, and peptide chain 3 are shown in Figure 2B.
  • Peptide chain 1 includes a heavy chain variable domain, an immunoglobulin CH1 domain, and an Fc domain from the N-terminus to the C-terminus.
  • Chain 2 contains the light chain variable domain and immunoglobulin CL domain in sequence from the N-terminus to the C-terminus
  • the peptide chain 3 contains the heavy chain variable domain, Fc domain and scFv domain in sequence from the N-terminus to the C-terminus.
  • the heavy chain variable domain of peptide 1 and the light chain variable domain of peptide 2 are paired to form the first antigen recognition site of BCMA.
  • the pairing of the light chain variable domains forms the second antigen recognition site of BCMA
  • the scFv of peptide 3 forms the antigen recognition site of CD3.
  • the respective Fc domains of peptide chain 1 and peptide chain 3 interact to form an Fc region.
  • the respective Fc domains of Peptide 1 and Peptide 3 contain a stabilizing interaction mutation, eg, a "knob-in-hole" mutation.
  • bispecific antibodies in 1+1 format are ADI-46757, ADI-46758, ADI-46759, ADI-46760, ADI-46761, ADI-46762.
  • bispecific antibodies in 2+1 format The antibodies were ADI-46780, ADI-46781, ADI-46782, ADI-46783, ADI-46784, ADI-46785.
  • Exemplary antibodies utilize Fc's "knob-in-hole" technique to address the heavy chain mismatch problem of this asymmetric IgG-like bispecific antibody.
  • the Fc region of an exemplary antibody is the heavy chain constant region of IgG1, which includes L234A, L235A ("EU" numbering according to Kabat) amino acid mutations that attenuate effector function.
  • exemplary bispecific antibodies of the invention organized in 1+1 format and 2+1 format (ADI-46757, ADI-46758, ADI-46759, ADI-46760, ADI-46761, ADI-46762, ADI-46780 , ADI-46781, ADI-46782, ADI-46783, ADI-46784, ADI-46785)
  • the amino acid sequences of CDR region, peptide chain 1, peptide chain 2 and peptide chain 3 are shown in Table 1 and Table 2, in which underlined The CDR regions defined according to the Kabat protocol for each parental antibody are indicated.
  • Expi293F cells (purchased from Thermo Fisher scientific company) were subcultured in Expi293F cell culture medium (purchased from Thermo Fisher scientific company). The cell density was detected one day before transfection, and the cell density was adjusted to 2 ⁇ 10 6 cells/ml with fresh Expi293 cell culture medium, and the cell density was adjusted to 3 ⁇ 10 6 cells/ml on the day of transfection.
  • Opti-MEM medium purchased from Gibco
  • Opti-MEM medium purchased from Gibco
  • Opti-MEM medium 1/10 of the final volume of transfected Expi293F cells as the transfection buffer, and add 10 ⁇ g of 1:1:1 molar ratio of the above-prepared recombinant to each ml of the transfection buffer.
  • Plasmids each recombinant plasmid contains the nucleotide sequences encoding peptide chain 1, peptide chain 2 or peptide chain 3 of the corresponding bispecific antibody
  • mix well and then add 30ug polyethyleneimine per ml of transfection buffer ( polyethylenimine, PEI) (Polysciences), mixed well, incubated at room temperature for 20 minutes, then the PEI/DNA mixture was gently poured into the Expi293F cell suspension, mixed well, placed on a shaker for culture, and the culture conditions were 8% CO 2 , 36.5° C. , 120rpm.
  • Affinity chromatography purification Hitrap MabSelect SuRe (obtained from GE Healthcare) affinity chromatography column was used. Before purification, 0.1M NaOH was used to remove endotoxin from the affinity chromatography column and pipeline for 2 hours. Then, the pipeline and pipeline were washed with distilled water. Column.
  • the column was equilibrated with 5 column volumes of 1x PBS (Gibco); the collected culture supernatant was loaded onto the column, and the column was washed with 10 column volumes of 1x PBS to remove nonspecific binding Protein; rinse the column with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), collect the eluate, and adjust the pH of the collected eluate to 6.0 with 2M Tris for the next step ion exchange chromatography.
  • 1x PBS Gibco
  • elution buffer 100mM sodium citrate, pH 3.5
  • Ion-exchange chromatography purification Mono S 5/50 GL (obtained from GE Healthcare) ion-exchange chromatography column was selected and placed in an AKTApure system (obtained from GE healthcare). An AKTApure system equipped with a Mono S 5/50 GL ion-exchange chromatography column was subjected to endotoxin removal with 0.5M NaOH for 2 hours, then the system and the column were rinsed with distilled water.
  • the purified bispecific antibody solution was centrifuged at 4500 rpm for 30 minutes in a 15ml ultrafiltration centrifuge tube, and the protein was diluted with PBS and then centrifuged at 4500 rpm for 30 minutes. Repeat this operation several times to replace the buffer. liquid.
  • the antibodies after the buffer exchange were combined, and the antibody concentration was measured.
  • the composition and content of bispecific antibodies were further characterized and quantified by the combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).
  • human BCMA-Fc antigen and cynomolgus monkey BCMA-Fc antigen were diluted in 10mM Acetate (pH 5.0), the final concentration of dilution was 5 ⁇ g/mL, and they were coupled to channels 2 and 4 of the chip respectively, and the coupling height was about 50RU. .
  • the remaining activated sites were then blocked by injecting 1 M ethanolamine at a flow rate of 10 ⁇ L/min for 120 s.
  • the buffer used in the experiment is HBS-EP+ solution with pH 7.4.
  • the high performance mode is adopted.
  • the antibody after gradient dilution is injected from low concentration to high concentration at a flow rate of 30 ⁇ l/min, and a concentration is measured in each cycle. Inject into channels 1, 2, 3, and 4 of the chip in sequence, with a binding time of 180s and a dissociation time of 600s.
  • the affinities of ADI-46757, ADI-46758, ADI-46759, ADI-46760, ADI-46761 and human BCMA are shown in Table 3 in experiments performed as described in the above assays.
  • the affinities of ADI-46757, ADI-46758, ADI-46759, ADI-46760, ADI-46761 and cynomolgus BCMA are shown in Table 4.
  • SA streptavidin protein
  • NHS N-hydroxysuccinimide
  • EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • the flow rate activates the chip for 420s.
  • streptavidin protein was diluted in 10 mM Acetate (pH 5.0), the dilution concentration was 50 ⁇ g/mL, and it was coupled to the 1, 2, 3, and 4 channels of the chip respectively, and the coupling height was about 3000RU.
  • the remaining activated sites were then blocked by injecting 1 M ethanolamine at a flow rate of 10 ⁇ L/min for 420 s.
  • the biotin-labeled human CD3D&E heterodimer antigen (R&D) and cynomolgus monkey CD3D&E heterodimer antigen (R&D) were coupled at the concentration of 0.05ug/ml to channels 2 and 4 of the chip, respectively.
  • Channel, coupling height is about 30RU.
  • the affinities of ADI-46757, ADI-46758, ADI-46759, ADI-46760, ADI-46761 and human CD3D&E are shown in Table 5 in experiments performed as described in the assays above.
  • the affinities of ADI-46757, ADI-46758, ADI-46759, ADI-46760, ADI-46761 and cynomolgus CD3D&E are shown in Table 6.
  • BCMA ⁇ CD3 bispecific antibody to BCMA-positive multiple myeloma (MM) cells was detected by flow cytometry.
  • NCl-H929 cells (Nanjing Kebai Biology, Item No. CBP60243), L363 cells (Nanjing Kebai Biology, Item No. CBP60240), and RPMI8226 cells (American ATCC, Item No. CCL-155) with different BCMA expression levels were cultured and passaged according to routine operations. After the cells were centrifuged and resuspended, the cells were counted, and the cell density was adjusted to 4 ⁇ 10 5 cells/ml, poured into the sample addition tank, and seeded in a 96-well plate with 50ul per well. Add 50ul of serially diluted antibody samples to 50ul of cells per well, and then put them into a cell incubator at 37°C and 5% CO2 for 60 minutes.
  • BCMA ⁇ CD3 bispecific antibody simultaneously binds to BCMA on the surface of multiple myeloma cells and CD3 on the surface of primary T cells, activates T cells through BCMA-dependent CD3 cross-linking, and mediates T cells against BCMA-positive tumor cells. kill.
  • the lactate dehydrogenase (LDH) assay was used to detect LDH released from dead cells in the supernatants collected 24 hours after the addition of the example antibodies under the co-culture conditions of peripheral blood mononuclear cells (PBMCs) and BCMA-positive multiple myeloma cells. The level of T cells to evaluate the killing ability of BCMA tumor cells.
  • LDH lactate dehydrogenase
  • the PBMC cells were taken out from the liquid nitrogen tank, thawed rapidly at 37°C, and added dropwise to the preheated 1640 medium (containing 0.1% DNase) to obtain 10 ml of the mixed solution. Centrifuge at 400 g for 5 minutes, resuspend with 10 ml of 1640 medium, add 10 ⁇ l DNase, and culture overnight at 37° C., 5% CO 2 , and adherent. Pipette the suspended T cells and centrifuge at 400g for 5 minutes, resuspend in 1640 medium, count, and adjust the cell density to 4 ⁇ 10 6 cells/ml as effector cells.
  • 1640 medium containing 0.1% DNase
  • BCMA-positive multiple myeloma cells were used as target cells, centrifuged at 400 g for 5 minutes, resuspended in 1640 medium, counted, and adjusted to a cell density of 2 ⁇ 10 5 cells/ml as target cells.
  • the target cells were added to the 96-well plate, 100 ⁇ l per well; the serially diluted antibody was added to the 96-well plate, 50 ⁇ l per well; and the effector cell PBMC was added to the 96-well plate, 50 ⁇ l per well.
  • the final effect-to-target ratio was 10:1.
  • the above 96-well plate was placed in a 37°C, 5% CO 2 incubator for 24 hours, and the supernatant was taken, and the amount of dead cells released into the supernatant was detected according to the instructions of the LDH detection kit (CytoTox96 non-radioacitive cytotoxicity kit, Promega). The content of LDH, and then the killing ratio of the example antibodies to multiple myeloma cells was calculated.
  • the exemplary antibodies were able to dose-dependently induce killing of L363 cells by PBMCs derived from different donors (see Figures 6A, 6B).
  • exemplary antibodies were compared in inducing killing of PBMCs derived from the same donor against multiple myeloma cell lines with different levels of BCMA expression (see Figures 7 and 8): wherein, See Figures 7A and 8A for the killing effect of PBMC induced by antibody on the surface of NCl-H929 cells, see 7B and 8B for the killing effect of PBMC induced by antibody on L363 cells, and see 7C and 8C for the killing effect of PBMC induced by antibody on RPMI8226 cells for example Antibody-induced killing of PBMCs against BCMA-negative NUGC4 cells (JCRB cell bank, JCRB834) is shown in 7D and 8D. It can be seen that, for PBMCs from different sources, the example antibodies can dose-dependently induce the killing effect of PBMCs from the same donor on multiple myeloma cells with different expression levels of BCMA.
  • Example 5 Concomitant T cell activation and cytokine release levels during the killing of human multiple myeloma cells by the anti-BCMA ⁇ CD3 antibodies of the present invention
  • the death percentage of multiple myeloma cells was detected by LDH method, and multiple cytokines were simultaneously detected by multi-factor detection kit (Human Th1/Th2/Th17, BD).
  • the percentages of CD25 and CD69 positive cells in T cells were detected by flow cytometry, and the corresponding functions of the exemplary antibodies of the present invention were studied.
  • Exemplary antibodies were tested for killing of target cells as described in Example 4.
  • Example 4 The supernatant collected after 24 hours as described in Example 4 was tested for the contents of cytokines IL-2, TNF ⁇ , IFN ⁇ and IL-6 according to the instructions of the detection kit (Human Th1/Th2/Th17kit, BD).
  • the killing effect of PBMC induced by the exemplary antibody on the surface of NCl-H929 cells is shown in Figure 8A, and the release levels of various cytokines accompanying the killing process are shown in Figure 9; the killing effect of PBMC induced by the exemplary antibody on L363 cells is shown in Figure 8B, and the killing process
  • the accompanying release levels of various cytokines are shown in Figure 10; the killing effect of PBMCs on RPMI8226 cells induced by an example antibody is shown in 8C, and the release levels of various cytokines accompanying the killing process are shown in Figure 11; an example antibody-induced PBMC is shown in Figure 11 on BCMA-negative NUGC4 cells See 8D for the killing effect.
  • FIGS 12A and 12B The extent of activation of CD8+ and CD4+ T cells accompanying the killing of NCl-H929 cells by exemplary antibody-induced PBMCs in experiments performed as described in the assay above is shown in Figures 12A and 12B, respectively;
  • the degree of activation of CD8+ and CD4+ T cells accompanying the killing process of cells is shown in Figure 12C, 12D respectively;
  • the degree of activation of CD8+ and CD4+ T cells accompanying the killing process of RPMI822626 cells by example antibody-induced PBMC is shown in Figure 12E, 12F respectively .
  • the example antibodies can dose-dependently activate CD8+ T cells and CD4+ T cells isolated from PBMC, and the activation degree of T cells has a certain correlation with the affinity of CD3 : The higher the anti-CD3 affinity, the stronger the ability to activate T cells.
  • CellTracker TM Deep Red was used to label CD8+ T cells isolated from PBMC, and then the labeled CD8+ T cells and L363 cells were co-cultured, and sample antibodies diluted in concentration gradients were added, and CD8+ T cells were detected by flow cytometry 96 hours later. proliferation.
