WO2022143870A1 - 一种特异性结合bcma的抗体及其应用 - Google Patents

一种特异性结合bcma的抗体及其应用 Download PDF

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WO2022143870A1
WO2022143870A1 PCT/CN2021/142886 CN2021142886W WO2022143870A1 WO 2022143870 A1 WO2022143870 A1 WO 2022143870A1 CN 2021142886 W CN2021142886 W CN 2021142886W WO 2022143870 A1 WO2022143870 A1 WO 2022143870A1
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amino acid
acid sequence
identity
antibody
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PCT/CN2021/142886
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French (fr)
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鲁薪安
何霆
齐菲菲
丁艳萍
刘光华
胡雪莲
许文平
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北京艺妙神州医药科技有限公司
北京艺妙医疗科技有限公司
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Priority to AU2021413526A priority Critical patent/AU2021413526A1/en
Priority to JP2023540655A priority patent/JP2024501888A/ja
Priority to EP21914601.6A priority patent/EP4273168A1/en
Publication of WO2022143870A1 publication Critical patent/WO2022143870A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present invention relates to an antibody that specifically binds to BCMA and a chimeric antigen receptor constructed based on the antibody, and discloses the amino acid sequence of the antibody, a cloning or expression vector, a host cell, a method for producing the antibody, and the antibody or chimeric antigen Use of receptors.
  • MM Multiple myeloma
  • MM is a malignant tumor characterized by massive proliferation of clonal plasma cells.
  • treatment options for multiple myeloma have advanced significantly in recent years, multiple myeloma remains a disease with high morbidity and mortality. Therefore, new therapeutic methods are urgently needed for the treatment of multiple myeloma.
  • Chimeric Antigen Receptor-T cell Chimeric Antigen Receptor-T cell
  • CAR-T Chimeric Antigen Receptor-T cell
  • scFv Single-chain antibody variable fragment
  • the scFv of CAR-T cells are generally derived from monoclonal antibodies, which can bypass the major histocompatibility complex (MHC) restriction to recognize intact cell surface proteins.
  • MHC major histocompatibility complex
  • BCMA B Cell Maturation Antigen
  • BCMA is mainly expressed by plasma cells and some mature B cells, but not expressed in most B cells and other tissues (B-cell Maturation Antigen Is a Promising Target for Adoptive T-cell Therapy of Multiple Myeloma, clinical cancer research,15 Apr 2013.). Therefore, BCMA can be used as a target antigen for CAR-T cell therapy in MM.
  • CAR-T cell therapy has achieved good therapeutic effects in the treatment of multiple myeloma.
  • most of the CAR-T cells currently used in clinical trials are mouse-derived single-chain antibody scFv.
  • CD19 chimeric antigen receptors with humanized scFv trigger leukemia cell killing, Cellular Immunology, 14 Mar 2016.; Chimeric Antigen Receptor T-cell Therapies for Multiple Myeloma, blood, 14 Dec 2017.).
  • scFv single-chain variable fragment
  • the affinity of scFv to target antigen is also an important factor to determine whether CAR-T can effectively kill target cells. Therefore, screening out scFvs that can specifically and effectively recognize BCMA is crucial for the construction of a safe and effective CAR-T for the treatment of MM.
  • the present invention discloses an isolated antibody that specifically binds to BCMA, comprising: a heavy chain variable domain (hereinafter abbreviated as VH) and a light chain variable domain (hereinafter abbreviated as VL), wherein
  • the VH includes: VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (GYTFX 1 X 2 YSMN), VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2 (RINTX 3 SG X 4 P X 5 YADDFKG) and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3 (SNDY X 6 YSLD X 7 ); wherein X 1 is selected from S, T, R; X 2 is selected from R, S, H; X 3 is selected from G, E , R; X 4 is selected from A, T, V; X 5 is selected from I, N; X 6 is selected from N, L; X 7 is selected from H, Y, F;
  • the VL includes: VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (RAS X 8 SV X 9 X 10 X 11 G X 12 X 13 X 14 X 15 Y), VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 5 (LASNVQT) VL-CDR2 of amino acid sequence and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6 (X 16 QSR X 17 IPRT); wherein X 8 is selected from R, E; X 9 is selected from T, S; X 10 is selected from I, V; X 11 is selected from A, L; X 12 is selected from S, N; X 13 is selected from P, H; X 14 is selected from I, L; X 15 is selected from I, V; X 16 is selected from L, F; X 17 is selected from T, S.
  • VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4
  • VL-CDR1
  • the VH of the antibody comprises: VH-CDR1 as set forth in the amino acid sequence of SEQ ID NO:1, VH-CDR2 as set forth in the amino acid sequence of SEQ ID NO:2, and VH-CDR2 as set forth in the amino acid sequence of SEQ ID NO:2 : amino acid sequence VH-CDR3 shown in 3;
  • the VL of the antibody includes: VL-CDR1 as shown in the amino acid sequence of SEQ ID NO:4, VL-CDR2 as shown in the amino acid sequence of SEQ ID NO:5 and as shown in the amino acid sequence of SEQ ID NO:6 VL-CDR3.
  • the present invention discloses an isolated antibody that specifically binds to BCMA, comprising VH and VL, said VH comprising VH-CDR1, VH-CDR2 and VH-CDR3, said VL comprising VL-CDR1, VL-CDR2 and VL -CDR3; wherein the VH-CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8 and 9; the VH-CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12 and 13
  • the VH-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15 and 16; the VL-CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 19
  • the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:20; the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22.
  • the VH-CDR1 is selected from the amino acid sequence of the group consisting of SEQ ID NOs: 7, 8 and 9; the VH-CDR2 is selected from the group consisting of SEQ ID NOs: 10, 11, 12 and 13 the amino acid sequence of the group consisting of; the VH-CDR3 is selected from the amino acid sequence of the group consisting of SEQ ID NO: 14, 15 and 16; the VL-CDR1 is selected from the group consisting of SEQ ID NO: 17, 18 and 19
  • the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:20; the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22.
  • the antibody :
  • VH comprising a VH-CDR1 as shown in SEQ ID NO:7, a VH-CDR2 as shown in SEQ ID NO:10, and a VH-CDR3 as shown in SEQ ID NO:14; and a VH comprising a VH-CDR3 as shown in SEQ ID NO:14 VL-CDR1 shown in: 17, VL-CDR2 shown in SEQ ID NO:20 and VL of VL-CDR3 shown in SEQ ID NO:21;
  • VH-CDR1 shown in SEQ ID NO:8 VH-CDR2 shown in SEQ ID NO:11 and VH-CDR3 shown in SEQ ID NO:15
  • VH comprising SEQ ID NO:15 VL-CDR1 shown in: 18, VL-CDR2 shown in SEQ ID NO:20 and VL of VL-CDR3 shown in SEQ ID NO:21;
  • VH-CDR1 as shown in SEQ ID NO:8, VH-CDR2 as shown in SEQ ID NO:12 and VH-CDR3 as shown in SEQ ID NO:14; and a VH comprising as SEQ ID NO:14 VL-CDR1 shown in: 17, VL-CDR2 shown in SEQ ID NO:20 and VL of VL-CDR3 shown in SEQ ID NO:21;
  • VH comprising the VH-CDR1 shown in SEQ ID NO:7, the VH-CDR2 shown in SEQ ID NO:10, and the VH-CDR3 shown in SEQ ID NO:14; and a VH comprising a VH-CDR3 shown in SEQ ID NO:14 VL-CDR1 shown in: 17, VL-CDR2 shown in SEQ ID NO:20 and VL of VL-CDR3 shown in SEQ ID NO:22;
  • VH comprising the VH-CDR1 shown in SEQ ID NO:9, the VH-CDR2 shown in SEQ ID NO:13, and the VH-CDR3 shown in SEQ ID NO:16; and a VH comprising a VH-CDR3 shown in SEQ ID NO:16 VL-CDR1 shown in SEQ ID NO:19, VL-CDR2 shown in SEQ ID NO:20 and VL of VL-CDR3 shown in SEQ ID NO:21.
  • the VH in the above-described antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32, or comprises An amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, further preferably at least 99% identity thereto.
  • the VL in the above-described antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 35, 36, 37, 38, 39, 40, 41, 42, and 43, or comprises An amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, further preferably at least 99% identity thereto.
  • the antibody :
  • the antibody described above comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 56, or an amino acid sequence that is at least 85%, preferably at least 90%, more preferably at least 95%, further preferably at least 99% identical to it; or
  • the antibody consists of or consists essentially of the above sequence.
  • the antibody of the present invention is a single-chain antibody scFv; its VH and VL are linked by a linker peptide chain rich in glycine and serine, and the sequences of VH and VL are interchangeable.
  • the antibodies of the invention are chimeric or humanized antibodies.
  • the present invention further discloses a chimeric antigen receptor targeting BCMA, which comprises: 1) the scFv of the present invention; 2) a hinge region; 3) a transmembrane region; 4) a costimulatory domain 5) CD3 ⁇ intracellular signal transduction domain.
  • the costimulatory domain is selected from CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD-2, CD7, LIGHT, NKG2C, B7-H3 or its any combination; preferably, the costimulatory domain is selected from 4-1BB, and the 4-1BB comprises the amino acid sequence as described in SEQ ID NO.57; the CD3 ⁇ intracellular signal transduction domain comprises as SEQ ID NO. The amino acid sequence described in NO.58.
  • the hinge region and and the transmembrane domain are selected from IgGl, IgG4, CD8 ⁇ , CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1 or CD34 hinge and transmembrane domains.
  • the present invention discloses an isolated nucleic acid molecule encoding an antibody or chimeric antigen receptor as described above.
