WO2022135468A1 - Anticorps bispécifique anti-bcma×cd3 et son utilisation - Google Patents

Anticorps bispécifique anti-bcma×cd3 et son utilisation Download PDF

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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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seq
peptide
peptide chain
chain cdrs
chain
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PCT/CN2021/140450
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Chinese (zh)
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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/fr

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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

L'invention concerne un nouvel anticorps bispécifique qui se lie spécifiquement à BCMA et à CD3, un acide nucléique codant l'anticorps bispécifique anti-BCMA×CD3, un vecteur contenant l'acide nucléique, une cellule hôte contenant l'acide nucléique ou le vecteur, et une composition pharmaceutique contenant l'anticorps ou un fragment de liaison à l'antigène associé. De plus, l'invention concerne en outre l'utilisation de l'anticorps bispécifique anti-BCMA×CD3 et de la composition pharmaceutique en immunothérapie, et pour la prévention et/ou le diagnostic de maladies.
PCT/CN2021/140450 2020-12-23 2021-12-22 Anticorps bispécifique anti-bcma×cd3 et son utilisation WO2022135468A1 (fr)

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Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140248A1 (fr) * 2013-03-15 2014-09-18 Amgen Research (Munich) Gmbh Molécules liantes pour bcma et cd3
CN104114578A (zh) * 2011-11-15 2014-10-22 安进研发（慕尼黑）股份有限公司 Bcma和cd3的结合分子
CN104968682A (zh) * 2013-02-05 2015-10-07 英格玛布股份公司 针对CD3ε和BCMA的双特异性抗体
WO2016020332A1 (fr) * 2014-08-04 2016-02-11 Engmab Ag Anticorps bispécifiques anti cd3epsilon et bcma
WO2016079177A1 (fr) * 2014-11-20 2016-05-26 Engmab Ag Anticorps bispécifiques anti-cd3epsilon et bcma
WO2018083204A1 (fr) * 2016-11-02 2018-05-11 Engmab Sàrl Anticorps bispécifique contre bcma et cd3 et médicament immunologique pour une utilisation combinée dans le traitement du myélome multiple
CN108350076A (zh) * 2015-08-17 2018-07-31 詹森药业有限公司 抗-bcma抗体，结合bcma和cd3的双特异性抗原结合分子及其用途
CN108659112A (zh) * 2017-03-30 2018-10-16 上海市同济医院 一种非对称双特异性抗体
WO2019149269A1 (fr) * 2018-02-01 2019-08-08 信达生物制药(苏州)有限公司 Fragment variable à chaîne unique anti-antigène de maturation des lymphocytes b (bcma) entièrement humain, et application associée
WO2019149250A1 (fr) * 2018-02-01 2019-08-08 南京驯鹿医疗技术有限公司 Récepteur antigénique chimérique (car) se liant à bcma et ses applications
CN110229232A (zh) * 2019-06-19 2019-09-13 北京智仁美博生物科技有限公司 双特异性抗体及其用途
WO2019237081A1 (fr) * 2018-06-07 2019-12-12 Cullinan Management, Inc. Protéines de liaison multi-spécifiques et procédés d'utilisation associés

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104114578A (zh) * 2011-11-15 2014-10-22 安进研发（慕尼黑）股份有限公司 Bcma和cd3的结合分子
CN104968682A (zh) * 2013-02-05 2015-10-07 英格玛布股份公司 针对CD3ε和BCMA的双特异性抗体
WO2014140248A1 (fr) * 2013-03-15 2014-09-18 Amgen Research (Munich) Gmbh Molécules liantes pour bcma et cd3
WO2016020332A1 (fr) * 2014-08-04 2016-02-11 Engmab Ag Anticorps bispécifiques anti cd3epsilon et bcma
WO2016079177A1 (fr) * 2014-11-20 2016-05-26 Engmab Ag Anticorps bispécifiques anti-cd3epsilon et bcma
CN108350076A (zh) * 2015-08-17 2018-07-31 詹森药业有限公司 抗-bcma抗体，结合bcma和cd3的双特异性抗原结合分子及其用途
WO2018083204A1 (fr) * 2016-11-02 2018-05-11 Engmab Sàrl Anticorps bispécifique contre bcma et cd3 et médicament immunologique pour une utilisation combinée dans le traitement du myélome multiple
CN108659112A (zh) * 2017-03-30 2018-10-16 上海市同济医院 一种非对称双特异性抗体
WO2019149269A1 (fr) * 2018-02-01 2019-08-08 信达生物制药(苏州)有限公司 Fragment variable à chaîne unique anti-antigène de maturation des lymphocytes b (bcma) entièrement humain, et application associée
WO2019149250A1 (fr) * 2018-02-01 2019-08-08 南京驯鹿医疗技术有限公司 Récepteur antigénique chimérique (car) se liant à bcma et ses applications
WO2019237081A1 (fr) * 2018-06-07 2019-12-12 Cullinan Management, Inc. Protéines de liaison multi-spécifiques et procédés d'utilisation associés
CN110229232A (zh) * 2019-06-19 2019-09-13 北京智仁美博生物科技有限公司 双特异性抗体及其用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FRERICHS, K. A. ET AL.: "Preclinical Activity of JNJ-7957, a Novel BCMA×CD3 Bispecific Antibody for the Treatment of Multiple Myeloma, Is Potentiated by Daratumumab", CLINICAL CANCER RESEARCH, vol. 26, no. 9, 1 May 2020 (2020-05-01), pages 2203 - 2215, XP055826554, DOI: 10.1158/1078-0432.CCR-19-2299 *
S HIPP, Y-T TAI, D BLANSET, P DEEGEN, J WAHL, O THOMAS, B RATTEL, P J ADAM, K C ANDERSON, M FRIEDRICH: "A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo", LEUKEMIA, vol. 31, no. 8, 1 August 2017 (2017-08-01), London, pages 1743 - 1751, XP055547607, ISSN: 0887-6924, DOI: 10.1038/leu.2016.388 *

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