  • PBMC recovery Take out PBMC from the liquid nitrogen tank and thaw quickly at 37°C, slowly add the cells to 10ml of AIM V medium (Gibco TM ) at 37°C (containing 0.1% DNase), 300 g for 8 minutes, Centrifuge at 25°C, remove the supernatant, and resuspend in a T75 culture flask with 10ml of AIM V medium at 37°C (containing 0.1% DNase) in a 37°C 5% CO 2 incubator for 3 hours.
  • AIM V medium Gibco TM
  • NUGC4 cells were removed from the cell culture medium, 2ml of 0.25% Trypsin-EDTA 5ml was digested at room temperature for 5 minutes, then the cell culture medium was added to neutralize trypsin, centrifuged at 400g for 5 minutes, the supernatant was removed, and CTS TM AIM V TM SFM medium was used Resuspend the cells, count the cells, and adjust the cell density to 1 x 10 5 cells/ml.
  • L363 cells were centrifuged at 400g for 5 minutes, the supernatant was removed, the cells were resuspended in CTS TM AIM V TM SFM medium, the cells were counted, and the cell density was adjusted to 1 ⁇ 10 5 cells/ml.
  • the exemplified antibodies were effective in stimulating CD8+ T cell proliferation in vitro in the presence of BCMA-positive L363 cells (see Figure 13A); and in the presence of BCMA-negative NUGC4 cells Exemplary antibodies did not show BCMA-independent, nonspecific proliferation of CD8+ T cells (see Figure 13B).
  • Exemplary antibodies can dose-dependently stimulate CD4+ T cell proliferation in vitro in the presence of BCMA-positive L363 cells ( Figure 14).
  • the exemplified antibody did not exhibit BCMA-independent and non-specific proliferation of CD4+ T cells.
  • Steps (1)-(5) were performed using steps similar to those disclosed in Example 7, except that CD8+ T cells were replaced with CD4+ T cells.
  • Example 9 In vivo efficacy test of the anti-BCMA ⁇ CD3 bispecific antibody of the present invention in animals
  • human multiple myeloma cells H929 cells were used to inoculate the PBMC model of NOG mice, and human BCMA-expressing Daudi cells (BCMA-Daudi) were used to inoculate NOG mice with two models to determine the antitumor effect of the example antibodies.
  • mice Female NOG mice (35-41 days old) were purchased from Beijing Weitongda Laboratory Animal Technology Co., Ltd. The grade is SPF grade. Mice were acclimated and quarantined for 7 days upon arrival before the study began.
  • NCl-H929 cells were routinely subcultured for subsequent in vivo experiments, the cells were collected by centrifugation, and the NCl-H929 cells were dispersed with PBS.
  • the right back and abdomen of 70 NOG mice were shaved and inoculated with NCl-H929 cells, 5 ⁇ 10 6 cells/mice, and the inoculation volume was 200ul/mice.
  • mice were intravenously injected with PBMC cells, 5 ⁇ 10 6 cells/mice, and the inoculation volume was 200ul/mice.
  • mice On the 3rd day after inoculation of PBMC cells, mice were grouped according to tumor volume (7 mice in each group) for administration. Administered once every 7 days for a total of 3 doses. An irrelevant hIgG antibody was used as a control.
  • TGI% relative tumor inhibition rate
  • TGI% 100% ⁇ (tumor volume in the hIgG control group-tumor volume in the treatment group)/(tumor volume in the hIgG control group-initial tumor volume in the hIgG control group), and the initial tumor volume in the control group was ⁇ 80mm 3 .
  • Body weight was measured using an electronic balance.
  • mice were euthanized throughout the study when tumors reached endpoints or when mice had >20% body weight loss. The tumor size was counted, and the tumor inhibition rate (TGI%) was calculated.
  • the tumor growth curve is shown in Figure 15.
  • the example antibody can significantly inhibit the growth of HCl-H929 cells.
  • the tumor size was counted on the 22nd day, and the tumor inhibition rate was calculated.
  • the tumor inhibition rates of exemplary antibodies 46757 and 46758 were 80.8% and 95.9%, respectively, compared to hIgG.
  • the tumor inhibition rates of exemplary antibodies ADI-46757 and ADI-46758 were 84.9% and 85.9%, respectively, compared to hIgG.
  • no weight loss was found in the administered mice groups.
  • mice Female NOG mice (9-16 g) were purchased from Beijing Weitongda Laboratory Animal Technology Co., Ltd. The grade was SPF, and the mice were acclimated and quarantined for 7 days upon arrival, and then began the study.
  • Daudi-BCMA cells were routinely subcultured for subsequent in vivo experiments. Daudi-BCMA cells were dispersed with PBS and Matrigel at a ratio of 1:1 to prepare a cell suspension with a cell concentration of 25 ⁇ 10 6 cells/mL. The right back of NOG mice was shaved, and 25 ⁇ 10 6 cells/mL Daudi-BCMA cell suspension was subcutaneously injected at 0.2 mL/mice, that is, the inoculation amount was 5 ⁇ 10 6 cells/mice.
  • PBMC cells Five days after tumor cell inoculation, PBMC cells were recovered with RPMI-1640 medium pre-warmed with 0.1% DNase, and then PBMC cells were dispersed with PBS to prepare a cell suspension with a cell concentration of 25 ⁇ 10 6 cells/mL. Mice were injected with PBMC cell suspension, 0.2 mL/mice, that is, the inoculation amount was 5 ⁇ 10 6 cells/mice.
  • mice Eight days after tumor cell inoculation, mice were administered into groups (7 mice in each group) according to tumor volume, once every 7 days, and administered twice in a row.
  • the mode of administration was intraperitoneal injection, and the administration volume was 10ml/kg/time.
  • the tumor volume and body weight of mice were monitored twice a week until the end of 29 days.
  • An irrelevant hIgG antibody was used as a control.
  • TGI% The relative tumor inhibition rate (TGI%) was calculated on the 29th day after inoculation, and the calculation formula was as follows:
  • TGI% 100% ⁇ (tumor volume of hIgG control group-tumor volume of treatment group)/(tumor volume of hIgG control group-tumor volume of hIgG control group before administration).
  • Body weight was measured using an electronic balance.
  • the tumor growth curve is shown in Figure 16.
  • the example antibody can significantly inhibit the growth of BCMA-overexpressing Daudi cells.
  • the tumor size was counted on the 29th day, and the tumor inhibition rate was calculated.
  • the tumor inhibition rates of exemplary antibodies 46757 and 46758 were 62% and 108%, respectively, compared to hIgG. At the same time, no weight loss was found in the administered mice groups.

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Abstract

提供了特异性结合BCMA和CD3的新型双特异性抗体、编码所述抗BCMA×CD3双特异性抗体的核酸、包含所述核酸的载体、包含所述核酸或载体的宿主细胞、包含所述抗体或其抗原结合片段的药物组合物。此外，还提供了这些抗BCMA×CD3双特异性抗体、药物组合物等在疾病的免疫治疗、预防和/或诊断上的应用。

Description

抗BCMA×CD3双特异性抗体及其用途 技术领域
本发明总体上涉及免疫学和抗体工程领域。具体而言，本发明涉及特异性结合BCMA和CD3的新型双特异性抗体。此外，本发明涉及编码所述抗BCMA×CD3双特异性抗体的核酸、包含所述核酸的载体、包含所述核酸或载体的宿主细胞、包含所述抗体或其抗原结合片段的药物组合物。此外，本发明涉及这些抗BCMA×CD3双特异性抗体、药物组合物等在疾病的免疫治疗、预防和/或诊断上的应用。
背景技术
B细胞成熟抗原(BCMA)，又名CD269或TNFRSF17，是肿瘤坏死因子受体家族成员。研究表明，BCMA可以结合B细胞激活因子受体(BAFF)和B细胞增殖诱导配体(APRIL)，促进B细胞分化、增殖、存活。而异常的信号传导可促使B细胞异常增殖，导致自身免疫疾病和肿瘤形成，参见Rickert等人,Immunological Reviews2011,第244卷：115-133。
在非肿瘤细胞中，BCMA主要表达在浆细胞和成熟B细胞亚群中。而在60-70％的多发性骨髓瘤(MM)患者中，BCMA也在癌化的浆细胞表面表达。在多发性骨髓瘤患者中血清BCMA水平升高，并且升高的水平与疾病状况、治疗反应、和整体存活相关。BCMA基因缺陷小鼠有着正常的B细胞水平，但是其浆细胞的生存周期会显著缩短。因此，BCMA是多发性骨髓瘤免疫治疗的理想靶点。
多发性骨髓瘤仍然是一种难以治疗的疾病。其通常伴随复发过程，许多患者需要多线疗法。诸如化学疗法和干细胞移植方法的疗法,但这些通常带来不想要的副作用。针对多发性骨髓瘤的免疫疗法目前取得了一定的成绩，但是仍然需要新的特异性结合分子，尤其是靶向癌细胞的BCMA并且可以在癌细胞周围募集T细胞的双特异性抗体分子。
本发明通过提供以高靶特异性和高亲合性结合BCMA和CD3的双特异性抗体，尤其是通过与肿瘤细胞表面上表达的BCMA结合而使得T细胞可以募集到肿瘤细胞周围的双特异性抗体，满足了这方面的需求。
发明内容
本发明公开了一种新的同时靶向BCMA和CD3的双特异性抗体、编码所述双特异性抗体的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、以及所述双特异性抗体在个体中治疗、预防和/或诊断与BCMA活性相关的疾病中的用途。
因此，在一个方面，本发明提供了特异性结合BCMA且结合CD3的双特异性抗体(抗 BCMA×CD3双特异性抗体)，其包含(i)抗BCMA抗体或其片段，和(ii)抗CD3抗体或其片段。在一个实施方案中，本发明提供了1+1格式的抗BCMA×CD3双特异性抗体，如图1所示，其由左右非对称的三条多肽链组成，其中左半部分由肽链1和肽链2组成，右半部分由肽链3组成，肽链1和肽链2分别是靶向BCMA或CD3的抗体重链和轻链，肽链3由靶向CD3或BCMA的scFv和Fc区组成。
在另一个实施方案中，本发明提供了2+1格式的抗BCMA×CD3双特异性抗体，如图2所示，其由左右非对称的四条多肽链组成，其中肽链1和肽链2分别是靶向BCMA或CD3的抗体重链和轻链，肽链3由靶向BCMA或CD3的抗体重链和靶向CD3或BCMA的scFv组成。
在一个实施方案中，本发明提供了1+1格式的抗BCMA×CD3双特异性抗体，其中肽链1和肽链2特异性结合BCMA，肽链3特异性结合CD3。在一个具体的实施方案中，本发明提供的抗BCMA×CD3双特异性抗体包含肽链1、肽链2和肽链3，其中肽链1包含选自SEQ ID NO:1，3或5所包含的3个重链CDR，肽链2包含选自SEQ ID NO:2，4或6所包含的3个轻链CDR，肽链3包含选自SEQ ID NO:7或9所包含的3个重链CDR和选自SEQ ID NO:8或10所包含的3个轻链CDR。