  • the present invention also discloses a cloning or expression vector, which contains the aforementioned nucleic acid molecule.
  • the vector is selected from one or more of DNA, RNA, plasmid, lentiviral vector, adenoviral vector and retroviral vector.
  • the present invention also discloses a host cell, comprising the aforementioned nucleic acid molecule or the aforementioned vector.
  • the host cells are T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, macrophages, cytotoxic T cells, tumor-infiltrating T cells or regulatory T cells.
  • the present invention also discloses a composition or kit comprising the aforementioned antibody, the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, the aforementioned carrier, the aforementioned At least one of the host cells, and a pharmaceutically acceptable excipient, diluent or carrier.
  • the present invention also discloses the aforementioned antibody, the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned host cell, or the aforementioned Use of the composition in the preparation of a medicament for preventing or treating B cell-related tumor diseases, autoimmune diseases, and infectious diseases caused by viruses or bacteria.
  • the present invention discloses a method of treating a disease, wherein an effective amount of the aforementioned antibody, the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, such as The aforementioned vector, the aforementioned host cell, or the aforementioned composition, wherein the subject suffers from a B cell-related tumor disease, an autoimmune disease, an infectious disease caused by a virus or bacteria at least one of.
  • the B cell associated neoplastic disease is selected from the group consisting of acute leukemias such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastoid, promyelocytic, myelomonocytic type , monocytic and erythroleukemia; chronic leukemias such as chronic myeloid (myeloid) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia; polycythemia vera, lymphoma, mantle cell lymphoma, diffuse large B-cell Lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myeloid development at least one of the bad.
  • acute leukemias such as acute lymphoblastic leukemia, acute myeloid
  • Figure 1 shows a schematic diagram of a CAR molecule.
  • Figure 2 shows a schematic diagram of a plasmid vector.
  • the plasmid used for the preparation of lentivirus is pLenti plasmid
  • the upstream promoter of the target gene is EF-1 ⁇
  • the downstream of the promoter is the signal peptide, Linker region and CAR structural sequence.
  • Figure 3 shows the transduction efficiency of lentiviruses with CARs with scFvs specifically recognizing BCMA in T cells.
  • Figure 4 shows flow cytometry plots of BCMA expression on the surface of NCI H929 cells and K562 cells.
  • Figure 5 shows the killing effect of CAR-T containing scFv-01 (01-CAR-T) on target cells, where 01-CAR-T represents CAR-T cells containing scFv-01, and C-CAR-T is Positive control, T cell treatment group was negative control.
  • Figure 6 shows a graph showing the killing effect of CAR-T containing scFv-04 (04-CAR-T) on target cells.
  • Figure 7 shows the killing effect of CAR-T containing scFv-05 (05-CAR-T) on target cells.
  • Figure 8 shows a graph of the killing effect of CAR-T containing scFv-06 (06-CAR-T) on target cells.
  • Figure 9 shows a graph of the killing effect of CAR-T containing scFv-07 (07-CAR-T) on target cells.
  • Figure 10 shows a graph of the killing effect of CAR-T containing scFv-08 (08-CAR-T) on target cells.
  • Figure 11 shows a graph of the killing effect of CAR-T containing scFv-09 (09-CAR-T) on target cells.
  • Figure 12 shows a graph showing the killing effect of scFv-10-containing CAR-T (10-CAR-T) on target cells.
  • Figure 13 shows a graph showing the killing effect of CAR-T containing scFv-11 (11-CAR-T) on target cells.
  • Figure 14 shows a graph of the killing effect of CAR-T containing scFv-12 (12-CAR-T) on target cells.
  • Figure 15 shows a graph of the killing effect of CAR-T containing scFv-13 (13-CAR-T) on target cells.
  • Figure 16 shows a graph of the killing effect of CAR-T containing scFv-14 (14-CAR-T) on target cells.
  • Figure 17 shows a graph of the killing effect of CAR-T containing scFv-15 (15-CAR-T) on target cells.
  • Figure 18 shows a normalized graph of the killing effect of CAR-T in the present invention on target cells.
  • Figure 19 shows a graph of cytokine expression of scFv-01-containing CAR-T (01-CAR-T) upon target cell stimulation.
  • antigen refers to a molecule that elicits an immune response, which may involve antibody production, or activation of specific immunocompetent cells.
  • any macromolecule including all proteins or peptides, can be used as an antigen.
  • Antigens can be derived from recombinant or genomic DNA.
  • DNA that includes a nucleotide sequence or part of a nucleotide sequence encoding a protein that elicits an immune response, encoding the term "antigen" as used herein.
  • the antigen need not be encoded solely by the full-length nucleotide sequence of the gene.
  • the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in different combinations to elicit a desired immune response.
  • the antigen need not be encoded by a "gene” at all, the antigen may be generated, synthesized or derived from a biological sample.
  • biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.
  • antibody refers to an immunoglobulin molecule that specifically binds to an antigen.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources, and can be immunoreactive portions of intact immunoglobulins. Antibodies are usually tetramers of immunoglobulin molecules.
  • Antibodies of the present invention may exist in various forms, including polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant , hybrid, mutated, and grafted antibodies; antibody formats in the present invention also include full-length antibodies, antibody fragments, such as Fab, Fab', F(ab')2, Fv, scFv, di-scFv, tri-scFv, Fd and other antibody fragments that retain antigen binding function; also can be dimeric structure Diabody or trimeric structure Triabody.
  • the fragment should include an antigen-binding fragment, which typically includes both VL and VH, however, it does not necessarily have to include both.
  • Fd antibody fragments consist only of the VH and CH1 domains, but still retain some of the antigen-binding functions of the intact antibody.
  • antibody as an immunoglobulin or fragment or derivative thereof includes any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo.
  • the VH or VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), and interspersed more conserved regions called framework regions (FWRs).
  • CDRs complementarity determining regions
  • FWRs framework regions
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • VH-CDRs CDRs in heavy chains
  • VL-CDRs CDRs in light chains
  • the CDRs of the disclosed antibodies and antigen-binding fragments are defined or identified by Kabat numbering.
  • single-chain variable region fragment refers to an antibody formed by recombinant DNA techniques in which the immunoglobulin heavy and light chain variable regions are formed by amino acid peptides Segments (linkers) are connected.
  • Various methods of generating single chain antibodies are known, including in U.S. Patent No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883 ; those described in Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.
  • variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • CDRs in heavy chains are abbreviated as VH-CDRs, eg, VH-CDR1, VH-CDR2, VH-CDR3, and CDRs in light chains are abbreviated as VL-CDRs, eg, VL-CDR1, VL-CDR2, VL-CDR3.
  • the CDRs of the antibodies and antigen-binding fragments disclosed herein are defined or identified by Kabat numbering.
  • chimeric refers to an antibody or antibody polypeptide in which a portion of the heavy chain is derived from one species and the other portion of the heavy chain is derived from another species.
  • a chimeric antibody may comprise constant regions derived from humans and variable regions derived from non-human animals such as camelids.
  • the non-human animal is a mammal, such as a camelid, mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.
  • humanized refers to an antibody or antibody polypeptide comprising CDRs derived from non-human animals, FWR regions derived from humans, and, where applicable, constant regions derived from humans.
  • BCMA can be derived from a human, and an exemplary sequence of human BCMA includes the human BCMA protein (Genbank accession No.: BAB60895.1).
  • BCMA as used herein is intended to encompass any form of BCMA, eg, 1) native unprocessed BCMA molecules, "full length” BCMA chains or naturally occurring BCMA variants, including, eg, splice variants or the like allelic variants; 2) any form of BCMA produced after intracellular processing; or 3) full-length, fragments (eg, truncated forms, extracellular/transmembrane domains) of BCMA subunits produced by recombinant methods, or Modified forms thereof (eg mutant forms, glycosylation/pegylation, His-tag/immunofluorescence fusion forms).
  • targeting BCMA refers to a chimeric antigen receptor capable of specifically binding to BCMA (eg, human BCMA), for example, a BCMA-targeting chimeric antigen receptor refers to a chimeric antigen receptor capable of specifically binding BCMA; for example, targeting BCMA An scFv refers to a single-chain antibody that can specifically bind to BCMA (eg, human BCMA).
  • the term “specifically binds” or “specifically binds” refers to a non-random binding reaction between two molecules, eg, a binding reaction between an antibody and an antigen.
  • the antibody polypeptides provided by the present disclosure specifically bind human BCMA with binding affinity (K D ) ⁇ 10 -6 M (eg, ⁇ 5x10 -7 M, ⁇ 2x10 -7 M, ⁇ 10 -7 M, ⁇ 5x10 -8 M, ⁇ 2x10 -8 M, ⁇ 10 -8 M, ⁇ 5x10 -9 M, ⁇ 4x10 -9 M, ⁇ 3x10 -9 M, ⁇ 2x10 -9 M or ⁇ 10 -9 M) .
  • K D refers to the ratio of dissociation rate to association rate (K off /K on ), which can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance methods, microscale thermal Electrophoresis, high performance liquid chromatography-mass spectrometry and flow cytometry (eg FACS). In certain embodiments, KD values can be appropriately determined using a flow cytometer.
  • vector refers to a molecular vehicle for the transport, transduction and expression in a target cell of a contained exogenous gene of interest (eg, a polynucleotide according to the present invention) that provides a suitable The nucleotide sequence that initiates transcription, the promoter.
  • Vectors include isolated nucleic acids, and which can be used to deliver isolated nucleic acids to the interior of cells. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes autonomously replicating plasmids or viruses.
  • viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • an "expression vector” refers to a vector that includes a recombinant polynucleotide that includes expression control sequences operably linked to the nucleotide sequence to be expressed.
  • the expression vector includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art that incorporate recombinant polynucleotides, such as plasmids (eg, naked or contained in liposomes) and viruses (eg, Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus).
  • a "cloning vector” refers to a DNA molecule such as a plasmid, cosmid, or phage that is capable of autonomous replication in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites and marker genes where foreign DNA sequences are inserted in a defined manner without losing the essential biology of the vector function, the marker gene is suitable for the identification and selection of cells transformed with the cloning vector.
  • Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
  • the host cell can be any prokaryotic or eukaryotic cell that contains a cloning vector or expression vector, including those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene in the chromosome or genome of the host cell.
  • Suitable mammalian host cells include myeloma cells, such as SP2/0 cells and NSO cells, as well as Chinese hamster ovary (CHO) cells, hybridoma cell lines, and other mammalian host cells that can be used to express antibodies. Special transgenic animals with modified immune systems can also be used to make antibodies.
  • identity refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions in each sequence aligned for comparison purposes. When a position in the compared sequences is occupied by the same base, then the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. The identity between two sequences can be calculated using various alignment algorithms and/or programs, including those available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used, for example, with default settings FASTA or BLAST.
  • polypeptides that are at least 70%, 85%, 90%, 95%, 98%, or 99% identical to a particular polypeptide described herein and preferably exhibit substantially the same function, and polynucleotides encoding the above-mentioned polypeptides.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or peptide that occurs naturally in a living animal is not “isolated”, but the same nucleic acid or peptide that is partially or completely separated from coexisting materials in its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-natural environment, such as a host cell.
  • nucleotide sequences "encoding" a nucleic acid molecule of a protein or an amino acid sequence of a protein as used herein include degeneracy of each other and all nucleotide sequences encoding the same amino acid sequence.
  • the nucleotide sequence may also include one or more introns.
  • variant means a variant of a polypeptide obtained by substituting at least one residue in the amino acid sequence of the parent molecule or by adding at least one amino acid residue to the N- or C-terminus.
  • the term “subject” includes any human or non-human animal.
  • the term “non-human animal” includes all vertebrates such as mammals and non-mammals such as non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, amphibians, reptiles Wait. Unless otherwise indicated, the terms “patient” or “subject” are used interchangeably. In the present invention, the preferred subject is a human.
  • beneficial or desired clinical outcomes include, but are not limited to, reduction in one or more symptoms, reduction in disease severity, stabilization (ie, no worsening) of disease state, delay or reduction in disease progression. Slowness, improvement or remission of disease state, and remission (whether partial or complete remission), whether detectable or undetectable.
  • Treatment may refer to prolonging survival compared to expected survival if not receiving treatment.
  • treatment includes prophylaxis.
  • the treatment is “effective” if the progression of the disease is reduced or stopped.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Patients in need of treatment include those who have been diagnosed with disorders associated with the expression of polynucleotide sequences, and those who may develop such disorders due to genetic predisposition or other factors.
  • autoimmune disease as used herein is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive responses to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes (type 1), dystrophic epidermia bullosa lysis, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy,
  • the nucleic acid encoding the scFv shown in Table 2 can be synthesized by the method of genetic engineering.
  • the nucleic acid fragment encoding scFv of the present invention was synthesized in Beijing Biomed Gene Technology Co., Ltd.
  • the scFv fragment contains VH, VL and a linker region.
  • the linker region links the two domains together between VH and VL to form an antigen-binding site that specifically recognizes BCMA.
  • a single chain antibody is generally an amino acid chain sequence encoded by a nucleotide chain, and the scFv can be further modified by amino acid deletions, insertions, substitutions, additions, and other modifications known in the art.
  • the single-chain antibody scFv in Example 1 forms a CAR structure together with the hinge region, the transmembrane domain, the CD28 or 4-1BB costimulatory domain, and the CD3 ⁇ domain. wherein the hinge region and the transmembrane region are selected from CD8 ⁇ .
  • the scFv gene targeting BCMA was synthesized by the method of gene synthesis (Beijing Biomed Gene Technology Co., Ltd.). 2) Using the existing CAR plasmid as a template, PCR was used to clone out the CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, the intracellular region of 4-1BB (corresponding to NP_001552.2), and CD3 ⁇ (corresponding to NP_000725.2) in the CAR molecule. 1) Nucleic acid fragments in the intracellular region. 3) Using the gene obtained in step 1) and the nucleic acid fragment obtained in step 2) as a template, a complete nucleic acid fragment targeting BCMA CAR is cloned by PCR.
  • step 4) Insert the complete nucleic acid fragment obtained in step 3) into the lentiviral vector pLenti6.3/V5 (Thermo Fisher, Waltham, MA, USA) by the method of restriction enzyme cleavage and ligation, and obtain the lentiviral vector carrying the targeting BCMCAR gene.
  • Viral transfer plasmid ( Figure 2).
  • the lentiviral packaging plasmids pLP/VSVG, pLP1/MDK, pLP2/RSK (Thermo Fisher, Waltham, MA, USA) and the transfer plasmid obtained in step 4) were mixed with Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA) After transfection into HEK293T cells, the culture medium was collected after 48 hours, centrifuged at 300 g to remove cell debris, and centrifuged at 25,000 rpm for 3 hours with an ultracentrifuge. Dissolve the pellet with 1 mL of normal saline, which is the desired lentiviral vector.
  • T cells were isolated from peripheral blood mononuclear cells (Miaotong (Shanghai) Biotechnology Co., Ltd., China) of healthy volunteers using CD3/CD28 Dynabeads (Thermo Fisher).
  • the isolated and purified T cells (the T cells at this time were linked to CD3/CD28Dynabeads) were seeded and cultured in X-VIVO 15 medium (Lonza, Switzerland) at 1.0 ⁇ 10 6 cells/mL, and IL- 2 (Shandong Jintai Bioengineering Co., Ltd., China) (500IU/mL) After culturing for 48 hours, T cells were infected with the above lentiviral vector.
  • the cells were centrifuged to change the medium, and fresh X-VIVO 15 containing IL-2 (500 IU/mL) was added to continue the culture. After 8 days of cell culture, all cells in the culture system were collected, and the Dynabeads in the culture system were removed with a magnetic stand, and the T cells were centrifuged and counted.
  • This example takes the detection of the expression of BCMA on the surface of the human myeloma cell line NCI H929 and the human leukemia cell line K562 as an example.
  • NCI H929 and K562 cells were purchased from ATCC. NCI H929 cells and K562 cells were cultured to the vigorous proliferation stage, 1.0 ⁇ 10 6 cells were taken from each, the cells were resuspended in 200 ⁇ L of DPBS, and the supernatant was removed after centrifugation at 1000 g. The cell pellet was resuspended in 100 ⁇ L DPBS, 5 ⁇ L fluorescently labeled BCMA antibody (Miltenyi Biotec, USA) was added, incubated at room temperature for 20 minutes, the cells were resuspended in 200 ⁇ L DPBS, and the supernatant was removed after centrifugation at 1000 g.
  • BCMA antibody Miltenyi Biotec, USA
  • the cells were resuspended in 100 ⁇ L of DPBS, and the ratio of BCMA expression in the two cells was detected by flow cytometer (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA). As shown in Figure 4, the expression level of BCMA on the surface of NCI H929 cells was high, while K562 cells hardly expressed BCMA.
  • the killing efficiency of CAR-T containing scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain, 4-1BB costimulatory domain and CD3 ⁇ domain that specifically recognizes BCMA is detected on BCMA-positive target cells as an example.
  • NCI H929 target cells ATCC
  • BCMA negative K562 cells Resuspend 2 ⁇ 10 6 NCI H929 target cells (ATCC) or BCMA negative K562 cells in 1 mL of normal saline, add 5 ⁇ L Calcein-AM (concentration 1 ⁇ g/ ⁇ L, ThermoFisher, USA), mix gently, and then put in 37 Incubate in a water bath for 5 min to label target cells, wash twice with 10 mL of normal saline to remove excess dye, and resuspend cells in 1 mL of normal saline for counting.
  • Calcein-AM Concentration 1 ⁇ g/ ⁇ L, ThermoFisher, USA
  • the fluorescence value of the cell supernatant was detected with a fluorescence microplate reader (Varioscan Lux, ThermoFisher) (excitation wavelength: 495 nm, emission wavelength: 515 nm).
  • FIGS 5 to 17 show that CAR-Ts with BCMA-targeting scFv, co-stimulatory signaling domains and CD3 ⁇ signaling domains can effectively kill NCI H929 cells by recognizing BCMA target proteins, and are more effective than Bluebird Bio's BCMA-targeting CAR-Ts.
  • CAR-T (C-CAR-T, sequence reference Seq No.15 in patent WO2016/014789A2) has higher killing efficiency on NCI H929 cells, but cannot effectively kill BCMA negative K562 cells, indicating that these CAR-Ts can effectively kill NCI H929 cells.
  • BCMA is specifically recognized by scFv, and CAR-T is activated, causing CAR-T to kill tumor cells.
  • Figure 18 shows that compared with C-CAR-T, most CAR-T cells (except 05-CAR-T) have higher killing efficiency on NCI H929 cells, indicating that these CAR-T cells have stronger effect on tumor cells reactivity.
  • CAR-T containing scFv-01, CD8 ⁇ hinge region, CD8 ⁇ transmembrane domain, 4-1BB costimulatory domain and CD3 ⁇ domain that specifically recognizes BCMA in BCMA-positive target cells (NCI H929) and BCMA
  • T cells and CAR-T cells were co-incubated with NCI H929 cells or K562 cells for 6 hours in a 37°C, 5% CO2 incubator (the ratio of effector cells to target cells was 10:1, 5:1, 1:1, respectively). 1), collect the supernatant, and use the CBA Human Th1/Th2/Th17 Cytokine Kit (BD Biosciences) to detect cytokine expression.