在一个具体的实施方案中，本发明提供的抗BCMA×CD3双特异性抗体包含肽链1、肽链2和肽链3，其中:
1)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR，肽链3包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
2)肽链1包含来自SEQ ID NO:3所包含的3个重链CDR，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR，肽链3包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
3)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR，肽链3包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
4)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR，肽链3包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR；
5)肽链1包含来自SEQ ID NO:3包含的3个重链CDR，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR，肽链3包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR；或
6)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR，肽链2包含来自SEQ ID NO:6 所包含的3个轻链CDR，肽链3包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR。
在一个具体的实施方案中，本发明提供的抗BCMA×CD3双特异性抗体包含肽链1、肽链2和肽链3，其中:
1)肽链1包含SEQ ID NO:47-49所示的3个重链CDR，肽链2包含SEQ ID NO:50-52所示的3个轻链CDR，肽链3包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
2)肽链1包含SEQ ID NO:53-55所示的3个重链CDR，肽链2包含SEQ ID NO:56-58所示的3个轻链CDR，肽链3包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
3)肽链1包含SEQ ID NO:59-61所示的3个重链CDR，肽链2包含SEQ ID NO:62-64所示的3个轻链CDR，肽链3包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
4)肽链1包含SEQ ID NO:47-49所示的3个重链CDR，肽链2包含SEQ ID NO:50-52所示的3个轻链CDR，肽链3包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76所示的3个轻链CDR；
5)肽链1包含SEQ ID NO:53-55所示的3个重链CDR，肽链2包含SEQ ID NO:56-58所示的3个轻链CDR，肽链3包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76所示的3个轻链CDR；或
6)肽链1包含SEQ ID NO:59-61所示的3个重链CDR，肽链2包含SEQ ID NO:62-64所示的3个轻链CDR，肽链3包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76所示的3个轻链CDR。
在一个具体的实施方案中，本发明提供的抗BCMA×CD3双特异性抗体包含肽链1、肽链2和肽链3，其中肽链1包含选自SEQ ID NO:1，3或5所包含的3个重链CDR且与SEQ ID NO:1，3或5具有至少90％同一性的VH，肽链2包含选自SEQ ID NO:2，4或6所包含的3个轻链CDR且与SEQ ID NO:2，4或6具有至少90％同一性的VL，肽链3包含选自SEQ ID NO:7或9所包含的3个重链CDR且与SEQ ID NO:7或9具有至少90％同一性的VH和选自SEQ ID NO:8或10所包含的3个轻链CDR且与SEQ ID NO:8或10具有至少90％同一性的VL。
在一个具体的实施方案中，本发明提供的抗BCMA×CD3双特异性抗体包含肽链1、肽链2和肽链3，其中:
1)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR且与SEQ ID NO:1具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR且与SEQ ID NO:2 具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:7所包含的3个重链CDR且与SEQ ID NO:7具有至少90％同一性的VH和来自SEQ ID NO:8所包含的3个轻链CDR且与SEQ ID NO:8具有至少90％同一性的VL；
2)肽链1包含来自SEQ ID NO:3所包含的3个重链CDR且与SEQ ID NO:3具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR且与SEQ ID NO:4具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:7所包含的3个重链CDR且与SEQ ID NO:7具有至少90％同一性的VH和来自SEQ ID NO:8所包含的3个轻链CDR且与SEQ ID NO:8具有至少90％同一性的VL；
3)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR且与SEQ ID NO:5具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR且与SEQ ID NO:6具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:7所包含的3个重链CDR且与SEQ ID NO:7具有至少90％同一性的VH和来自SEQ ID NO:8所包含的3个轻链CDR且与SEQ ID NO:8具有至少90％同一性的VL；
4)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR且与SEQ ID NO:1具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR且与SEQ ID NO:2具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:9所包含的3个重链CDR且与SEQ ID NO:9具有至少90％同一性的VH和来自SEQ ID NO:10所包含的3个轻链CDR且与SEQ ID NO:10具有至少90％同一性的VL；
5)肽链1包含来自SEQ ID NO:3所包含的3个重链CDR且与SEQ ID NO:3具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR且与SEQ ID NO:4具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:9所包含的3个重链CDR且与SEQ ID NO:9具有至少90％同一性的VH和来自SEQ ID NO:10所包含的3个轻链CDR且与SEQ ID NO:10具有至少90％同一性的VL；或
6)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR且与SEQ ID NO:5具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR且与SEQ ID NO:6具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:9所包含的3个重链CDR且与SEQ ID NO:9具有至少90％同一性的VH和来自SEQ ID NO:10所包含的3个轻链CDR且与SEQ ID NO:10具有至少90％同一性的VL。
在另一个实施方案中，本发明提供的抗BCMA×CD3双特异性抗体包含肽链1、肽链2和肽链3，其中肽链1包含选自SEQ ID NO:1，3或5所示的VH，肽链2包含选自SEQ ID NO:2，4或6所示的VL，肽链3包含选自SEQ ID NO:7或9所示的VH和选自SEQ ID NO:8或10所示的VL。
在一个具体的实施方案中，本发明提供的抗BCMA×CD3双特异性抗体包含肽链1、肽链2和肽链3，其中:
1)肽链1包含SEQ ID NO:1所示VH，肽链2包含SEQ ID NO:2所示VL，肽链3包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
2)肽链1包含SEQ ID NO:3所示VH，肽链2包含SEQ ID NO:4所示VL，肽链3包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
3)肽链1包含SEQ ID NO:5所示VH，肽链2包含SEQ ID NO:6所示VL，肽链3包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
4)肽链1包含SEQ ID NO:1所示VH，肽链2包含SEQ ID NO:2所示VL，肽链3包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL；
5)肽链1包含SEQ ID NO:3所示VH，肽链2包含SEQ ID NO:4所示VL，肽链3包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL；或
6)肽链1包含SEQ ID NO:5所示VH，肽链2包含SEQ ID NO:6所示VL，肽链3包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL。
在另一个实施方案中，本发明提供的抗BCMA×CD3双特异性抗体包含肽链1、肽链2和肽链3，其中肽链1和肽链3包含相同或者不同的Fc区。在另一个优选的实施方案中，肽链1和肽链3所包含的Fc区分别具有“杵”和“臼”结构，所述杵臼结构相互作用从而稳定双特异性抗体的空间结构。
在一个具体实施方案中，在本发明的抗BCMA×CD3双特异性抗体中，肽链1和肽链3中包含的可变区与Fc区是同源的或者异源的。在另一个实施方案中，肽链1和肽链3中包含的可变区与Fc区直接连接或者通过接头连接。在一个具体实施方案中，所述接头是本领域通用的柔性接头。在另一个实施方案中，所述接头是(G4S)n，其中n＝1-6，优选n＝1,2,3或4。
在一个具体实施方案中，在本发明抗BCMA×CD3双特异性抗体的肽链3中，重链可变区和轻链可变区通过接头形成scFv。连接重链可变区和轻链可变区的接头是本领域通用的柔性接头。
在另一方面，本发明提供了抗BCMA×CD3双特异性抗体，其包含肽链1、肽链2和肽链3，其中：
1)肽链1包含与SEQ ID NO:11具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:11组成；肽链2包含与SEQ ID NO:12具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:12组成；肽链3包含与SEQ ID  NO:13具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:13组成；
2)肽链1包含与SEQ ID NO:14具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:14组成；肽链2包含与SEQ ID NO:15具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:15组成；肽链3包含与SEQ ID NO:16具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:16组成；
3)肽链1包含与SEQ ID NO:17具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:17组成；肽链2包含与SEQ ID NO:18具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:18组成；肽链3包含与SEQ ID NO:19具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:19组成；
4)肽链1包含与SEQ ID NO:20具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:20组成；肽链2包含与SEQ ID NO:21具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:21组成；肽链3包含与SEQ ID NO:22具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:22组成；
5)肽链1包含与SEQ ID NO:23具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:23组成；肽链2包含与SEQ ID NO:24具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:24组成；肽链3包含与SEQ ID NO:25具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:25组成；或
6)肽链1包含与SEQ ID NO:26具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:26组成；肽链2包含与SEQ ID NO:27具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:27组成；肽链3包含与SEQ ID NO:28具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:28组成。
在另一方面，本发明提供了2+1格式的抗BCMA×CD3双特异性抗体，其由肽链1，肽链3和2条肽链2组成，包含两个识别BCMA的抗原识别位点和一个识别CD3的抗原识别位点，如图2所示。在一个实施方案中，本发明提供了2+1格式的抗BCMA×CD3双特异性抗体，其中特异性结合CD3的scFv结构域直接或者通过接头连接到肽链1的C末端，从而构成肽链3。在一个具体实施方案中，通过本领域已知的适当接头连接特异性结合CD3的重链可变区和轻链可变区，从而形成所述scFv。
在一个实施方案中，肽链1的重链可变结枃域和肽链2的轻链可变结构域配对形成BCMA的第一个抗原识别位点，肽链3的重链可变结枃域和肽链2的轻链可变结构域配对形成BCMA的第二个抗原识别位点，肽链3的scFv形成CD3的抗原识别位点。肽链1和肽链3各自的Fc结构域相互作用，形成Fc区。在一个具体的实施方案中，肽链1和肽链3各自的Fc结构域中含有稳定相互作用的突变，例如含有“杵入臼”突变。
在一个具体的实施方案中，本发明提供的2+1格式的抗BCMA×CD3双特异性抗体包含肽链1、2条肽链2和肽链3，其中:
1)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:1所包含的3个重链CDR并且C末端包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
2)肽链1包含来自SEQ ID NO:3所包含的3个重链CDR，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:3所包含的3个重链CDR并且C末端包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
3)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:5所包含的3个重链CDR并且C末端包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
4)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:1所包含的3个重链CDR并且C末端包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR；
5)肽链1包含来自SEQ ID NO:3包含的3个重链CDR，肽链2包含来自SEQ ID NO:4 所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:3所包含的3个重链CDR并且C末端包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR；或
6)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:5所包含的3个重链CDR并且C末端包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR。
在一个具体的实施方案中，本发明提供的2+1格式的抗BCMA×CD3双特异性抗体包含肽链1、2条肽链2和肽链3，其中:
1)肽链1包含SEQ ID NO:47-49所示的3个重链CDR，肽链2包含SEQ ID NO:50-52所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:47-49所示的3个重链CDR并且C末端包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
2)肽链1包含SEQ ID NO:53-55所示的3个重链CDR，肽链2包含SEQ ID NO:56-58所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:53-55所示的3个重链CDR并且C末端包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
3)肽链1包含SEQ ID NO:59-61所示的3个重链CDR，肽链2包含SEQ ID NO:62-64所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:59-61所示的3个重链CDR并且C末端包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
4)肽链1包含SEQ ID NO:47-49所示的3个重链CDR，肽链2包含SEQ ID NO:50-52所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:47-49所示的3个重链CDR并且C末端包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76所示的3个轻链CDR；
5)肽链1包含SEQ ID NO:53-55所示的3个重链CDR，肽链2包含SEQ ID NO:56-58所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:53-55所示的3个重链CDR并且C末端包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76所示的3个轻链CDR；或
6)肽链1包含SEQ ID NO:59-61所示的3个重链CDR，肽链2包含SEQ ID NO:62-64所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:59-61所示的3个重链CDR并且C末端包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76所示的3 个轻链CDR。
在一个具体的实施方案中，本发明提供的2+1格式的抗BCMA×CD3双特异性抗体包含肽链1、2条肽链2和肽链3，其中:
1)肽链1包含SEQ ID NO:1所示VH，肽链2包含SEQ ID NO:2所示VL，肽链3的N末端包含SEQ ID NO:1所示VH并且C末端包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
2)肽链1包含SEQ ID NO:3所示VH，肽链2包含SEQ ID NO:4所示VL，肽链3的N末端包含SEQ ID NO:3所示VH并且C末端包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
3)肽链1包含SEQ ID NO:5所示VH，肽链2包含SEQ ID NO:6所示VL，肽链3的N末端包含SEQ ID NO:5所示VH并且C末端包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
4)肽链1包含SEQ ID NO:1所示VH，肽链2包含SEQ ID NO:2所示VL，肽链3的N末端包含SEQ ID NO:1所示VH并且C末端包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL；
5)肽链1包含SEQ ID NO:3所示VH，肽链2包含SEQ ID NO:4所示VL，肽链3的N末端包含SEQ ID NO:3所示VH并且C末端包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL；或
6)肽链1包含SEQ ID NO:5所示VH，肽链2包含SEQ ID NO:6所示VL，肽链3的N末端包含SEQ ID NO:5所示VH并且C末端包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL。
在另一方面，本发明提供了2+1格式的抗BCMA×CD3双特异性抗体，其包含肽链1、2条肽链2和肽链3，其中：
1)肽链1包含与SEQ ID NO:29具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:29组成；肽链2包含与SEQ ID NO:30具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:30组成；肽链3包含与SEQ ID NO:31具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:31组成；
2)肽链1包含与SEQ ID NO:32具有至少90％、91％、92％、93％、94％、95％、96％、 97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:32组成；肽链2包含与SEQ ID NO:33具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:33组成；肽链3包含与SEQ ID NO:34具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:34组成；
3)肽链1包含与SEQ ID NO:35具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:35组成；肽链2包含与SEQ ID NO:36具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:36组成；肽链3包含与SEQ ID NO:37具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:37组成；
4)肽链1包含与SEQ ID NO:38具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:38组成；肽链2包含与SEQ ID NO:39具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:39组成；肽链3包含与SEQ ID NO:40具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:40组成；
5)肽链1包含与SEQ ID NO:41具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:41组成；肽链2包含与SEQ ID NO:42具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:42组成；肽链3包含与SEQ ID NO:43具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:43组成；或
6)肽链1包含与SEQ ID NO:44具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:44组成；肽链2包含与SEQ ID NO:45具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:45组成；肽链3包含与SEQ ID NO:46具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:46组成。
在一个方面，本发明还提供了编码本发明抗BCMA×CD3双特异性抗体的多核苷酸(核酸)、包含所述多核苷酸的载体，优选地表达载体。
在另一个方面，本发明提供了包含本发明多核苷酸或载体的宿主细胞。本发明也提供了一种用于产生本发明抗BCMA×CD3双特异性抗体的方法，包括步骤(i)在适于表达本发明抗BCMA×CD3双特异性抗体的条件下培养本发明的宿主细胞，和(ii)回收本发明的抗BCMA×CD3双特异性抗体。
在一个方面，本发明提供了一种包含本发明抗BCMA×CD3双特异性抗体的诊断试剂盒和药物组合物。进一步地，还提供了本发明的抗BCMA×CD3双特异性抗体、诊断试剂盒或药物组合物的用途，用于治疗、预防和/或诊断与BCMA活性相关的疾病，特别地用于治疗、预防和/或诊断多发性骨髓瘤。
在另一个方法，本发明提供了治疗与BCMA活性相关的疾病的方法，包括将治疗有效量的本发明的抗BCMA×CD3双特异性抗体，或本发明的药物组合物施用给有需要的患者。优选地，所述疾病是过量表达BCMA的癌症，更优选所述疾病是多发性骨髓瘤。
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
附图说明
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1. 1+1格式双特异性抗体结构的示意图，其中图1B显示组成1+1格式双特异性抗体结构各条链的结构。
图2. 2+1格式结构示意图。
图3.示例抗体与NCl-H929细胞的结合能力。
图4.示例抗体与L363细胞的结合能力。
图5.示例抗体与RPMI8226细胞的结合能力。
图6.示例抗体介导的PBMC对L363细胞的杀伤作用，6A.示例抗体介导的PBMC(供者：2001030)对L363细胞的杀伤作用；6B.示例抗体介导的PBMC(供者：2090306)对L363细胞的杀伤作用。
图7.示例抗体介导的同一供者来源的PBMC对BCMA不同表达水平的多发性骨髓瘤细胞的杀伤作用，7A.示例抗体介导的PBMC对NCl-H929细胞的杀伤作用，7B.示例抗体介导的PBMC，对L363细胞的杀伤作用，7C.示例抗体介导的PBMC对RPMI8226细胞的杀伤作用，7D.示例抗体介导的PBMC对NUGC4细胞的杀伤作用
图8.示例抗体介导的PBMC对多发性骨髓瘤细胞系的杀伤作用，8A.示例抗体介导的PBMC对NCL-H929细胞的杀伤作用，8B.示例抗体介导的PBMC对L363细胞的杀伤作用，8C.示例抗体介导的PBMC对RPMI8226细胞的杀伤作用，8D.示例抗体介导的PBMC对NUGC4细胞的杀伤作用。
图9.流式细胞技术检测示例抗体诱导的PBMC对NCl-H929细胞的杀伤过程伴随的细胞因子的释放量。
图10.流式细胞技术检测示例抗体诱导的PBMC对L363细胞的杀伤过程伴随的细胞因子的释放量。
图11.流式细胞技术检测示例抗体诱导的PBMC对RPMI8226细胞的杀伤过程伴随的细胞因子的释放量。
图12.示例抗体介导的PBMC中T细胞的激活，12A.示例抗体介导的PBMC对NCl-H929细胞杀伤过程中CD8+T细胞中CD25+/CD69+双阳性细胞的百分比(反应CD8+T细胞的激活程度)；12B.示例抗体介导的PBMC对NCl-H929细胞杀伤过程中CD4+T细胞中CD25+/CD69+双阳性细胞的百分比(反应CD4+T细胞的激活程度)；12C.示例抗体介导的PBMC对L363细胞杀伤过程中CD8+T细胞中CD25+/CD69+双阳性细胞的百分比(反应CD8+T细胞的激活程度)；12D.示例抗体介导的PBMC对L363细胞杀伤过程中CD4+T细胞中CD25+/CD69+双阳性细胞的百分比(反应CD4+T细胞的激活程度)；12E.示例抗体介导的PBMC对RPPMI8226细胞杀伤过程中CD8+T细胞中CD25+/CD69+双阳性细胞的百分比(反应CD8+T细胞的激活程度)；12F.示例抗体介导的PBMC对RPPMI8226细胞杀伤过程中CD4+T细胞中CD25+/CD69+双阳性细胞的百分比(反应CD4+T细胞的激活程度)。
图13.示例抗体促进CD8+T细胞增殖，13A.示例抗体在L363细胞存在时可剂量依赖性地促进CD8+T细胞增殖，13B.示例抗体在BCMA阴性的NUGC4细胞存在时无非特异性的CD8+T细胞增殖。
图14.示例抗体促进CD4+T细胞增殖，14A.示例抗体在L363细胞存在时可剂量依赖性地促进CD4+T细胞增殖，14B.示例抗体在BCMA阴性的NUGC4细胞存在时无非特异性的CD4+T细胞增殖。
图15.示例抗体在NCl-H929荷瘤人源化小鼠模型中的抑瘤作用。
图16.示例抗体在Daudi-BCMA荷瘤人源化小鼠模型中的抑瘤作用。
具体实施方式
I.定义
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”意指当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。
术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其它要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组合的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“抗体”在本文中以最广意义使用并且涵盖多种抗体结构物，包括但不限于单克隆抗体、多克隆抗体、重组抗体、人源化抗体、嵌合抗体、多特异性抗体(例如，双特异性抗体)、单链抗体、完整抗体或其显示出所需的抗原结合活性的抗体片段。完整抗体通常将包含至少两条全长重链和两条全长轻链，但在某些情况下可包括较少的链，例如骆驼中天然存在的抗体可仅包含重链。
术语“抗原结合片段”(在本文中可与“抗体片段”和“抗原结合部分”互换使用)，指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗原结合片段的例子包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’) 2；双抗体(diabodies,dAb)；线性抗体；单链抗体(例如scFv)；单结构域抗体(单域抗体)；双价或双特异性抗体的抗原结合片段；骆驼科抗体；和表现出所需的结合抗原(例如BCMA和/或CD3)能力的其它片段。
如本文所用，术语“结合”和“特异性结合”意指抗体的结合作用对抗原是选择性的，并且可以与不想要的或非特异的相互作用区别开。抗体与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)、表面等离子共振法(SPR)或生物膜层光学干涉技术(ForteBio)或本领域已知的其它常规结合测定法测定。例如在SPR中，抗体以大约1×10 -7或更低的KD，大约 1×10 -8或更低的KD，大约1×10 -9或更低的KD，大约1×10 -10或更低的KD、大约1×10 -11或更低的KD与BCMA或CD3结合，则该抗体是“与BCMA或CD3特异性结合”的抗体。然而，特异性结合人BCMA或CD3的抗体可以与来自其它物种的BCMA或CD3蛋白具有交叉反应性。例如，特异于人BCMA或CD3的抗体，在一些实施方案中，可以与食蟹猴BCMA或CD3发生交叉反应。测定交叉反应性的方法包括实施例中所述的方法以及本领域已知的标准测定法，例如通过使用生物光干涉，或流式细胞术技术。
术语“单链可变片段”或“scFv”是一种小分子的基因工程抗体，它是在DNA水平上利用基因工程方法将天然抗体的重链可变区(VH)和轻链可变区(VL)连接(通常通过一段人工合成的连接肽(或“接头”)连接)而成的小分子重组抗体。与完整抗体分子相比，scFv单链抗体具有以下优点：含有完整的抗体可变区，保留原抗体的抗原特异性和结合活性；不含有抗体分子的Fc区，因而免疫原性弱，用于人体不易产生免疫反应；易于操作，适用于作为基因工程构件，制备具有新性质的其它抗原特异性结合分子，例如全长抗体、scFv-Fc等。
术语“Fc区”在本文中用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。在某些实施方案中，人IgG重链Fc区通常从Cys226或Pro230延伸至重链的羰基端。然而，Fc区的C端赖氨酸(Lys447)可以存在或者可以不存在。除非另外说明，Fc区或恒定区中的氨基酸残基的编号是根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5 th Ed.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。
术语“杵入臼”指在本申请所述的双特异性抗体分子的一条Fc链上产生“杵”结构，而在另一条链上产生“臼”结构，从而使得该臼与该杵具有相同或相似的大小，适宜地放置，使得在两个Fc相互作用时，一个Fc的杵可定位在另一个Fc对应的臼中，从而稳定了异源多聚体的结构(参见例如美国专利号5,731,168)。
在一些实施方案中，根据本领域现有技术，杵可以通过用较大的侧链取代小的氨基酸侧链来构建。在一些实施方案中，臼可以通过用较小的侧链取代大的氨基酸侧链来构建。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的框架区(FR)和三个互补决定区(参见，例如，Kindt等Kuby Immunology，6 th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。
“互补决定区”或“CDR区”或“CDR”或“高变区”，是抗体可变结构域中在序列上高度可变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR从N-端开始顺序编号，通常称作CDR1、CDR2和CDR3。位于抗体重链可变结构域内的CDR也称作HCDR1、HCDR2和 HCDR3，而位于抗体轻链可变结构域内的CDR则称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，可以采用本领域公知的多种方案确定其CDR序列，例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(国际免疫遗传学信息系统,万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义(North等，“A New Clustering of Antibody CDR Loop Con格式ions”，Journal of Molecular Biology，406，228-256(2011))。
例如，使用Kabat和Chothia编号的CDR区域的不同定义范围。
Figure PCTCN2021140450-appb-000001
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
CDR也可以基于与参考CDR序列具有相同的Kabat编号位置而确定。除非另有说明，否 则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest,5 th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。
然而，应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia,AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
术语“细胞毒性剂”用在本发明中指抑制或防止细胞功能和/或引起细胞死亡或破坏的物质。
术语“化疗剂”包括在治疗癌症中有用的化学化合物。