  • Figure 19 shows that, compared with T cells, BCMA CAR-T cells co-incubated with BCMA-positive target cells (NCI H929) significantly enhanced the induction effect of IFN- ⁇ , and at the same time, with the increase of the ratio of effector cells to NCI H929 cells , the cytokine release effect was enhanced.
  • BCMA CAR-T cells co-incubated with BCMA-positive target cells (NCI H929) significantly enhanced the induction effect of IFN- ⁇ , and at the same time, with the increase of the ratio of effector cells to NCI H929 cells , the cytokine release effect was enhanced.
  • the induction effect of IFN- ⁇ was not significantly different from that of the T cell group.
  • the above experimental results show that the CAR-T cells of the present invention can specifically recognize the BCMA target antigen, thereby producing cytotoxicity-related cytokines.

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Abstract

本发明公开了一种特异性结合BCMA的抗体，包括重链可变结构域VH和轻链可变结构域VL，以及包含其序列的嵌合抗原受体，进一步公开了本发明抗体或嵌合抗原受体的氨基酸序列、载体、宿主细胞、组合物。本发明还公开了所述抗体或嵌合抗原受体在制备用于预防或治疗疾病的药物中的用途。

Description

一种特异性结合BCMA的抗体及其应用 技术领域
本发明涉及一种特异性结合BCMA的抗体和基于该抗体构建的嵌合抗原受体，并公开了抗体的氨基酸序列、克隆或表达载体、宿主细胞、生产抗体的方法以及该抗体或嵌合抗原受体的用途。
背景技术
多发性骨髓瘤(Multiple Myeloma,MM)是以克隆性浆细胞大量增生为特征的恶性肿瘤。尽管多发性骨髓瘤的治疗方案近年来已经得到了很大的进步，但是多发性骨髓瘤仍然是一种高发病率和死亡率的疾病。因此，急需新的治疗方法对多发性骨髓瘤进行救治。
近年来，嵌合抗原受体T细胞(Chimeric Antigen Receptor-T cell,CAR-T)免疫疗法在恶性血液肿瘤治疗中取得了较快的发展，是目前较为有效的恶性肿瘤治疗方式之一。嵌合抗原受体(Chimeric Antigen Receptors,CAR)是一种融合蛋白，由单链抗体可变片段(Single-Chain Fragment Variable,scFv)、铰链区、跨膜结构域、胞内信号结构域(共刺激结构域及T细胞激活结构域)组成。CAR-T细胞的scFv一般来源于单克隆抗体，可以绕过主要组织相容性复合物(Major Histocompatibility Complex,MHC)限制来识别完整的细胞表面蛋白。
多发性骨髓瘤免疫治疗中的一种候选靶点是B细胞成熟抗原(B Cell Maturation Antigen,BCMA)。BCMA主要由浆细胞和一部分成熟B细胞表达，而在大部分B细胞以及其它组织中都不表达(B-cell Maturation Antigen Is a Promising Target for Adoptive T-cell Therapy of Multiple Myeloma,clinical cancer research,15 Apr 2013.)。因此，BCMA可以作为CAR-T细胞疗法治疗MM的靶抗原。目前国内外的临床试验证实CAR-T细胞疗法在多发性骨髓瘤治疗中已经取得了较好的治疗效果，但是目前用于临床试验的CAR-T细胞，大多为鼠源的单链抗体scFv，这可能会导致CAR-T细胞在体内产生抗CAR的免疫反应，导致CAR-T细胞在体内的扩增能力 降低、CAR-T在体内难以持续及令人失望的临床疗效等(The novel anti-CD19 chimeric antigen receptors with humanized scFv(single-chain variable fragment)trigger leukemia cell killing,Cellular Immunology,14 Mar 2016.；Chimeric Antigen Receptor T-cell Therapies for Multiple Myeloma,blood,14 Dec 2017.)。此外，scFv与靶抗原的亲和力强弱也是决定CAR-T是否能有效杀伤靶细胞的重要因素。因此，筛选出能特异有效识别BCMA的scFv对构建安全有效治疗MM的CAR-T至关重要。
发明内容
本发明公开了一种特异性结合BCMA的分离的抗体，其包括：重链可变结构域(以下缩写为VH)和轻链可变结构域(以下缩写为VL)，其中
所述VH包括：包含SEQ ID NO:1(GYTFX 1X 2YSMN)的氨基酸序列的VH-CDR1，包含SEQ ID NO:2(RINTX 3SG X 4P X 5YADDFKG)的氨基酸序列的VH-CDR2和包含SEQ ID NO:3(SNDY X 6YSLD X 7)的氨基酸序列的VH-CDR3；其中X 1选自S、T、R；X 2选自R、S、H；X 3选自G、E、R；X 4选自A、T、V；X 5选自I、N；X 6选自N、L；X 7选自H、Y、F；
所述VL包括：包含SEQ ID NO:4(RAS X 8SV X 9X 10X 11G X 12X 13X 14X 15Y)的氨基酸序列的VL-CDR1，包含SEQ ID NO:5(LASNVQT)的氨基酸序列的VL-CDR2和包含SEQ ID NO:6(X 16QSR X 17IPRT)的氨基酸序列的VL-CDR3；其中X 8选自R、E；X 9选自T、S；X 10选自I、V；X 11选自A、L；X 12选自S、N；X 13选自P、H；X 14选自I、L；X 15选自I、V；X 16选自L、F；X 17选自T、S。
在一个具体实施方案中，所述抗体的VH包括：如SEQ ID NO:1的氨基酸序列所示的VH-CDR1、如SEQ ID NO:2的氨基酸序列所示的VH-CDR2和如SEQ ID NO:3所示的氨基酸序列VH-CDR3；
所述抗体的VL包括：如SEQ ID NO:4的氨基酸序列所示的VL-CDR1，如SEQ ID NO:5的氨基酸序列所示的VL-CDR2和如SEQ ID NO:6的氨基酸序列所示的VL-CDR3。
本发明公开了一种特异性结合BCMA的分离的抗体，其包括VH和VL，所述VH包括VH-CDR1、VH-CDR2和VH-CDR3，所述VL包括VL-CDR1、VL-CDR2和VL-CDR3；其中，所述VH-CDR1包含选自由SEQ ID NO:7、8和9组成 的组中的氨基酸序列；所述VH-CDR2包含选自由SEQ ID NO:10、11、12和13组成的组中的氨基酸序列；所述VH-CDR3包含选自由SEQ ID NO:14、15和16组成的组中的氨基酸序列；所述VL-CDR1包含选自由SEQ ID NO:17、18和19组成的组中的氨基酸序列；所述VL-CDR2包含SEQ ID NO:20的氨基酸序列；所述VL-CDR3包含SEQ ID NO:21或SEQ ID NO:22的氨基酸序列。
在一个具体实施方案中，所述VH-CDR1选自由SEQ ID NO:7、8和9组成的组中的氨基酸序列；所述VH-CDR2选自由SEQ ID NO:10、11、12和13组成的组中的氨基酸序列；所述VH-CDR3选自由SEQ ID NO:14、15和16组成的组中的氨基酸序列；所述VL-CDR1选自由SEQ ID NO:17、18和19组成的组中的氨基酸序列；所述VL-CDR2包含SEQ ID NO:20的氨基酸序列；所述VL-CDR3包含SEQ ID NO:21或SEQ ID NO:22的氨基酸序列。
在一个具体实施方案中，所述抗体：
1)包含如SEQ ID NO:7所示的VH-CDR1、如SEQ ID NO:10所示的VH-CDR2和如SEQ ID NO:14所示的VH-CDR3的VH；和包含如SEQ ID NO:17所示的VL-CDR1、如SEQ ID NO:20所示的VL-CDR2和如SEQ ID NO:21所示的VL-CDR3的VL；
2)包含如SEQ ID NO:8所示的VH-CDR1、如SEQ ID NO:11所示的VH-CDR2和如SEQ ID NO:15所示的VH-CDR3的VH；和包含如SEQ ID NO:18所示的VL-CDR1、如SEQ ID NO:20所示的VL-CDR2和如SEQ ID NO:21所示的VL-CDR3的VL；
3)包含如SEQ ID NO:8所示的VH-CDR1、如SEQ ID NO:12所示的VH-CDR2和如SEQ ID NO:14所示的VH-CDR3的VH；和包含如SEQ ID NO:17所示的VL-CDR1、如SEQ ID NO:20所示的VL-CDR2和如SEQ ID NO:21所示的VL-CDR3的VL；
4)包含如SEQ ID NO:7所示的VH-CDR1、如SEQ ID NO:10所示的VH-CDR2和如SEQ ID NO:14所示的VH-CDR3的VH；和包含如SEQ ID NO:17所示的VL-CDR1、如SEQ ID NO:20所示的VL-CDR2和如SEQ ID NO:22所示的VL-CDR3的VL；或
5)包含如SEQ ID NO:9所示的VH-CDR1、如SEQ ID NO:13所示的VH-CDR2 和如SEQ ID NO:16所示的VH-CDR3的VH；和包含如SEQ ID NO:19所示的VL-CDR1、如SEQ ID NO:20所示的VL-CDR2和如SEQ ID NO:21所示的VL-CDR3的VL。
在一个具体实施方案中，上述抗体中的所述VH包含选自由SEQ ID NO:23、24、25、26、27、28、29、30、31和32组成的组中的氨基酸序列，或包含与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列。
在一个具体实施方案中，上述抗体中的所述VL包含选自由SEQ ID NO:33、35、36、37、38、39、40、41、42和43组成的组中的氨基酸序列，或包含与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列。
在一个具体实施方案中，所述抗体：
1)包含如SEQ ID NO:23所示的VH和包含如SEQ ID NO:33所示的VL；
2)包含如SEQ ID NO:24所示的VH和如包含SEQ ID NO:33所示的VL；
3)包含如SEQ ID NO:25所示的VH和如包含SEQ ID NO:35所示的VL；
4)包含如SEQ ID NO:26所示的VH和如包含SEQ ID NO:36所示的VL；
5)包含如SEQ ID NO:27所示的VH和如包含SEQ ID NO:37所示的VL；
6)包含如SEQ ID NO:28所示的VH和如包含SEQ ID NO:38所示的VL；
7)包含如SEQ ID NO:29所示的VH和如包含SEQ ID NO:33所示的VL；
8)包含如SEQ ID NO:30所示的VH和如包含SEQ ID NO:39所示的VL；
9)包含如SEQ ID NO:31所示的VH和如包含SEQ ID NO:40所示的VL；
10)包含如SEQ ID NO:32所示的VH和如包含SEQ ID NO:41所示的VL；
11)包含如SEQ ID NO:28所示的VH和如包含SEQ ID NO:42所示的VL；或
12)包含如SEQ ID NO:29所示的VH和如包含SEQ ID NO:43所示的VL.
在一个具体实施方案中，上述抗体包含选自由SEQ ID NO:44、45、46、47、48、49、50、51、52、53、54、55和56组成的组中的氨基酸序列，或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列；或者
所述抗体由上述序列组成或基本上由上述序列组成。
在一个具体实施方案中，本发明所述抗体为单链抗体scFv；其VH和VL由富含甘氨酸和丝氨酸的接头肽链相连，VH和VL的顺序可互换。
在一个具体实施方案中，本发明所述抗体为嵌合的或人源化抗体。
另一方面，本发明进一步公开了一种靶向BCMA的嵌合抗原受体，其包含：1)本发明所述的scFv；2)铰链区；3)跨膜区；4)共刺激结构域；5)CD3ζ胞内信号转导结构域。其中所述共刺激结构域选自CD27、CD28、4-1BB、OX-40、CD30、CD40、PD-1、ICOS、LFA-1、CD-2、CD7、LIGHT、NKG2C、B7-H3或其任意组合；优选地，所述共刺激结构域选自4-1BB，所述4-1BB包含如SEQ ID NO.57所述的氨基酸序列；所述CD3ζ胞内信号转导结构域包含如SEQ ID NO.58所述的氨基酸序列。
在一个具体实施方案中，所述铰链区和和跨膜结构域选自IgG1、IgG4、CD8α、CD28、IL-2受体、IL-7受体、IL-11受体、PD-1或CD34的铰链区和跨膜结构域。
所述示例性抗体的序列如表1和序列表所示。
表1示例性抗BCMA scFv的可变区序列
Figure PCTCN2021142886-appb-000001
所述示例性抗体的CDR序列如表2和序列表所示。
表2示例性抗体的CDR序列
Figure PCTCN2021142886-appb-000002
Figure PCTCN2021142886-appb-000003
Figure PCTCN2021142886-appb-000004
Figure PCTCN2021142886-appb-000005
Figure PCTCN2021142886-appb-000006
本发明公开了一种分离的核酸分子，其编码如前所述的抗体或嵌合抗原受体。
本发明还公开了一种克隆或表达载体，其含有如前所述的核酸分子。其中所述载体选自DNA、RNA、质粒、慢病毒载体、腺病毒载体和逆转录病毒载体中的一种或多种。
本发明还公开了一种宿主细胞，包括如前所述的核酸分子或如前所述的载体。其中所述宿主细胞为T淋巴细胞、B淋巴细胞、自然杀伤细胞、树突状细胞、巨噬细胞、细胞毒性T细胞、肿瘤浸润T细胞或调节性T细胞。
本发明还公开了一种组合物或试剂盒，其包含如前所述的抗体、如前所述的嵌合抗原受体、如前所述的核酸分子、如前所述的载体、如前所述的宿主细胞中的至少一种，以及药学可接受的赋形剂、稀释剂或载体。
本发明还公开了如前所述的抗体、如前所述的嵌合抗原受体、如前所述的核酸分子、如前所述的载体、如前所述的宿主细胞、或如前所述的组合物在制备预防或治疗用于治疗B细胞相关肿瘤疾病、自身免疫疾病、病毒或细菌引起的感染性疾病的药物中的用途。
本发明公开了一种治疗疾病的方法，其中向需要治疗的受试者施用有效量的如前所述的抗体、如前所述的嵌合抗原受体、如前所述的核酸分子、如前所述的载体、如前所述的宿主细胞、或如前所述的组合物，其中所述受试者患有B细胞相关肿瘤疾病、自身免疫疾病、病毒或细菌引起的感染性疾病中的至少一种。
在一个实施方案中，所述B细胞相关肿瘤疾病选自：急性白血病诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病；慢性白血病诸如慢性髓细胞(粒细胞性) 白血病、慢性骨髓性白血病和慢性淋巴细胞白血病；真性红细胞增多症、淋巴瘤、套细胞淋巴瘤、扩散大B-细胞淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良中的至少一种。
附图说明
图1示出CAR分子模式图。
图2示出质粒载体模式图，制备慢病毒所使用的质粒为pLenti质粒，目的基因上游启动子为EF-1α，启动子下游依次为信号肽、Linker区及CAR结构序列。
图3示出带有特异识别BCMA的scFv的CAR的慢病毒在T细胞中的转导效率。
图4示出NCI H929细胞和K562细胞表面BCMA表达的流式检测图。
图5示出含有scFv-01的CAR-T(01-CAR-T)对靶细胞的杀伤效果图，其中01-CAR-T表示含有scFv-01的CAR-T细胞，C-CAR-T为阳性对照，T细胞处理组为阴性对照。
图6示出含有scFv-04的CAR-T(04-CAR-T)对靶细胞的杀伤效果图。
图7示出含有scFv-05的CAR-T(05-CAR-T)对靶细胞的杀伤效果图。
图8示出含有scFv-06的CAR-T(06-CAR-T)对靶细胞的杀伤效果图。
图9示出含有scFv-07的CAR-T(07-CAR-T)对靶细胞的杀伤效果图。
图10示出含有scFv-08的CAR-T(08-CAR-T)对靶细胞的杀伤效果图。
图11示出含有scFv-09的CAR-T(09-CAR-T)对靶细胞的杀伤效果图。
图12示出含有scFv-10的CAR-T(10-CAR-T)对靶细胞的杀伤效果图。
图13示出含有scFv-11的CAR-T(11-CAR-T)对靶细胞的杀伤效果图。
图14示出含有scFv-12的CAR-T(12-CAR-T)对靶细胞的杀伤效果图。
图15示出含有scFv-13的CAR-T(13-CAR-T)对靶细胞的杀伤效果图。
图16示出含有scFv-14的CAR-T(14-CAR-T)对靶细胞的杀伤效果图。
图17示出含有scFv-15的CAR-T(15-CAR-T)对靶细胞的杀伤效果图。
图18示出本发明中的CAR-T对靶细胞的杀伤效果的归一化图。
图19示出含有scFv-01的CAR-T(01-CAR-T)在靶细胞刺激下的细胞因子表达图。
具体实施方式
下面通过具体实施方式对本发明作进一步的说明。除非另有限定，本文中所使用的术语具有与本领域普通技术人员通常理解相同的含义。
本文所用的术语“抗原”是指引起免疫应答的分子，该免疫应答可涉及抗体产生，或特异性免疫活性细胞的活化。本领域技术人员均可理解任何大分子包括所有的蛋白质或肽，可用作抗原。抗原可源自重组或基因组DNA。本领域技术人员均可理解任何DNA其包括编码引起免疫应答的蛋白质的核苷酸序列或部分核苷酸序列，编码如本文使用的术语“抗原”。此外，本领域技术人员均可理解抗原不必单独地由基因的全长核苷酸序列编码。容易显而易见的是本公开包括但不限于，多于一个的基因的部分核苷酸序列的用途，并且这些核苷酸序列以不同的组合进行布置，以引起期望的免疫应答。此外，领域技术人员均可理解抗原根本不必由“基因”进行编码，抗原可被产生、合成或可源自生物学样本。这种生物学样本可包括但不限于组织样本、肿瘤样本、细胞或生物学流体。
如本文所用的，术语“抗体”指的是与抗原特异性结合的免疫球蛋白分子。抗体可为源于天然来源或源于重组来源的完整的免疫球蛋白，并可为完整免疫球蛋白的免疫反应部分。抗体通常为免疫球蛋白分子的四聚物。本发明中的抗体可以以多种形式存在，包括多克隆、单克隆、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂合的、突变的、以及嫁接的抗体；本发明中的抗体形式还包括全长抗体、抗体片段，例如Fab、Fab’、F(ab’)2、Fv、scFv、di-scFv、tri-scFv、Fd和其它保留抗原结合功能的抗体片段；还可以是二聚体结构Diabody或三聚体结构Triabody。通常情况下，所述片段应当包括抗原结合片段，抗原结合片段通常包括VL和VH，然而，它不一定必须包括两者。例如，所谓的Fd抗体片段仅由VH和CH1结构域组成，但仍保留了完整抗体的一些抗原结合功能。术语“抗体”作为免疫球蛋白或其片段或它们的衍生物，其包括包含抗原结合位点的任何多肽，而不论其是否是在体外或体内产生的。VH或VL区可以进一步细分成：称为互补决定区(CDR)的高变区，以及穿插分布的称为框架区(FWR)的更保守区域。重链和轻链的可变区包含与抗原相互作用的结合结构域。重链中的CDR缩写为VH-CDR，例如VH-CDR1、VH-CDR2、VH-CDR3，轻链中的CDR缩写为VL-CDR，例如VL-CDR1、VL-CDR2、VL-CDR3。本发明公开的抗体和抗原 结合片段的CDR由Kabat编号所定义或识别。
本文使用的术语“单链可变区片段”、“单链抗体”或“scFv”是指通过重组DNA技术形成的抗体，其中免疫球蛋白重链可变区和轻链可变区通过氨基酸肽段(linker)连接而成。生成单链抗体的多种方法是已知的，包括在美国专利号4,694,778；Bird(1988)Science 242:423-442；Huston等(1988)Proc.Natl.Acad.Sci.USA 85:5879-5883；Ward等(1989)Nature 334:54454；Skerra等(1988)Science 242:1038-1041中描述的那些。重链和轻链的可变区包含与抗原相互作用的结合结构域。重链中的CDR缩写为VH-CDR，例如VH-CDR1、VH-CDR2、VH-CDR3，轻链中的CDR缩写为VL-CDR，例如VL-CDR1、VL-CDR2、VL-CDR3。本发明公开的抗体和抗原结合片段的CDR由Kabat编号所定义或识别。
本公开所用术语“嵌合的”是指一部分重链来源于一个物种而重链其他部分来源于其他物种的抗体或抗体多肽。在说明性示例中，嵌合抗体可包含源于人类的恒定区域和来自诸如驼科的非人动物的可变区域。在某些实施方案中，非人动物是一种哺乳动物，例如驼科、小鼠、大鼠、兔、山羊、绵羊、豚鼠或仓鼠。
本公开所用术语“人源化的”是指抗体或抗体多肽包括来源于非人动物的CDRs、来源于人类的FWR区以及在适用的情况下来源于人类的恒定区。
本公开所用“BCMA”可来源于人类，人BCMA的示例性序列包括人BCMA蛋白(Genbank accession No.:BAB60895.1)。
本发明所用术语“BCMA”旨在包含任何形式的BCMA，例如，1)天然未经处理的BCMA分子、“全长”BCMA链或天然产生的BCMA变体，包括，例如，剪接变体或等位基因变体；2)细胞内处理后产生的任何形式的BCMA；或3)通过重组方法产生的BCMA亚单位的全长、片段(例如，截短形式，细胞外/跨膜结构域)或其修饰形式(如突变形式、糖基化/聚乙二醇化、His-tag/免疫荧光融合形式)。