术语“小分子药物”是指低分子量的能够调节生物过程的有机化合物。“小分子”被定义为分子量小于10kD、通常小于2kD和优选小于1kD的分子。小分子包括但不限于无机分子、有机分子、含无机组分的有机分子、含放射性原子的分子、合成分子、肽模拟物和抗体模拟物。作为治疗剂，小分子可以比大分子更能透过细胞、对降解更不易感和更不易于引发免疫应答。
术语“功能性Fc区”指这样的Fc区，其拥有天然序列Fc区的“效应器功能”。例示性的“效应器功能”包括C1q结合；CDC；Fc受体结合；ADCC；吞噬作用；细胞表面受体(例如B细胞受体；BCR)下调等。此类效应器功能一般要求Fc区与结合结构域(例如抗体可变域)联合，而且可以使用多种测定法来评估，例如本文所公开的那些。
本文所述的术语“治疗剂”涵盖在预防或治疗肿瘤(例如癌症)中有效的任何物质，包括化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂、小分子药物或免疫调节剂。
本文使用的术语“免疫调节剂”指抑制或调节免疫应答的天然或合成活性剂或者药物。免疫应答可以是体液应答或细胞应答。
术语“有效量”指本发明的抗体或其片段或缀合物或组合物这样的量或剂量，其以单一或 多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。针对治疗或预防的目的，可以将“有效量”区分为“治疗有效量”和“预防有效量”。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素而容易地确定：诸如哺乳动物的物种、大小、年龄和一般健康、涉及的具体疾病、疾病的程度或严重性、个体患者的应答、施用的具体抗体、施用模式、施用制剂的生物利用率特征、选择的给药方案、和任何伴随疗法的使用。
在一个实施方式中，相比较于对照，有效量的本发明双特异性抗体优选地抑制可度量参数(例如肿瘤生长率，肿瘤体积等)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％或90％。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括最初原代转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
如本文所用，术语“多特异性”抗体指具有至少两个不同抗原结合位点的抗体，所述至少两个不同抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。多特异性抗体是对至少两个不同抗原表位具有结合特异性的抗体。在一个实施方案中，本文提供了这样的双特异性抗体，其具有针对第一抗原或靶标(BCMA)和第二抗原靶标(CD3)的结合特异性。鉴于根据本发明的抗体构建体是(至少)双特异性的，其不是天然存在的并且其与天然存在的产物明显不同。因此，“双特异性”抗体或免疫球蛋白是具有至少两个具有不同特异性的不同结合侧的人工杂交抗体或免疫球蛋白。
术语“靶标”表示BCMA或CD3。术语“第一靶标和第二靶标”表示BCMA作为第一个靶标和CD3作为第二个靶标或反之亦然。
术语“细胞因子”是由一种细胞群释放，作为细胞间介质作用于另一细胞的蛋白质的通称。此类细胞因子的例子有淋巴因子、单核因子、白介素(IL)，诸如IL-1，IL-1α，IL-2，IL-3，IL-4，IL-5，IL-6，IL-7，IL-8，IL-9，IL-11，IL-12，IL-15；肿瘤坏死因子，诸如TNF-α或TNF-β；及其它多肽因子，包括LIF和kit配体(KL)和γ-干扰素。如本文中使用的，术语细胞因子包括来自天然来源或来自重组细胞培养物的蛋白质及天然序列细胞因子的生物学活性等效物，包括通过人工合成产生的小分子实体，及其药剂学可接受的衍生物和盐。
术语“免疫缀合物”是与一个或多个其它物质(包括但不限于细胞毒性剂或标记)缀合的抗体。
术语“个体”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如， 小鼠和大鼠)。在一些实施方案中，个体或受试者是人。
术语“分离的”抗体是这样的抗体，其已经与其天然环境的组分分离。在一些实施方案中，将抗体纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。对于用于评估抗体纯度的方法的综述，参见，例如，Flatman等，J.Chromatogr.B848：79-87(2007)。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其它家族成员序列或相关序列。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、载体或稳定剂等。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
术语“组合疗法”或“联合疗法”是指施用两种或更多种治疗剂以治疗如本公开所述的癌 症或感染。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如以具有固定比例的活性成分的单一胶囊。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如片剂、胶囊、粉末和液体)中的共同施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。此外，这种施用还包括以大致相同的时间或在不同的时间以顺序的方式使用每种类型的治疗剂。在任一情况下，治疗方案将提供药物组合在治疗本文所述的病症或病状中的有益作用。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其可操作相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
II.抗体
如本文所用的术语“BCMA、靶BCMA、人BCMA”指人B细胞成熟靶标，也称为BCMA，TR17_人,TNFRSF17(UniProt Q02223)。BCMA的胞外结构域根据UniProt由氨基酸1-54(或5-51)组成。本文所述及的“针对BCMA的抗体”、“抗BCMA抗体”指特异性结合BCMA的抗体。在一些实施方案中，如例如通过表面等离子体共振(SPR)所测量，抗BCMA抗体与不相关的非BCMA蛋白的结合程度比抗体与BCMA的结合低约10倍、优选>100倍。在一个实施方案中，结合至BCMA的抗体具有10 -7M或更低、优选10 -8M至10 -13M、优选10 -9M至10 -13M的解离常数(Kd)。在一个实施方案中，所述抗BCMA抗体结合至BCMA的表位，所述表位在来自不同物种的BCMA中、优选在人和食蟹猴中是保守的。
在一个实施方案中，本文述及的“抗BCMA抗体”包含重链可变区和轻链可变区，重链可变区包含来自SEQ ID NO:1，3或5的CDR，轻链可变区包含来自SEQ ID NO:2，4或6的CDR。
T细胞或T淋巴细胞是在细胞介导的免疫中发挥核心作用的一类淋巴细胞。T细胞应答的特异性由TCR对抗原(在主要组织相容性复合体MHC的环境中展示)的识别来介导。作为TCR的一部分，CD3受体复合物是一种蛋白质复合物，包含CD3γ(gamma)链、CD3δ(delta)链和存在于细胞表面的两条CD3ε(epsilon)链，参与激活细胞毒性T细胞(CD8+幼稚T细胞)和T辅助细胞(CD4+幼稚T细胞)。CD3在T细胞上诸如通过固定化抗CD3抗体的簇集，导致T细胞活化，这类似于T细胞受体的接合，但不依赖于其克隆特有的特异性。
发现CD3与所有成熟T细胞的膜结合，这种高特异性，再加上在T细胞发育各个阶段都存在CD3，使其成为组织切片中T细胞有用的免疫组织化学标记。设想根据本发明的抗体构建体通常并且有利地显示更少的非特异性T细胞活化，其在特异性免疫疗法中是不需要的。这意味着副作用的风险降低。
本文所述及的“抗CD3抗体”指结合CD3的抗体。在一个实施方案中，本文述及“抗CD3抗体"包含重链可变区和轻链可变区，重链可变区包含来自SEQ ID NO:7或9的CDR，轻链可变区包含来自SEQ ID NO:8或10的CDR。
本文述及的“抗BCMA和CD3的双特异性抗体”、“特异性结合BCMA和CD3的双特异性抗体”、“抗BCMA×CD3双特异性抗体”、“BCMA/CD3双抗”等术语指能够以足够亲和力结合靶标BCMA和CD3的双特异性抗体，所述双特异性抗体能够募集T细胞，对靶细胞进行重定向溶解。接合的T细胞能够连续靶细胞溶解，并且不受干扰肽抗原加工和呈递或克隆T细胞分化免疫逃逸机制的影响。在一个实施方案中，本文述及的“抗BCMA和CD3的双特异性抗体”包含靶向BCMA的重链可变区和轻链可变区，重链可变区包含来自SEQ ID NO:1，3或5的CDR，轻链可变区包含来自SEQ ID NO:2，4或6的CDR，以及靶向CD3的重链可变区和轻链可变区，重链可变区包含来自SEQ ID NO:7或9的CDR，轻链可变区包含来自SEQ ID NO:8或10的CDR。所述抗体可以用作靶向表达BCMA的癌症的诊断剂和/或治疗剂。
本发明提供的抗BCMA×CD3双特异性抗体具有如下优点：
在一些实施方案中，本发明的抗BCMA×CD3双特异性抗体，通过将不同的抗BCMA抗体与具有不同亲和力的抗CD3抗体组装形成，通过在抗BCMA×CD3双特异性抗体诱导的PBMC对肿瘤细胞的杀伤实验中引入对T细胞激活程度和多种细胞因子释放水平的检测，有利于早期(体外筛选阶段)对BCMA/CD3双抗的药效和安全性进行综合性评估。筛选体外筛选阶段的最大杀伤类似而细胞因子释放水平较低的差异化的分子进行体内实验和毒理实验，有助于降低此类双抗临床应用过程中细胞因子风暴的风险。
在一些实施方案中，本发明的BCMA/CD3双抗同时结合多发性骨髓瘤细胞表面的BCMA和原代T细胞表面的CD3，介导T细胞对BCMA阳性肿瘤细胞的杀伤。在一些实施方案中，本发明的双特异性抗体能够剂量依赖性地诱导PBMCs对具有不同BCMA表达水平的多发性骨髓瘤细胞的杀伤作用。
在一些实施方案中，本发明的抗BCMA×CD3抗体介导人多发性骨髓瘤细胞的杀伤，并剂量依赖性地激活从PBMC分离的CD8+T细胞和CD4+T细胞。在一些实施方案中，本发明的抗体促进人CD8+T、CD4+T细胞的增殖能力。
在一些实施方案中，本发明的抗体能有效地抑制肿瘤的生长，相比较于对照，肿瘤抑制率可以达到62％，甚至108％。
在本发明的一个实施方案中，本文涵盖具有氨基酸改变的抗BCMA×CD3双特异性抗体，其中所述的氨基酸改变包括氨基酸的置换、插入或缺失。优选的，本文所述的氨基酸改变为氨基酸置换，优选地保守置换。
在优选的实施方案中，本发明所述的氨基酸改变发生在CDR外的区域(例如在FR中)。更优选地，本发明所述的氨基酸改变发生在重链可变区外和/或轻链可变区外的区域。
在一些实施方案中，置换为保守性置换。保守置换是指一个氨基酸经相同类别内的另一氨基酸置换，例如一个酸性氨基酸经另一酸性氨基酸置换，一个碱性氨基酸经另一碱性氨基酸置换，或一个中性氨基酸经另一中性氨基酸置换。示例性的置换如下表所示：
原始残基 示例性置换 优选的保守氨基酸置换
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) Val、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
在某些实施方案中，置换发生在抗体的CDR区。通常，获得的变体相对于亲本抗体在某些生物学特性方面(例如，增加的亲和力)具有修饰(例如，改善)和/或将具有亲本抗体的基本上保留的某些生物学特性。示例性置换变体是亲和力成熟抗体。
在某些实施方案中，本文中所提供的抗体经改变以增加或降低抗体经糖基化的程度。对抗体的糖基化位点的添加或缺失可通过改变氨基酸序列以便产生或移除一或多个糖基化位点而方便地实现。当抗体包含Fc区时，可以改变附着于其的糖类。在一些应用中，除去不想要的糖基化位点的修饰可以是有用的，例如除去岩藻糖模块以提高抗体依赖性细胞介导的细胞毒性细胞毒性(ADCC)功能(参见Shield等(2002)JBC277:26733)。在其它应用中，可以进行半乳糖苷化修饰以修饰补体依赖性细胞毒性(CDC)。
在某些实施方案中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包括在一或多个氨基酸位置处包含氨基酸修饰(例如置换)的人Fc区序列(例如人IgGl、IgG2、IgG3或IgG4Fc区)。关于Fc变体的实例参见美国专利号7,332,581，美国专利号6,737,056，美国专利号6,737,056；WO 2004/056312和Shields等人，J.Biol.Chem.9(2):6591-6604(2001)，美国专利号6,194,551、WO99/51642和Idusogie等人J.Immunol.164:4178-4184(2000)，美国专利号7,371,826，Duncan&Winter,Nature 322:738-40(1988)；美国专利号5,648,260；美国专利号5,624,821；和WO 94/29351。
在某些实施方案中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。可以如，例如美国专利号7,521,541中所述地生成半胱氨酸改造的抗体。
在某些实施方案中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其它非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1,3-二烷、聚-1,3,6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
III.本发明的核酸以及包含其的载体和宿主细胞
在一方面，本发明提供了编码以上任何抗BCMA、抗CD3和抗BCMA×CD3抗体或其抗原结合片段的核酸。本发明还涵盖与上述核酸在严格性条件下杂交的核酸，与上述核酸相比具有一个或多个置换(例如保守性置换)、缺失或插入的核酸，或与上述核酸相比具有至少80％，至少85％，至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同一性的核酸序列。
在另一方面，本发明提供了包含上述核酸的载体。在一个优选的实施方案中，所述载体是表达载体。本领域技术人员完全可以理解，在本发明所属的技术领域中通常采用的载体可以应用于本发明。
在一个实施方案中，本发明提供了包含所述核酸或所述载体的宿主细胞。用于克隆或表达编码抗体的载体的适当宿主细胞包括本文描述的原核或真核细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。
IV.药物组合物
在一些实施方案中，本发明提供包含本文所述的任何抗BCMA×CD3抗体或其抗原结合片段的组合物，优选地组合物为药物组合物。在一个实施方案中，所述组合物还包含药用辅料。在一个实施方案中，组合物(例如，药物组合物)包含本发明的抗BCMA×CD3抗体或其抗原结合片段，以及一种或多种其它治疗剂(例如化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂、小分子药物或免疫调节剂)的组合。
本发明的药物组合物还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地影响彼此活性的那些活性成分。例如，理想的是还提供其它抗癌活性成分，例如化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂、小分子药物或免疫调节剂等。所述活性成分以对于目的应用有效的量合适地组合存在。
实施例
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围，根据本申请说明书的描述，本领域技术人员可以进行多种修改。
除非明确指明相反，否则本发明的实施将采用本领域技术内的常规化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学和细胞生物学的方法。这些方法的描述可以参见，例如，Sambrook等人，Molecular Cloning:A Laboratory Manual(第3版，2001)；Sambrook等人，Molecular Cloning:A Laboratory Manual(第2版,1989)；Maniatis等人，Molecular Cloning:A Laboratory Manual(1982)；Ausubel等人，Current Protocols in Molecular Biology(John Wiley和Sons,2008年7月更新)；Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates和Wiley-Interscience；Glover,DNA Cloning:A Practical Approach,vol.I&II(IRL Press,Oxford,1985)；Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992)；Transcription and Translation(B.Hames&S.Higgins,Eds.,1984)；Perbal,A Practical Guide to  Molecular Cloning(1984)；Harlow和Lane,Antibodies,(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1998)Current Protocols in Immunology Q.E.Coligan,A.M.Kruisbeek,D.H.Margulies,E.M.Shevach和W.Strober,eds.,1991)；Annual Review of Immunology；以及期刊专著如Advances in Immunology。
本发明抗体的分子结构设计
以三个由Adimab筛选并进行亲和力成熟的抗BCMA亲本抗体(ADI-38447，ADI-38456和ADI-38476)和两个不同亲和力的抗CD3亲本抗体(Adimab CD3bispecific platform提供序列)为基础，设计同时靶向BCMA和CD3的双特异性抗体。
设计两种不同格式的抗BCMA和抗CD3双特异性抗体分子：1+1格式和2+1格式，其中数字分别表示结合抗原BCMA和CD3的抗原识别位点数。例如1+1格式表示双特异抗体具有一个结合抗原BCMA的抗原识别位点和一个结合CD3的抗原识别位点。