本发明所用术语“靶向BCMA”是指能够特异性结合BCMA(如人BCMA)，例如靶向BCMA的嵌合抗原受体是指能够特异性结合BCMA的嵌合抗原受体；例如靶向BCMA的scFv是指能够特异性结合BCMA(如人BCMA)的单链抗体。
本公开所用术语“特异性结合”或“特异性地结合”是指两个分子之间的非随机结合反应，例如抗体和抗原之间的结合反应。在某些实施方案中，本公开提供的抗体多肽特异性结合人BCMA，其结合亲和力(K D)≤10 -6M(例如，≤5x10 -7M、≤2x10 -7M、≤10 -7M、≤5x10 -8M、≤2x10 -8M、≤10 -8M、≤5x10 -9M、≤4x10 - 9M、≤3x10 -9M、≤2x10 -9M或≤10 -9M)。本公开所用K D指解离率与结合率之比(K off/K on)，其可通过使用本领域已知的任何常规方法来测定，包括但不限于表面等离子体共振方法、微尺度热泳法、高效液相色谱-质谱法和流式细胞术(如FACS)法。在某些实施方案中，可以用流式细胞仪适当测定K D值。
本文所用的术语“载体”是指运输、转导和在靶细胞表达被包含的外源目的基因(例如本发明所述的多核苷酸)的分子工具，所述工具提供合适的在靶细胞中起始转录的核苷酸序列，即启动子。载体其包括分离的核酸，并且其可以用于递送分离的核酸至细胞内部。众多载体在本领域是已知的，包括但不限于线性多核苷酸、与离子或两亲性化合物相关联的多核苷酸、质粒和病毒。因而，术语“载体”包括自主复制的质粒或病毒。该术语也应当解释为包括便于将核酸转移入细胞的非质粒和非病毒化合物，例如，聚赖氨酸化合物、脂质体等。病毒载体的实例包括但不限于仙台病毒载体、腺病毒载体、腺伴随病毒载体、逆转录病毒载体、慢病毒载体等。
本文使用的“表达载体”是指包括重组多核苷酸的载体，所述重组多核苷酸包括可操作地连接至待表达的核苷酸序列的表达控制序列。表达载体包括足够的用于表达的顺式作用元件；用于表达的其它元件可以由宿主细胞供应或在体外表达系统中供应。表达载体包括所有本领域已知的并入重组多核苷酸的那些，比如质粒(例如，裸露或包含在脂质体中)和病毒(例如，仙台病毒、慢病毒、逆转录病毒、腺病毒和腺伴随病毒)。
本文使用的“克隆载体”是指在宿主细胞中具有自主复制能力的DNA分子例如质粒、粘粒或噬菌体。克隆载体通常含有一个或少量限制内切核酸酶识别位点以及标记基因，其中在所述限制内切核酸酶识别位点将外源DNA序列以确定的方式插入而不会丧失载体的必需生物学功能，所述标记基因适合用于对用克隆载体转化的细胞的鉴定和选择。标记基因通常包括提供四环素抗性或氨苄青霉素抗性的基因。
宿主细胞可以是含有克隆载体或表达载体的任何原核细胞或真核细胞，包括已 经进行基因工程改造以在宿主细胞的染色体或基因组中含有克隆化基因的那些原核细胞或真核细胞。合适的哺乳动物宿主细胞包括骨髓瘤细胞，例如SP2/0细胞和NS0细胞以及中国仓鼠卵巢(CHO)细胞、杂交瘤细胞系及其它可用于表达抗体的哺乳动物宿主细胞。具有经修饰的免疫系统的特殊转基因动物也可用于制备抗体。
本文使用的“同一性”是指在两种核酸分子或多肽之间的序列一致性。可以通过比较为比较目的而对齐的每个序列中位置来确定同一性。当比较的序列中的位置被相同碱基占据时，那么在该位置处分子是相同的。在核酸或氨基酸序列之间相似性或同一性的程度是在由核酸序列共享的位置处相同或匹配核苷酸的数目的函数。可使用各种比对算法和/或程序来计算两个序列之间的同一性，包括可获得为GCG序列分析包(University of Wisconsin，Madison，Wis.)的一部分，并且可以以例如默认设置使用的FASTA或BLAST。例如，本领域技术人员可以预期与在本文中描述的特定多肽具有至少70％、85％、90％、95％、98％或99％的同一性并且优选展现出基本上相同的功能的多肽，以及编码上述多肽的多核苷酸。
本文使用的“分离的”是指从自然状态改变或移出。例如，天然存在于活动物中的核酸或肽不是“分离的”，但是部分或完全与它的自然状态的共存物质分开的相同的核酸或肽是“分离的”。分离的核酸或蛋白质可以以基本上纯化的形式存在，或可以存在于非自然环境，比如，宿主细胞中。
除非另外规定，本文使用的“编码”某蛋白的核酸分子或某蛋白的氨基酸序列的核苷酸序列包括彼此的简并形式(Degeneracy)并且编码相同的氨基酸序列的所有的核苷酸序列。该核苷酸序列还可以包括一个或多个内含子。
本文使用的术语“变体”意指通过在母体分子的氨基酸序列中替代至少一个残基或者在N端或C端添加至少一个氨基酸残基所获得的多肽变体。
如本文所用，术语“受试者”包括任何人或非人动物。术语“非人类动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，例如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、大鼠、小鼠、两栖动物、爬行动物等。除非另有说明，否则术语“患者”或“受试者”可互换使用。在本发明中，优选的受试者是人类。
如本文所用，术语“治疗”是指向受试者施用有效量的具有根据本文所述的方法离体改变的靶基因的多核苷酸序列的细胞，以使得所述受试者具有所述疾病的至 少一种症状的减少或所述疾病的改善，例如，有益的或所需的临床结果。出于本公开的目的，有益的或所需的临床结果包括但不限于一种或多种症状的减轻、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和，以及缓解(无论是部分缓解还是全部缓解)，无论是可检测的或是不可检测的。治疗可指与未接受治疗情况下的预期存活期相比，延长存活期。因此，本领域的技术人员意识到治疗可改善疾病状况，但可能不是疾病的完全治愈。如本文所用，术语“治疗”包括预防。或者，治疗在疾病的进展减少或停止的情况下是“有效的”。“治疗”还可意指与在未接受治疗情况下的预期存活期相比，延长存活期。需要治疗的病人包括已经被诊断具有与多核苷酸序列的表达相关的病症，以及由于遗传易感性或其他因素可能发展这种病症。
如本文所用的术语“自身免疫疾病”被定义为由自身免疫应答产生的紊乱。自体免疫疾病是对自身抗原的不适当和过度应答的结果。自身免疫疾病的例子包括但不限于阿狄森氏疾病、斑秃、强直性脊柱炎、自身免疫肝炎、自身免疫腮腺炎、克罗恩氏疾病、糖尿病(1型)、营养不良性大疱性表皮松解症、附睾炎、肾小球性肾炎、格雷夫斯氏疾病、吉兰-巴雷综合征、桥本氏疾病、溶血性贫血、系统性红斑狼疮、多发性硬化症、重症肌无力、寻常型天疱疮、牛皮癣、风湿热、类风湿性关节炎、结节病、硬皮病、斯耶格伦氏综合征、脊椎关节病变、甲状腺炎、血管炎、白癜风、粘液性水肿、恶性贫血、溃疡性结肠炎等等。
在本文中陈述数值极限或范围的情况下，包括端点。此外，具体包括在数值极限或范围内的所有值和子范围。
实施例
实施例1.scFv基因合成
在本发明的具体实施方案中，编码如表2所示的scFv的核酸可以通过基因工程的方法进行合成。本发明的编码scFv的核酸片段在北京博迈德基因技术有限公司合成。scFv片段包含VH、VL及接头区，接头区在VH和VL之间将两个结构域连接在一起形成一个特异性识别BCMA的抗原结合位点。单链抗体一般是由一条核苷酸链编码的一条氨基酸链序列，所述scFv可通过本领域已知的氨基酸缺失、插入、取代、增加及其它修饰方式进行进一步修饰。此外，根据某种氨基酸序列在 其核酸序列中引入上述修饰方法也是本领域技术人员已知的，所以，对scFv进行修饰优选在核酸水平上进行。
实施例2.CAR分子的构建和慢病毒包装
如图1所示，实施例1中的单链抗体scFv和铰链区、跨膜结构域、CD28或4-1BB共刺激结构域及CD3ζ结构域共同形成CAR结构。其中铰链区和跨膜区选自CD8α。
1)靶向BCMA的scFv基因通过基因合成的方法合成(北京博迈德基因技术有限公司)。2)以已有的CAR质粒为模板，利用PCR克隆出CAR分子中含有CD8α铰链区、CD8α跨膜区、4-1BB(对应于NP_001552.2)的胞内区、以及CD3ζ(对应于NP_000725.1)胞内区的核酸片段。3)以步骤1)获得的基因和步骤2)获得的核酸片段为模板，通过PCR克隆出靶向BCMA CAR的完整核酸片段。4)通过限制性酶切和连接的方法，将步骤3)获得的完整核酸片段插入慢病毒载体pLenti6.3/V5(Thermo Fisher，Waltham，MA，USA)，获得载带靶向BCMCAR基因的慢病毒转移质粒(图2)。5)将慢病毒包装质粒pLP/VSVG,pLP1/MDK,pLP2/RSK(Thermo Fisher，Waltham，MA，USA)与步骤4)获得的转移质粒，用Lipofectamine 3000(Thermo Fisher，Waltham，MA，USA)转染至HEK293T细胞，48小时后收集培养基，300g离心去除细胞碎片后，用超速离心机25000rpm离心3小时。将沉淀用1mL生理盐水溶解，即为所需的慢病毒载体。
实施例3.CAR-T细胞制备
使用CD3/CD28Dynabeads(Thermo Fisher)从健康志愿者的外周血单个核细胞(妙通(上海)生物科技有限公司，中国)中分离T细胞。将分离纯化后的T细胞(此时的T细胞与CD3/CD28Dynabeads连在一起)以1.0×10 6个细胞/mL接种培养至X-VIVO 15培养基(Lonza，Switzerland)中，并加入IL-2(山东金泰生物工程有限公司，中国)(500IU/mL)培养48小时后，用上述慢病毒载体感染T细胞。病毒感染细胞24小时后，细胞离心换液，并加入含IL-2(500IU/mL)的新鲜X-VIVO 15继续培养。细胞培养8天后，收集培养体系中的所有细胞，并用磁力架去除培养体系中的Dynabeads，T细胞离心并计数。
实施例4.慢病毒转导效率检测
从制备好的CAR-T细胞中取1.0×10 6个细胞，用200μL DPBS重悬细胞，1000g离心后去上清。将细胞沉淀用100μL DPBS重悬，加入3μL抗体，室温孵育20分钟，用200μL DPBS重悬细胞，1000g离心后去上清。再用100μL DPBS重悬细胞，用流式细胞仪(NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA)检测各组细胞中CAR含量。如图3显示，在慢病毒感染MOI＝0.5时，各CAR基因的转导效率均在35-65％之间。
实施例5.靶细胞表面BCMA表达的检测
本实施例以检测人骨髓瘤细胞系NCI H929和人白血病细胞系K562表面BCMA的表达为例。
NCI H929和K562细胞均购自ATCC。将NCI H929细胞和K562细胞培养至旺盛增殖期，各取1.0×10 6个细胞，分别用200μL DPBS重悬细胞，1000g离心后去上清。将细胞沉淀用100μL DPBS重悬，加入5μL荧光标记的BCMA抗体(Miltenyi Biotec，USA)，室温孵育20分钟，用200μL DPBS重悬细胞，1000g离心后去上清。再用100μL DPBS重悬细胞，用流式细胞仪(NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA)检测两种细胞中BCMA表达的比例。如图4显示，NCI H929细胞表面的BCMA表达水平高，而K562细胞几乎不表达BCMA。
实施例6.BCMA CAR-T杀伤靶细胞的效率
本实施例以检测包含特异识别BCMA的scFv、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激结构域及CD3ζ结构域的CAR-T对表达BCMA阳性的靶细胞的杀伤效率为例。
利用1mL生理盐水重悬2×10 6NCI H929靶细胞(ATCC)或BCMA表达阴性的K562细胞，加入5μL Calcein-AM(浓度1μg/μL，ThermoFisher，USA)，轻轻混匀，然后放入37℃水浴中孵育5min，以标记靶细胞，使用10mL生理盐水洗涤两次，去除多余染料，细胞用1mL生理盐水重悬计数。向48孔细胞培养板(Corning Incorporated，Corning，NY，USA)每孔中加入1×10 5个上述标记的NCI  H929或K562细胞，按E:T＝5:1加入各种CAR-T细胞，置于37℃、5％CO2细胞培养箱中孵育6h，取培养上清进行检测。用荧光酶标仪(Varioscan Lux，ThermoFisher)检测细胞上清的荧光值(激发波长:495nm，发射波长:515nm)。
图5至图17显示，具有靶向BCMA的scFv、共刺激信号域和CD3ζ信号域设计的CAR-T均能通过识别BCMA靶蛋白有效杀伤NCI H929细胞，且比蓝鸟生物的靶向BCMA的CAR-T(C-CAR-T，序列参考专利WO2016/014789A2中的Seq No.15)对NCI H929细胞的杀伤效率更高，但无法有效杀伤BCMA表达阴性的K562细胞，说明这些CAR-T能通过scFv特异识别BCMA，并激活CAR-T，引起CAR-T杀伤肿瘤细胞。
图18显示，与C-CAR-T相比，大部分CAR-T细胞(05-CAR-T除外)对NCI H929细胞的杀伤效率更高，说明这些CAR-T细胞对肿瘤细胞有更强的反应性。
实施例7.BCMA CAR-T在靶细胞刺激下的细胞因子表达
本实施例以检测包含特异识别BCMA的scFv-01、CD8α铰链区、CD8α跨膜结构域、4-1BB共刺激结构域及CD3ζ结构域的CAR-T在BCMA阳性靶细胞(NCI H929)和BCMA阴性细胞(K562)刺激后细胞因子干扰素γ(IFN-γ)的表达为例。
将T细胞和CAR-T细胞分别与NCI H929细胞或K562细胞于37℃、5％CO2培养箱中共同孵育6小时(效应细胞与靶细胞的比例分别为10:1、5:1、1:1)，收集上清液，利用CBA人Th1/Th2/Th17细胞因子试剂盒(BD Biosciences)检测细胞因子表达。
图19显示，与T细胞相比，BCMA CAR-T细胞与BCMA阳性靶细胞(NCI H929)共同孵育后，IFN-γ诱导效应显著增强，同时，随着效应细胞与NCI H929细胞比例的增大，细胞因子释放效应随之增强。但是，BCMA CAR-T细胞与BCMA阴性细胞K562共同孵育后，IFN-γ诱导效应与T细胞组相比无显著差异。以上实验结果表明，本发明的CAR-T细胞可以特异性地识别BCMA靶抗原，进而产生细胞毒性相关的细胞因子。

Claims (15)

  1. 一种特异性结合BCMA的分离的抗体，包括重链可变结构域VH和轻链可变结构域VL，
    所述VH包括：包含SEQ ID NO:1的VH-CDR1、包含SEQ ID NO:2的VH-CDR2和包含SEQ ID NO:3的VH-CDR3；
    所述VL包括：包含SEQ ID NO:4的VL-CDR1，包含SEQ ID NO:5的VL-CDR2和包含SEQ ID NO:21的VL-CDR3。
  2. 如权利要求1所述的抗体，其中，所述VH-CDR1包含选自由SEQ ID NO:7、8和9组成的组中的氨基酸序列；所述VH-CDR2包含选自由SEQ ID NO:10、11、12和13组成的组中的氨基酸序列；所述VH-CDR3包含选自由SEQ ID NO:14、15和16组成的组中的氨基酸序列；所述VL-CDR1包含选自由SEQ ID NO:17、18和19组成的组中的氨基酸序列；所述VL-CDR2包含SEQ ID NO:20的氨基酸序列；所述VL-CDR3包含SEQ ID NO:21的氨基酸序列。
  3. 如权利要求1所述的抗体，其中，所述抗体包括：
    1)VH，其包括：包含SEQ ID NO:7的VH-CDR1，包含SEQ ID NO:10的VH-CDR2和包含SEQ ID NO:14的VH-CDR3；VL，其包括：包含SEQ ID NO:17的VL-CDR1，包含SEQ ID NO:20的VL-CDR2和包含SEQ ID NO:21的VL-CDR3；
    2)VH，其包括：包含SEQ ID NO:8的VH-CDR1，包含SEQ ID NO:11的VH-CDR2和包含SEQ ID NO:15的VH-CDR3；VL，其包括：包含SEQ ID NO:18的VL-CDR1，包含SEQ ID NO:20的VL-CDR2和包含SEQ ID NO:21的VL-CDR3；；
    3)VH，其包括：包含SEQ ID NO:8的VH-CDR1，包含SEQ ID NO:12的VH-CDR2和包含SEQ ID NO:14的VH-CDR3；VL，其包括：包含SEQ ID NO:17的VL-CDR1，包含SEQ ID NO:20的VL-CDR2和包含SEQ ID NO:21的VL-CDR3；；
    4)VH，其包括：包含SEQ ID NO:9的VH-CDR1，包含SEQ ID NO:13的VH-CDR2和包含SEQ ID NO:16的VH-CDR3；VL，其包括：包含SEQ ID NO:19的VL-CDR1，包含SEQ ID NO:20的VL-CDR2和包含SEQ ID NO:21的VL-CDR3。
  4. 如权利要求1所述的抗体，其中所述VH包含选自由SEQ ID NO:23、24、 25、26、27、28、29、30、31和32组成的组中的氨基酸序列，或包含与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列；所述VL包含选自由SEQ ID NO:33、35、36、37、38、39、40、41、42和43组成的组中的氨基酸序列，或包含与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列。
  5. 如权利要求1所述的抗体，包括：
    1)包含SEQ ID NO:23或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:33或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL；
    2)包含SEQ ID NO:24或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:33或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL；
    3)包含SEQ ID NO:25或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:35或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL；
    4)包含SEQ ID NO:26或包含与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:36或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL；
    5)包含SEQ ID NO:27或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:37或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL；
    6)包含SEQ ID NO:28或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:38或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL；
    7)包含SEQ ID NO:29或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:33或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL；
    8)包含SEQ ID NO:30或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:39或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL；
    9)包含SEQ ID NO:31或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:40或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL；
    10)包含SEQ ID NO:32或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:41或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL；
    11)包含SEQ ID NO:28或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:42或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL；或