1+1格式双特异性抗体的结构示意图如图1A所示，其由左右非对称的三条多肽链组成，其中左半部分由肽链1和肽链2组成，右半部分由肽链3组成。其中肽链1、肽链2和肽链3的结构如图1B所示，肽链1从N端到C端依次包含重链可变结构域、免疫球蛋白CH1结构域和Fc结构域，肽链2从N端到C端依次包含轻链可变结构域、免疫球蛋白CL结构域，肽链3从N端到C端依次包含由抗体重链可变区与轻链可变区通过人工合成的接头连接而成的单链抗体(single chain Fv，scFv)和Fc结构域。肽链1的重链可变结构域和肽链2的轻链可变结构域配对形成BCMA的抗原识别位点，肽链3的scFv形成CD3的抗原识别位点。肽链1和肽链3各自的Fc结构域相互作用，形成Fc区。在一个具体的实施方案中，肽链1和肽链3各自的Fc结构域中含有稳定相互作用的突变，例如含有“杵入臼”突变。
2+1格式双特异性抗体的结构如图2A所示，其由左右非对称的四条多肽链组成，其中左半部分由肽链1和肽链2组成，右半部分由肽链2和肽链3组成。其中肽链1、肽链2和肽链3的结构如图2B所示，肽链1从N端到C端依次包含重链可变结构域、免疫球蛋白CH1结构域和Fc结构域，肽链2从N端到C端依次包含轻链可变结构域、免疫球蛋白CL结构域，肽链3从N端到C端依次包含重链可变结构域、Fc结构域和scFv结构域。肽链1的重链可变结枃域和肽链2的轻链可变结构域配对形成BCMA的第一个抗原识别位点，肽链3的重链可变结枃域和肽链2的轻链可变结构域配对形成BCMA的第二个抗原识别位点，肽链3的scFv形成CD3的抗原识别位点。肽链1和肽链3各自的Fc结构域相互作用，形成Fc区。在一个具体的实施方案中，肽链1和肽链3各自的Fc结构域中含有稳定相互作用的突变，例如含有“杵入臼”突变。
1+1格式双特异性抗体的6个示例抗体是ADI-46757、ADI-46758、ADI-46759、ADI-46760、 ADI-46761、ADI-46762。2+1格式双特异性抗体的6个示例抗体是ADI-46780、ADI-46781、ADI-46782、ADI-46783、ADI-46784、ADI-46785。示例抗体利用Fc的“杵入臼”技术解决该非对称IgG样双特异性抗体的重链错配问题。示例抗体的Fc区为IgG1的重链恒定区，其中包括减弱效应子功能的L234A，L235A(根据Kabat的“EU”编号)氨基酸突变。
实施例1.本发明抗体的表达及纯化
三个抗BCMA亲本抗体(ADI-38447，ADI-38456和ADI-38476)和两个不同亲和力的抗CD3亲本抗体(Anti-CD3med，Anti-CD3low)的CDR区、轻链可变区和重链可变区的氨基酸序列如表1所示。
以1+1格式和2+1格式组建的本发明12个示例性双特异性抗体(ADI-46757，ADI-46758，ADI-46759，ADI-46760，ADI-46761，ADI-46762，ADI-46780，ADI-46781，ADI-46782，ADI-46783，ADI-46784，ADI-46785)的CDR区、肽链1、肽链2和肽链3的氨基酸序列如表1和表2所示，其中下划线标出了各个亲本抗体根据Kabat方案定义的CDR区。
表1.本申请涉及的亲本抗体、示例性双特异性抗体的氨基酸序列
Figure PCTCN2021140450-appb-000002
Figure PCTCN2021140450-appb-000003
Figure PCTCN2021140450-appb-000004
Figure PCTCN2021140450-appb-000005
Figure PCTCN2021140450-appb-000006
Figure PCTCN2021140450-appb-000007
表2
Figure PCTCN2021140450-appb-000008
Figure PCTCN2021140450-appb-000009
Figure PCTCN2021140450-appb-000010
Figure PCTCN2021140450-appb-000011
Figure PCTCN2021140450-appb-000012
委托苏州泓迅生物科技有限公司合成分别编码各个双特异抗体的肽链1、肽链2或肽链3的核苷酸序列，并将其分别插入载体pcDNA3.1中获得相应的重组质粒。所述重组质粒用于在宿主细胞中进行相应克隆和/或表达。
1.各个相应双特异性抗体在HEK293细胞中的表达和纯化
将Expi293F细胞(购自Thermo Fisher scientific公司)传代培养于Expi293F细胞培养基(购自Thermo Fisher scientific公司)中。转染前一天检测细胞密度，并用新鲜的Expi293细胞培养基将细胞密度调整为2×10 6个细胞/ml后继续培养，转染当天将细胞密度调整为3×10 6个细胞/ml。
取转染Expi293F细胞终体积1/10的Opti-MEM培养基(购自Gibco公司)作为转染缓冲液，每毫升转染缓冲液中加入10μg的1:1:1摩尔比率的上述制备的重组质粒(各个重组质粒分别包含编码相应双特异性抗体的肽链1、肽链2或肽链3的核苷酸序列)，混匀，每毫升转染缓冲液中再加入30ug聚乙烯亚胺(polyethylenimine，PEI)(Polysciences)，混匀，室温温育20分钟，然后将PEI/DNA混合物轻柔倒入Expi293F细胞悬浮液中混匀，置于摇床培养，培养条件为8％CO 2、36.5℃、120rpm。
培养16～18小时后，向培养瓶中补加转染后培养物体积1/50的浓度为200g/L的FEED(100g/L Phytone Peptone+100g/L Difco Select Phytone)、终浓度为4g/L的葡萄糖溶液和终浓度为2mM/L的VPA(Gibco)，轻轻混匀，置于8％CO 2、36.5℃、120rpm摇床继续培养。连续培养6天，收集培养物，以4000转/分钟离心30分钟，取细胞上清用0.45μM滤膜过滤，通过 亲和层析和离子交换层析进行纯化。
亲和层析纯化：选用Hitrap MabSelect SuRe(获自GE Healthcare)亲和层析柱，纯化前用0.1M NaOH对亲和层析柱及管路除内毒素2h，然后，用蒸馏水清洗管路以及层析柱。用5倍柱体积的1×PBS(Gibco)平衡层析柱；将收集的培养物上清液加载到层析柱，再用10倍柱体积的1×PBS清洗层析柱，去除非特异性结合蛋白；用5倍柱体积的洗脱缓冲液(100mM sodium citrate，pH 3.5)冲洗层析柱，收集洗脱液，然后用2M Tris调节收集的洗脱液的pH至6.0，以用于下一步的离子交换层析。
离子交换层析纯化：选用Mono S 5/50 GL(获自GE Healthcare)离子交换层析柱，并置于AKTApure系统(获自GE healthcare)内。用0.5M NaOH对装备有Mono S 5/50 GL离子交换层析柱的AKTApure系统除内毒素2小时，然后，用蒸馏水清洗系统以及层析柱。使用5-10倍柱体积的上样缓冲液(20mM柠檬酸+柠檬酸钠,pH 5.0)平衡层析柱，直至电导以及pH稳定；将亲和层析所获得的蛋白质溶液用上样缓冲液稀释10倍，然后上样；使用5倍柱体积的上样缓冲液再平衡；以洗脱缓冲液(20mM柠檬酸+柠檬酸钠,1M NaCl,pH 5.0)的含量为0-40％的梯度进行线性洗脱，共洗脱30个柱体积，根据紫外吸收值来收集样品。
利用大小排阻层析(size exclusion chromatography；SEC)检测收集的各级分管中样品的纯度。根据SEC结果将纯度大于95％的级分管中的样品合并。
将纯化后的双特异性抗体溶液使用15ml超滤离心管，4500转/分钟离心30分钟，使用PBS将蛋白稀释后继续离心，4500转/分钟离心30分钟，重复该操作几次，以更换缓冲液。将更换缓冲液后的抗体合并，测抗体浓度。进一步利用毛细管电泳(CE-SDS)和液相色谱-质谱联用技术(LC-MS)相结合对双特异性抗体的成分和含量进行定性和定量。
实施例2.本发明抗体亲和力测定
采用表面等离子共振法(Surface plasmon resonance)(Biacore T200)测定本发明抗BCMA×CD3的示例性双特异抗体与人BCMA、食蟹猴BCMA的结合动力学。
偶联人BCMA-Fc抗原(R&D),食蟹猴BCMA-Fc抗原(R&D)。将50mM N-羟基琥珀酰亚胺(NHS)与200mM 1-乙基-3-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)新鲜混匀，以10μL/分钟的流速活化芯片120s。然后将人BCMA-Fc抗原,食蟹猴BCMA-Fc抗原分别稀释于10mM Acetate(pH 5.0)中，稀释终浓度为5μg/mL，分别偶联在芯片的2,4通道，偶联高度约50RU。
然后以10μL/分钟的流速注入1M乙醇胺120s，对剩余的活化位点进行封闭。实验所用缓冲液为pH 7.4的HBS-EP+溶液，采用高性能(high performance)模式，将梯度稀释后的抗 体，从低浓度到高浓度以30μl/分钟的流速注入，每个循环测定一个浓度，依次注入芯片1,2,3,4通道，结合时间180s，解离时间600s。
在如以上测定法所述进行的实验中，ADI-46757、ADI-46758、ADI-46759、ADI-46760、ADI-46761和人BCMA的亲和力如表3所示。ADI-46757、ADI-46758、ADI-46759、ADI-46760、ADI-46761和食蟹猴BCMA的亲和力如表4所示。
表3.示例抗体与人BCMA的亲和力检测
Figure PCTCN2021140450-appb-000013
由以上图表结果可见，本发明上述示例抗体均显示与人BCMA极高的亲和力。
表4.示例抗体与食蟹猴BCMA的亲和力检测
Figure PCTCN2021140450-appb-000014
采用表面等离子共振法(Biacore T200)测定本发明抗BCMA×CD3的示例抗体与人CD3、食蟹猴CD3的结合动力学。
偶联链霉亲和素蛋白(streptavidin protein，SA)，固化生物素标记的CD3抗原。将50mM N-羟基琥珀酰亚胺(NHS)与200mM 1-乙基-3-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)新鲜混匀，以10μL/分钟的流速活化芯片420s。然后将链霉亲和素蛋白稀释于10mM Acetate(pH 5.0)中，稀释浓度为50μg/mL，分别偶联在芯片的1，2，3，4通道，偶联高度约3000RU。
然后以10μL/分钟的流速注入1M乙醇胺420s，对剩余的活化位点进行封闭。偶联结束后，将生物素标记的人CD3D&E异源二聚体抗原(R&D)和食蟹猴CD3D&E异源二聚体抗原(R&D)分别以0.05ug/ml的浓度偶联在芯片2通道和4通道，偶联高度约30RU。
在如以上测定法所述进行的实验中，ADI-46757、ADI-46758、ADI-46759、ADI-46760、ADI-46761和人CD3D&E的亲和力如表5所示。ADI-46757、ADI-46758、ADI-46759、ADI-46760、ADI-46761和食蟹猴CD3D&E的亲和力如表6所示。
表5.示例抗体与人CD3D&E的亲和力检测
Figure PCTCN2021140450-appb-000015
表6.示例抗体与猴CD3D&E的亲和力检测
Figure PCTCN2021140450-appb-000016
实施例3.本发明抗BCMA×CD3双特异性抗体与人多发性骨髓瘤细胞的结合实验
通过流式细胞技术检测BCMA×CD3双特异性抗体与BCMA阳性的多发性骨髓瘤(MM)细胞的结合能力。
将具有不同BCMA表达水平的NCl-H929细胞(南京科佰生物，货号CBP60243)、L363细胞(南京科佰生物，货号CBP60240)、RPMI8226细胞(美国ATCC，货号CCL-155)按照常规操作培养传代。细胞离心重悬后计数，将细胞密度调整至4×10 5个细胞/ml倒于加样槽中,以每孔50ul接种于96孔板中。每孔50ul细胞中加入50ul梯度稀释的抗体样品，然后放入37℃5％CO 2的细胞培养箱中孵育60分钟。之后400g离心5分钟除去液体，加入200ul PBS，再400g 5分钟离心除去液体,重复3次。加入山羊抗人IgG PE(1:200)室温避光孵育30分钟，200ul PBS洗涤2次；重悬于60ul PBS，流式细胞仪读数。拟合曲线，计算EC50值。
在如以上测定法所述进行的实验中，本发明示例抗体与BCMA高表达细胞系NCl-H929细胞表面的BCMA结合见图3；示例抗体与BCMA中等表达细胞系L363细胞表面的BCMA结合见图4；示例抗体与BCMA低表达细胞系RPMI8226细胞表面的BCMA结合见图5。
实施例4.本发明抗BCMA×CD3双特异性抗体对人多发性骨髓瘤细胞的杀伤实验
BCMA×CD3双特异性抗体同时与多发性骨髓瘤细胞表面的BCMA和原代T细胞表面的CD3结合，通过BCMA依赖性的CD3交联，激活T细胞，介导T细胞对BCMA阳性肿瘤细胞的杀伤。本研究利用乳酸脱氢酶(LDH)法检测外周血单核细胞(PBMC)与BCMA阳性的多发性骨髓瘤细胞共培养条件下，加入示例抗体24小时后收集的上清中死细胞释放的LDH的水平，从而评估T细胞对BCMA肿瘤细胞的杀伤能力。
从液氮罐中取出PBMC细胞于37℃快速融化，滴加入预热的1640培养基(含0.1％DNA酶)中，得混合液10ml。400g离心5分钟，用10ml的1640培养基重悬，并加入10μl DNA酶，37℃、5％CO 2、贴壁培养过夜。吸取悬浮T细胞400g离心5分钟，用1640培养基重悬，计数，并将细胞密度调整为4×10 6个细胞/ml，作为效应细胞。BCMA阳性多发性骨髓瘤细胞作为靶细胞，400g离心5分钟，用1640培养基重悬，计数，并将细胞密度调整为2×10 5个细胞/ml，作为靶细胞。将靶细胞加到96孔板中，每孔100μl；将梯度稀释好的抗体加到96孔板中，每孔50μl；再将效应细胞PBMC加到96孔板中，每孔50μl。最终效靶比为10:1。将上述96孔板置于37℃、5％CO 2培养箱中培养24小时后取上清，按照LDH检测试剂盒(CytoTox96 non-radioacitive cytotoxicity kit，Promega)说明书检测死细胞释放到上清中的LDH的含量，然后计算示例抗体对多发性骨髓瘤细胞的杀伤比例。
在如以上测定法所述进行的实验中，示例抗体能够剂量依赖性地诱导不同供者来源的PBMC对L363细胞的杀伤作用(见图6A，6B)。
在如以上测定法所述进行的实验中，比较了示例抗体在诱导同一供者来源的PBMC对BCMA表达水平不同的多发性骨髓瘤细胞系的杀伤作用(见图7和图8)：其中，示例抗体诱导PBMC对NCl-H929细胞表面的杀伤作用见图7A和8A，示例抗体诱导PBMC对L363细胞的杀伤作用见7B和8B，示例抗体诱导PBMC对RPMI8226细胞的杀伤作用见7C和8C，示例抗体诱导PBMC对BCMA阴性的NUGC4细胞(JCRB细胞库，JCRB834)的杀伤作用见7D和8D。可见，针对不同来源的PBMC，示例抗体都能够剂量依赖性地诱导同一供者PBMC对具有不同BCMA表达水平的多发性骨髓瘤细胞的杀伤作用。
实施例5.本发明抗BCMA×CD3抗体对人多发性骨髓瘤细胞的杀伤过程中伴随的T细胞激活和细胞因子释放水平
本实施例在多发性骨髓瘤细胞和PBMC共孵育体系中，通过LDH法检测多发性骨髓瘤细胞死亡百分比，利用多因子检测试剂盒(Human Th1/Th2/Th17,BD)同时检测多种细胞因子的水平；同时通过流式细胞技术检测T细胞中CD25和CD69阳性细胞的百分比，研究了本发明 示例抗体的相应功能。
如实施例4所述检测示例抗体对靶细胞的杀伤性。
将24小时后收取的如实施例4所述的上清液，按照检测试剂盒(Human Th1/Th2/Th17kit，BD)说明书检测细胞因子IL-2，TNFα，IFNγ，IL-6的含量。
其中，示例抗体诱导PBMC对NCl-H929细胞表面的杀伤作用见图8A，杀伤过程中伴随的多种细胞因子释放水平见图9；示例抗体诱导PBMC对L363细胞的杀伤作用见8B，杀伤过程中伴随的多种细胞因子释放水平见图10；示例抗体诱导PBMC对RPMI8226细胞的杀伤作用见8C，杀伤过程中伴随的多种细胞因子释放水平见图11；示例抗体诱导PBMC对BCMA阴性的NUGC4细胞的杀伤作用见8D。
在如以上测定法所述进行的实验中，示例抗体诱导的PBMC对NCl-H929细胞的杀伤过程伴随的CD8+及CD4+T细胞的激活程度分别见图12A和12B；示例抗体诱导的PBMC对L363细胞的杀伤过程伴随的CD8+及CD4+T细胞的激活程度分别见图12C，12D；示例抗体诱导的PBMC对RPMI822626细胞的杀伤过程伴随的CD8+及CD4+T细胞的激活程度分别见图12E，12F。在有BCMA阳性的多发性骨髓瘤细胞存在时，示例抗体均可剂量依赖性地激活从PBMC分离的CD8+T细胞核CD4+T细胞，并且T细胞的激活程度与CD3的亲和力存在一定的相关性：抗CD3亲和力越高则对T细胞激活能力越强。
实施例7.示例抗体促进人CD8+T细胞的增殖能力
利用CellTracker TM Deep Red标记PBMC分离的CD8+T细胞，然后将标记好的CD8+T细胞和L363细胞共培养，加入浓度梯度稀释的示例抗体，96小时后利用流式细胞计数检测CD8+T细胞的增殖情况。
实验步骤
(1)PBMC复苏:从液氮罐中取出PBMC于37℃快速融化，将细胞缓慢加入37℃(含0.1％DNA酶)的10ml的AIM V培养基(Gibco TM)中，300g，8分钟，25℃离心，去上清后，用37℃(含0.1％DNA酶)的10ml的AIM V培养基重悬于T75培养瓶中于37℃5％CO 2培养箱，静置3小时。
(2)纯化CD8+T细胞：用10ml移液管轻轻吹吸悬浮细胞，25℃，300g离心8分钟。去除上清后，使用1ml Robosep缓冲液重悬细胞，并加入100ul负性筛选抗体混合液以及100ul来自人AB血型的AB血清。混匀后，置于4℃冰箱孵育20分钟，每5分钟摇晃一次。向混合细胞悬液加入10ml Robosep缓冲液，300g 25℃离心8分钟以去除未吸附的抗体。重复该清洗步骤以完全去除未吸附的抗体。清洗细胞的同时，预洗负性筛选磁性微球(取出装有负性筛选微球的小瓶，涡旋振摇30秒后取1ml加入15ml离心管。加入7ml Robosep缓冲液后， 将离心管置于磁力架上1分钟。倒掉上清液后，使用新的1ml Robosep缓冲液重悬微球)。使用1ml新的Robosep缓冲液重悬细胞，加入已预洗好的磁性微球。将上述混合液置于选装摇床，10rpm室温旋转孵育30分钟。孵育完成后，加入新的6ml Robosep缓冲液，置于磁力架上1分钟，取出上清液并置于新15ml离心管中。重复该清洗步骤，以完全去除附着有非目的细胞的微球。上清300g、25℃离心8分钟后，使用预热的CTS TM AIM V TM SFM培养基(Gibco TM)重悬。
(3)将纯化的CD8+T细胞，400g离心5分钟，去除上清，使用CTS TM AIM V TM SFM培养基重悬细胞，细胞计数，调整细胞密度至1×10 6个细胞/ml，按照5ml细胞悬液加入1ul CellTracker TM Deep Red染液，于37℃着色30分钟，400g离心5分钟，去除上清，使用CTS TM AIM V TM SFM重悬细胞，细胞计数，调整细胞密度至1×10 6个细胞/ml。
(4)NUGC4细胞去除细胞培养基，2ml 0.25％Trypsin-EDTA 5ml室温消化5分钟，随即加入细胞培养基中和胰酶，400g离心5分钟，去除上清，使用CTS TM AIM V TM SFM培养基重悬细胞，细胞计数，调整细胞密度至1×10 5个细胞/ml。
(5)L363细胞，400g离心5分钟，去除上清，使用CTS TM AIM V TM SFM培养基重悬细胞，细胞计数，调整细胞密度至1×10 5个细胞/ml。
(6)96孔圆底培养板(有盖)第一竖排每孔加入150ul CD8+T细胞悬液和150ul L363细胞悬液，第二至第10竖排每孔加入100ul CD8+T细胞悬液和100ul L363细胞悬液，在第一竖排每孔加入待测抗体终浓度为200nM，取100ul混合液至第二孔，3倍稀释至第十孔，取第二块96孔圆底培养板(有盖)稀释至第十四孔，取第二块96孔加入200nM的IgG1作为对照孔，并进行相应梯度稀释，将板于37℃、5％CO 2培养箱放置96小时。
(7)取96孔圆底培养板(有盖)第一竖排每孔加入150ul CD8+T细胞悬液和150ul NUGC4细胞悬液，第二至第10竖排每孔加入100ul CD8+T细胞悬液和100ul NUGC4细胞悬液，在第一竖排每孔加入待测抗体终浓度200nM，取100ul混合液至第二孔，3倍稀释至第十孔。G12孔加入200nM的IgG1抗体作为对照孔，并进行相应梯度稀释，将板于37℃、5％CO 2培养箱放置96小时。
(8)400g离心5分钟，去除上清，每孔加入100ul PBS(含2ul抗人CD8-PE和抗-CD3(OKT3)-PerCp-Cy5.5)重悬细胞，4℃静置30分钟，400g离心5分钟，去除上清，每孔100ul PBS重悬细胞，流式细胞仪读数。
实验结果
在如以上测定法所述进行的实验中，示例抗体以在BCMA阳性的L363细胞存在时，可以在体外有效刺激CD8+T细胞增殖(见图13A)；而在BCMA阴性的NUGC4细胞存在时，示例抗体无BCMA非依赖性的非特异性的CD8+T细胞的增殖(见图13B)。
实施例8.示例抗体促进人CD4+T细胞的增殖能力
示例抗体在BCMA阳性的L363细胞存在时，在体外可以剂量依赖性地刺激CD4+T细胞增殖(图14)。而在BCMA阴性的NUGC4细胞存在时，示例抗体无BCMA非依赖性的非特异性的CD4+T细胞的增殖。
实验步骤
第(1)-(5)步骤采用与实施例7中公开的步骤相似的步骤进行，区别在于将CD8+T细胞替换为CD4+T细胞。
(6)取96孔圆底培养板(有盖)第一竖排每孔加入150ul CD4+T细胞悬液和150ul L363细胞悬液，第二至第11竖排每孔加入100ul CD4+T细胞悬液和100ul L363细胞悬液，在第一竖排每孔加入不同的待测抗体终浓度为200nM，取100ul混合液至第二孔，3倍稀释至第十二孔，IgG1孔作为对照孔，并进行相应梯度稀释，将板放入二氧化碳培养箱，于37℃刺激72小时。
(7)取96孔圆底培养板(有盖)第一竖排每孔加入150ul CD4+T细胞悬液和150ul NUGC4细胞悬液，第二至第11竖排每孔加入100ul CD4+T细胞悬液和100ul NUGC4细胞悬液，在第一竖排每孔加入不同的待测抗体终浓度为200nM，取100ul混合液至第二孔，3倍稀释至第九孔，IgG1孔作为对照孔，并进行相应梯度稀释，将板放入二氧化碳培养箱，于37℃刺激72小时。
(8)400g离心5分钟，去除上清，每孔100ul PBS(含2ul FITC抗人CD4和抗-CD3(OKT3)-PerCp-Cy5.5)重悬细胞，4℃静置30分钟,400g离心5分钟，去除上清，每孔100ul PBS重悬细胞，流式细胞仪读数。
实验结果
在如以上测定法所述进行的实验中，示例抗体在BCMA阳性的L363细胞存在时，在体外可以剂量依赖性地刺激CD4+T细胞增殖(见图14A)；而在BCMA阴性的NUGC4细胞存在时无BCMA非依赖性的非特异性的CD4+T细胞的增殖(见14B)。
实施例9.本发明抗BCMA×CD3双特异性抗体动物体内药效试验
本研究采用人多发性骨髓瘤细胞H929细胞接种NOG小鼠的PBMC模型，表达人BCMA的Daudi细胞(BCMA-Daudi)接种NOG小鼠两个模型测定示例抗体的抗肿瘤作用。
9.1示例抗体在NCl-H929荷瘤人源化小鼠模型体内的抗肿瘤药效
实验步骤
雌性NOG小鼠(35-41天)购自北京维通达实验动物技术有限公司。等级为SPF级。小 鼠在到达后驯化检疫7天，随后开始研究。
将NCl-H929细胞进行常规传代培养用于后续体内实验，离心收集细胞，以PBS分散NCl-H929细胞。将70只NOG小鼠右侧背腹部剃毛接种NCl-H929细胞，5x10 6个/只，接种体积200ul/只。NCl-H929细胞接种后第五天进行小鼠静脉注射PBMC细胞，5x10 6个/只，接种体积200ul/只。
给药：PBMC细胞接种后第3天，根据小鼠瘤体积进行分组(每组7只)给药。每7天给药一次,总共给药3次。以无关hIgG抗体作为对照。
每周2次监测小鼠瘤体积与体重。接种后第26天计算相对肿瘤抑制率(TGI％)，计算公式如下：
TGI％＝100％×(hIgG对照组肿瘤体积–治疗组肿瘤体积)/(hIgG对照组肿瘤体积–hIgG对照组初始肿瘤体积)，对照组初始肿瘤体～80mm 3。
肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W2/2。
采用电子天平测定体重。
在整个研究期间，当肿瘤达到端点时或当小鼠具有>20％体重减轻时，使小鼠安乐死。统计肿瘤大小，计算肿瘤抑制率(TGI％)。
实验结果
肿瘤生长曲线见图15，示例抗体可以显著抑制HCl-H929细胞的生长，在第22天统计肿瘤大小，并计算出肿瘤抑制率。在0.5mg/kg剂量组，与hIgG对比，示例抗体46757和46758的肿瘤抑制率分别为80.8％和95.9％。在1.0mg/kg剂量组，与hIgG对比，示例抗体ADI-46757和ADI-46758的肿瘤抑制率分别为84.9％和85.9％。同时在给药的小鼠组中，均没有发现体重减轻的现象。
9.2示例抗体在过表达BCMA的Daudi(Daudi-BCMA)荷瘤人源化小鼠模型体内的抗肿瘤药效
实验步骤
雌性NOG小鼠(9-16g)购自北京维通达实验动物技术有限公司。等级为SPF级，小鼠在到达后驯化检疫7天，随后开始研究。
将Daudi-BCMA细胞进行常规传代培养用于后续体内实验。用PBS与基质胶(Matrigel)以1:1的比例分散Daudi-BCMA细胞，制备成细胞浓度为25×10 6个细胞/mL的细胞悬液。NOG小鼠右侧背部剃毛，皮下注射25×10 6个细胞/mL Daudi-BCMA细胞悬液，0.2mL/只，即接种量为5×10 6个细胞/只小鼠。
肿瘤细胞接种5天后，用0.1％DNA酶预热过的RPMI-1640培养基复苏PBMC细胞，然 后用PBS分散PBMC细胞，制备成细胞浓度为25×10 6个细胞/mL的细胞悬液。小鼠脉注射PBMC细胞悬液，0.2mL/只，即接种量为5×10 6个细胞/只小鼠。
肿瘤细胞接种8天后，根据小鼠瘤体积进行分组(每组7只)给药，每7天一次，连续给药2次。给药方式为腹腔注射，给药体积为10ml/kg/次。每周2次监测小鼠瘤体积与体重，监测至29天结束。以无关hIgG抗体作为对照。
接种后第29天计算相对肿瘤抑制率(TGI％)，计算公式如下：
TGI％＝100％×(hIgG对照组肿瘤体积–治疗组肿瘤体积)/(hIgG对照组肿瘤体积–hIgG对照组给药前肿瘤体积)。
肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W2/2。
采用电子天平测定体重。
实验结果
肿瘤生长曲线见图16，示例抗体可以显著抑制BCMA过表达Daudi细胞的生长，在第29天统计肿瘤大小，并计算出肿瘤抑制率。在1.0mg/kg剂量组，与hIgG对比，示例抗体46757和46758的肿瘤抑制率分别为62％和108％。同时在给药的小鼠组中，均没有发现体重减轻的现象。

Claims (18)

  1. 一种特异性结合BCMA和CD3的抗体或其抗原结合片段，其包含肽链1、肽链2和肽链3，其中:
    1)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR，肽链3包含scFv，其包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
    2)肽链1包含来自SEQ ID NO:3所包含的3个重链CDR，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR，肽链3包含scFv，其包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
    3)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR，肽链3包含scFv，其包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
    4)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR，肽链3包含scFv，其包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR；
    5)肽链1包含来自SEQ ID NO:3包含的3个重链CDR，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR，肽链3包含scFv，其包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR；或
    6)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR，肽链3包含scFv，其包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR；
    或者所述抗体或其抗原结合片段包含4条链，其中包含1条肽链1，1条肽链3和2条肽链2，其中：
    7)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:1所包含的3个重链CDR并且C末端包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
    8)肽链1包含来自SEQ ID NO:3所包含的3个重链CDR，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:3所包含的3个重链CDR并且C末端包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
    9)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:5所包含的3个重链CDR并且C末端包含来自SEQ ID NO:7所包含的3个重链CDR和来自SEQ ID NO:8所包含的3个轻链CDR；
    10)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:1所包含的3个重链CDR并且C末端包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR；
    11)肽链1包含来自SEQ ID NO:3包含的3个重链CDR，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:3所包含的3个重链CDR并且C末端包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR；或
    12)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR，肽链3的N末端包含来自SEQ ID NO:5所包含的3个重链CDR并且C末端包含来自SEQ ID NO:9所包含的3个重链CDR和来自SEQ ID NO:10所包含的3个轻链CDR。
  2. 权利要求1的抗体或其抗原结合片段，其中
    1)肽链1包含SEQ ID NO:47-49所示的3个重链CDR，肽链2包含SEQ ID NO:50-52所示的3个轻链CDR，肽链3包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
    2)肽链1包含SEQ ID NO:53-55所示的3个重链CDR，肽链2包含SEQ ID NO:56-58所示的3个轻链CDR，肽链3包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
    3)肽链1包含SEQ ID NO:59-61所示的3个重链CDR，肽链2包含SEQ ID NO:62-64所示的3个轻链CDR，肽链3包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
    4)肽链1包含SEQ ID NO:47-49所示的3个重链CDR，肽链2包含SEQ ID NO:50-52所示的3个轻链CDR，肽链3包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76所示的3个轻链CDR；
    5)肽链1包含SEQ ID NO:53-55所示的3个重链CDR，肽链2包含SEQ ID NO:56-58所示的3个轻链CDR，肽链3包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76 所示的3个轻链CDR；或
    6)肽链1包含SEQ ID NO:59-61所示的3个重链CDR，肽链2包含SEQ ID NO:62-64所示的3个轻链CDR，肽链3包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76所示的3个轻链CDR，
    或者所述抗体或其抗原结合片段包含4条链，其中包含1条肽链1，1条肽链3和2条肽链2，其中：
    7)肽链1包含SEQ ID NO:47-49所示的3个重链CDR，肽链2包含SEQ ID NO:50-52所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:47-49所示的3个重链CDR并且C末端包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
    8)肽链1包含SEQ ID NO:53-55所示的3个重链CDR，肽链2包含SEQ ID NO:56-58所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:53-55所示的3个重链CDR并且C末端包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
    9)肽链1包含SEQ ID NO:59-61所示的3个重链CDR，肽链2包含SEQ ID NO:62-64所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:59-61所示的3个重链CDR并且C末端包含SEQ ID NO:65-67所示的3个重链CDR和SEQ ID NO:68-70所示的3个轻链CDR；
    10)肽链1包含SEQ ID NO:47-49所示的3个重链CDR，肽链2包含SEQ ID NO:50-52所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:47-49所示的3个重链CDR并且C末端包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76所示的3个轻链CDR；
    11)肽链1包含SEQ ID NO:53-55所示的3个重链CDR，肽链2包含SEQ ID NO:56-58所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:53-55所示的3个重链CDR并且C末端包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76所示的3个轻链CDR；或
    12)肽链1包含SEQ ID NO:59-61所示的3个重链CDR，肽链2包含SEQ ID NO:62-64所示的3个轻链CDR，肽链3的N末端包含SEQ ID NO:59-61所示的3个重链CDR并且C末端包含SEQ ID NO:71-73所示的3个重链CDR和SEQ ID NO:74-76所示的3个轻链CDR。。
  3. 权利要求1或2所述的抗体或其抗原结合片段，其中
    1)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR且与SEQ ID NO:1具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR且与SEQ ID NO:2具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:7所包含的3个重链CDR且与SEQ ID NO:7具有至少90％同一性的VH和来自SEQ ID NO:8所包含的3个轻链CDR且与SEQ ID NO:8具有至少90％同一性的VL；
    2)肽链1包含来自SEQ ID NO:3所包含的3个重链CDR且与SEQ ID NO:3具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR且与SEQ ID NO:4具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:7所包含的3个重链CDR且与SEQ ID NO:7具有至少90％同一性的VH和来自SEQ ID NO:8所包含的3个轻链CDR且与SEQ ID NO:8具有至少90％同一性的VL；
    3)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR且与SEQ ID NO:5具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR且与SEQ ID NO:6具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:7所包含的3个重链CDR且与SEQ ID NO:7具有至少90％同一性的VH和来自SEQ ID NO:8所包含的3个轻链CDR且与SEQ ID NO:8具有至少90％同一性的VL；
    4)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR且与SEQ ID NO:1具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR且与SEQ ID NO:2具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:9所包含的3个重链CDR且与SEQ ID NO:9具有至少90％同一性的VH和来自SEQ ID NO:10所包含的3个轻链CDR且与SEQ ID NO:10具有至少90％同一性的VL；
    5)肽链1包含来自SEQ ID NO:3所包含的3个重链CDR且与SEQ ID NO:3具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR且与SEQ ID NO:4具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:9所包含的3个重链CDR且与SEQ ID NO:9具有至少90％同一性的VH和来自SEQ ID NO:10所包含的3个轻链CDR且与SEQ ID NO:10具有至少90％同一性的VL；或
    6)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR且与SEQ ID NO:5具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR且与SEQ ID NO:6具有至少90％同一性的VL，肽链3包含来自SEQ ID NO:9所包含的3个重链CDR且与SEQ ID NO:9具有至少90％同一性的VH和来自SEQ ID NO:10所包含的3个轻链CDR且与SEQ ID NO:10具有至少90％同一性的VL，
    或者所述抗体或其抗原结合片段包含4条链，其中包含1条肽链1，1条肽链3和2条肽链2，其中：
    7)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR且与SEQ ID NO:1具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR且与SEQ ID NO:2具有至少90％同一性的VL，肽链3的N末端包含来自SEQ ID NO:1所包含的3个重链CDR且与SEQ ID NO:1具有至少90％同一性的VH并且C末端包含来自SEQ ID NO:7所包含的3个重链CDR且与SEQ ID NO:7具有至少90％同一性的VH和来自SEQ ID NO:8所包含的3个 轻链CDR且与SEQ ID NO:8具有至少90％同一性的VL；
    8)肽链1包含来自SEQ ID NO:3所包含的3个重链CDR且与SEQ ID NO:3具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR且与SEQ ID NO:4具有至少90％同一性的VL，肽链3的N末端包含来自SEQ ID NO:3所包含的3个重链CDR且与SEQ ID NO:3具有至少90％同一性的VH并且C末端包含来自SEQ ID NO:7所包含的3个重链CDR且与SEQ ID NO:7具有至少90％同一性的VH和来自SEQ ID NO:8所包含的3个轻链CDR且与SEQ ID NO:8具有至少90％同一性的VL；
    9)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR且与SEQ ID NO:5具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR且与SEQ ID NO:6具有至少90％同一性的VL，肽链3的N末端包含来自SEQ ID NO:5所包含的3个重链CDR且与SEQ ID NO:5具有至少90％同一性的VH并且C末端包含来自SEQ ID NO:7所包含的3个重链CDR且与SEQ ID NO:7具有至少90％同一性的VH和来自SEQ ID NO:8所包含的3个轻链CDR且与SEQ ID NO:8具有至少90％同一性的VL；
    10)肽链1包含来自SEQ ID NO:1所包含的3个重链CDR且与SEQ ID NO:1具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:2所包含的3个轻链CDR且与SEQ ID NO:2具有至少90％同一性的VL，肽链3的N末端包含来自SEQ ID NO:1所包含的3个重链CDR且与SEQ ID NO:1具有至少90％同一性的VH并且C末端包含来自SEQ ID NO:9所包含的3个重链CDR且与SEQ ID NO:9具有至少90％同一性的VH和来自SEQ ID NO:10所包含的3个轻链CDR且与SEQ ID NO:10具有至少90％同一性的VL；
    11)肽链1包含来自SEQ ID NO:3所包含的3个重链CDR且与SEQ ID NO:3具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:4所包含的3个轻链CDR且与SEQ ID NO:4具有至少90％同一性的VL，肽链3的N末端包含来自SEQ ID NO:3所包含的3个重链CDR且与SEQ ID NO:3具有至少90％同一性的VH并且C末端包含来自SEQ ID NO:9所包含的3个重链CDR且与SEQ ID NO:9具有至少90％同一性的VH和来自SEQ ID NO:10所包含的3个轻链CDR且与SEQ ID NO:10具有至少90％同一性的VL；或
    12)肽链1包含来自SEQ ID NO:5所包含的3个重链CDR且与SEQ ID NO:5具有至少90％同一性的VH，肽链2包含来自SEQ ID NO:6所包含的3个轻链CDR且与SEQ ID NO:6具有至少90％同一性的VL，肽链3的N末端包含来自SEQ ID NO:5所包含的3个重链CDR且与SEQ ID NO:5具有至少90％同一性的VH并且C末端包含来自SEQ ID NO:9所包含的3个重链CDR且与SEQ ID NO:9具有至少90％同一性的VH和来自SEQ ID NO:10所包含的3个轻链CDR且与SEQ ID NO:10具有至少90％同一性的VL。
  4. 权利要求1-3中任一项所述的抗体或其抗原结合片段，其包含：
    1)肽链1包含SEQ ID NO:1所示VH，肽链2包含SEQ ID NO:2所示VL，肽链3包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
    2)肽链1包含SEQ ID NO:3所示VH，肽链2包含SEQ ID NO:4所示VL，肽链3包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
    3)肽链1包含SEQ ID NO:5所示VH，肽链2包含SEQ ID NO:6所示VL，肽链3包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
    4)肽链1包含SEQ ID NO:1所示VH，肽链2包含SEQ ID NO:2所示VL，肽链3包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL；
    5)肽链1包含SEQ ID NO:3所示VH，肽链2包含SEQ ID NO:4所示VL，肽链3包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL；或
    6)肽链1包含SEQ ID NO:5所示VH，肽链2包含SEQ ID NO:6所示VL，肽链3包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL，
    或者所述抗体或其抗原结合片段包含4条链，其中包含1条肽链1，1条肽链3和2条肽链2，其中：
    7)肽链1包含SEQ ID NO:1所示VH，肽链2包含SEQ ID NO:2所示VL，肽链3的N末端包含SEQ ID NO:1所示VH并且C末端包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
    8)肽链1包含SEQ ID NO:3所示VH，肽链2包含SEQ ID NO:4所示VL，肽链3的N末端包含SEQ ID NO:3所示VH并且C末端包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
    9)肽链1包含SEQ ID NO:5所示VH，肽链2包含SEQ ID NO:6所示VL，肽链3的N末端包含SEQ ID NO:5所示VH并且C末端包含SEQ ID NO:7所示VH和SEQ ID NO:8所示VL；
    10)肽链1包含SEQ ID NO:1所示VH，肽链2包含SEQ ID NO:2所示VL，肽链3的N末端包含SEQ ID NO:1所示VH并且C末端包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL；
    11)肽链1包含SEQ ID NO:3所示VH，肽链2包含SEQ ID NO:4所示VL，肽链3的N末端包含SEQ ID NO:3所示VH并且C末端包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL；或
    12)肽链1包含SEQ ID NO:5所示VH，肽链2包含SEQ ID NO:6所示VL，肽链3的N末端包含SEQ ID NO:5所示VH并且C末端包含SEQ ID NO:9所示VH和SEQ ID NO:10所示VL。
  5. 权利要求1-4中任一项所述的抗体或其抗原结合片段，其中肽链1和肽链3还包含Fc区，优选地，所述Fc区与重链可变区同源。
  6. 权利要求5所述的抗体或其抗原结合片段，其中肽链3中的scFv区直接或者通过接头与Fc区连接。
  7. 权利要求6所述的抗体或其抗原结合片段，其中所述接头包含(G4S)n的序列，其中n＝1,2,3,4,5,6,7,8，优选地，n＝1,2,3,4。
  8. 权利要求5-7中任一项所述的抗体或其抗原结合片段，其中所述Fc区包含稳定双Fc片段的突变，优选地，所述突变是杵臼突变。
  9. 权利要求1-8中任一项所述的抗体或其抗原结合片段，其包含肽链1、肽链2和肽链3，其中：
    1)肽链1包含与SEQ ID NO:11具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:11组成；肽链2包含与SEQ ID NO:12具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:12组成；肽链3包含与SEQ ID NO:13具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:13组成；
    2)肽链1包含与SEQ ID NO:14具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:14组成；肽链2包含与SEQ ID NO:15具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:15组成；肽链3包含与SEQ ID NO:16具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:16组成；
    3)肽链1包含与SEQ ID NO:17具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:17组成；肽链2包含与SEQ ID NO:18具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:18组成；肽链3包含与SEQ ID NO:19具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:19组成；
    4)肽链1包含与SEQ ID NO:20具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:20组成；肽链2包含与SEQ ID NO:21具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:21组成；肽链3包含与SEQ ID NO:22具有至少90％、 91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:22组成；
    5)肽链1包含与SEQ ID NO:23具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:23组成；肽链2包含与SEQ ID NO:24具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:24组成；肽链3包含与SEQ ID NO:25具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:25组成；或
    6)肽链1包含与SEQ ID NO:26具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:26组成；肽链2包含与SEQ ID NO:27具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:27组成；肽链3包含与SEQ ID NO:28具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:28组成，
    或者所述双特异性抗体包含4条链，其中包含1条肽链1，1条肽链3和2条肽链2，其中：
    7)肽链1包含与SEQ ID NO:29具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:29组成；肽链2包含与SEQ ID NO:30具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:30组成；肽链3包含与SEQ ID NO:31具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:31组成；
    8)肽链1包含与SEQ ID NO:32具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:32组成；肽链2包含与SEQ ID NO:33具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:33组成；肽链3包含与SEQ ID NO:34具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:34组成；
    9)肽链1包含与SEQ ID NO:35具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:35组成；肽链2包含与SEQ ID NO:36具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:36组成；肽链3包含与SEQ ID NO:37具有至少90％、 91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:37组成；
    10)肽链1包含与SEQ ID NO:38具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:38组成；肽链2包含与SEQ ID NO:39具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:39组成；肽链3包含与SEQ ID NO:40具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:40组成；
    11)肽链1包含与SEQ ID NO:41具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:41组成；肽链2包含与SEQ ID NO:42具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:42组成；肽链3包含与SEQ ID NO:43具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:43组成；或
    12)肽链1包含与SEQ ID NO:44具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:44组成；肽链2包含与SEQ ID NO:45具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:45组成；肽链3包含与SEQ ID NO:46具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，或者由SEQ ID NO:46组成。
  10. 编码如权利要求1-9中任一项所述抗体或其抗原结合片段的多核苷酸。
  11. 包含权利要求10所述多核苷酸的载体，优选是表达载体。
  12. 包含权利要求10所述多核苷酸或者权利要求11所述载体的宿主细胞。
  13. 包含如权利要求1-9中任一项所述抗体或其抗原结合片段的药物组合物。
  14. 如权利要求1-9中所述抗体或其抗原结合片段或者如权利要求13中所述药物组合物在制备用于治疗与BCMA活性相关的疾病中的用途。
  15. 权利要求14所述的用途，其中所述疾病是癌症，优选是多发性骨髓瘤。
  16. 一种治疗与BCMA活性相关的疾病的方法，包括将治疗有效量的如权利要求1-9所述的抗体或其抗原结合片段，或如权利要求13所述的药物组合物施用给有需要的患者。
  17. 如权利要求1-9中所述抗体或其抗原结合片段或者如权利要求13中所述药物组合物在制备药物中的用途，所述药物与一种或者多种疗法联用以治疗与BCMA活性相关的疾病。
  18. 权利要求17所述的用途，其中所述一种或者多种疗法为治疗方式和/或其它治疗剂；优选地，所述治疗方式包括手术治疗和/或放射疗法；优选地,所述其它治疗剂选自化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂、小分子药物或免疫调节剂。
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