    12)包含SEQ ID NO:29或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VH；和包含SEQ ID NO:43或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列的VL。
  6. 如权利要求1所述的抗体，其包含SEQ ID NO:44、45、47、48、49、50、51、52、53、54、55或56所示的氨基酸序列或与其具有至少85％、优选至少90％、更优选至少95％、进一步优选至少99％同一性的氨基酸序列。
  7. 如权利要求1-6中任一项所述的抗体，其中所述抗体为scFv。
  8. 一种靶向BCMA的嵌合抗原受体，其包含如权利要求7所述的scFv，其可 选择地进一步包括铰链区、跨膜区、共刺激结构域、胞内信号转导结构域中的至少一种。
  9. 如权利要求8所述的嵌合抗原受体，其中所述共刺激结构域选自CD27、CD28、4-1BB、OX-40、CD30、CD40、PD-1、ICOS、LFA-1、CD-2、CD7、LIGHT、NKG2C、B7-H3或其任意组合；优选地，所述共刺激结构域选自4-1BB，所述4-1BB包含如SEQ ID NO:57所述的氨基酸序列；优选地，所述胞内信号转导结构域选自CD3ζ，包含如SEQ ID NO:58所述的氨基酸序列。
  10. 一种分离的核酸分子，其编码如权利要求1-7任一项所述的抗体或8-9任一项所述的嵌合抗原受体。
  11. 一种载体，其含有如权利要求10所述的核酸分子。
  12. 一种宿主细胞，包括如权利要求10所述的核酸分子或如权利要求11所述的载体。
  13. 一种组合物或试剂盒，其包含如权利要求1至7中任一项所述的抗体、如权利要求8-9任一项所述的嵌合抗原受体、如权利要求10所述的核酸分子、如权利要求11所述的载体、如权利要求12所述的宿主细胞中的至少一种，以及药学可接受的赋形剂、稀释剂或载体。
  14. 如权利要求1至7中任一项所述的抗体、如权利要求7-8任一项所述的嵌合抗原受体、如权利要求9所述的核酸分子、如权利要求10所述的载体、如权利要求11所述的宿主细胞、或如权利要求12所述的组合物在制备用于预防或治疗受试者的B细胞相关肿瘤疾病、自身免疫疾病、病毒或细菌引起的感染性疾病的药物中的用途。
  15. 如权利要求13所述的用途，其中所述的B细胞相关肿瘤疾病选自：急性白血病诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病；慢性白血病诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病；真性红细胞增多症、淋巴瘤、套细胞淋巴瘤、弥漫大B-细胞淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链病、骨髓增生异 常综合征、多毛细胞白血病和脊髓发育不良中的至少一种。
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Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113024671B (zh) * 2020-12-31 2022-05-24 北京艺妙神州医药科技有限公司 一种抗bcma的抗体及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4694778A (en) 1984-05-04 1987-09-22 Anicon, Inc. Chemical vapor deposition wafer boat
CN102421801A (zh) * 2009-03-10 2012-04-18 比奥根艾迪克Ma公司 抗-bcma抗体
CN104379179A (zh) * 2012-04-11 2015-02-25 美国卫生和人力服务部 靶向b-细胞成熟抗原的嵌合抗原受体
WO2016014789A2 (en) 2014-07-24 2016-01-28 Bluebird Bio, Inc. Bcma chimeric antigen receptors
CN109153731A (zh) * 2015-08-11 2019-01-04 南京传奇生物科技有限公司 靶向bcma的嵌合抗原受体及其使用方法
CN111787938A (zh) * 2017-11-15 2020-10-16 诺华股份有限公司 靶向bcma的嵌合抗原受体、靶向cd19的嵌合抗原受体及组合疗法
CN112321713A (zh) * 2020-12-31 2021-02-05 北京艺妙神州医药科技有限公司 一种抗bcma的抗体及其应用

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2017001011A (es) * 2014-07-21 2018-05-28 Novartis Ag Tratamiento de cancer de usando un receptor quimerico de antigeno anti-bcma.
PT3331910T (pt) * 2015-08-03 2020-03-24 Engmab Sarl Anticorpos monoclonais contra o antigénio de maturação de células b (bcma) humano
CN105777911B (zh) * 2016-04-12 2019-07-02 上海优卡迪生物医药科技有限公司 抗bcma嵌合抗原受体、编码基因、重组表达载体及其构建方法和应用
IL268206B2 (en) * 2017-01-23 2024-05-01 Carsgen Therapeutics Co Ltd Antibodies directed against BCMA and their use
CA3053006C (en) * 2017-02-08 2023-09-05 Dana-Farber Cancer Institute, Inc. Regulating chimeric antigen receptors
IL310031A (en) * 2017-05-01 2024-03-01 Juno Therapeutics Inc A combination of cellular therapy and an immune modulatory compound
EP3703688A2 (en) * 2017-11-01 2020-09-09 Juno Therapeutics, Inc. Antibodies and chimeric antigen receptors specific for b-cell maturation antigen
RU2020120411A (ru) * 2017-11-21 2021-12-23 Новартис Аг Триспецифические связывающие молекулы, направленные против опухолеассоциированных антигенов, и пути их применения
CN110372796B (zh) * 2018-04-12 2023-05-02 上海赛比曼生物科技有限公司 靶向bcma的嵌合抗原受体及其制法和应用
CN109134665B (zh) * 2018-08-24 2021-06-11 上海先博生物科技有限公司 一种基于单域抗体的bcma嵌合抗原受体及应用
CN112125974B (zh) * 2018-09-25 2022-04-15 上海邦耀生物科技有限公司 靶向bcma蛋白的抗体、嵌合抗原受体和药物
CN109678961A (zh) * 2019-01-15 2019-04-26 深圳市南科生物工程有限公司 一种基于bmca纳米抗体序列的嵌合抗原受体的构建方法及其应用
CA3098995A1 (en) * 2019-01-16 2020-07-23 Caribou Biosciences, Inc. Humanized bcma antibody and bcma-car-t cells
CN113784733A (zh) * 2019-05-07 2021-12-10 亘喜生物科技（上海）有限公司 靶向bcma的工程化免疫细胞及其用途
CN111944054B (zh) * 2019-05-15 2022-04-05 重庆精准生物技术有限公司 抗bcma的car及其表达载体和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4694778A (en) 1984-05-04 1987-09-22 Anicon, Inc. Chemical vapor deposition wafer boat
CN102421801A (zh) * 2009-03-10 2012-04-18 比奥根艾迪克Ma公司 抗-bcma抗体
CN104379179A (zh) * 2012-04-11 2015-02-25 美国卫生和人力服务部 靶向b-细胞成熟抗原的嵌合抗原受体
WO2016014789A2 (en) 2014-07-24 2016-01-28 Bluebird Bio, Inc. Bcma chimeric antigen receptors
CN109153731A (zh) * 2015-08-11 2019-01-04 南京传奇生物科技有限公司 靶向bcma的嵌合抗原受体及其使用方法
CN111787938A (zh) * 2017-11-15 2020-10-16 诺华股份有限公司 靶向bcma的嵌合抗原受体、靶向cd19的嵌合抗原受体及组合疗法
CN112321713A (zh) * 2020-12-31 2021-02-05 北京艺妙神州医药科技有限公司 一种抗bcma的抗体及其应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"Chimeric Antigen Receptor T-cell Therapies for Multiple Myeloma", BLOOD, 14 December 2017 (2017-12-14)
B-CELL MATURATION ANTIGEN IS A PROMISING TARGET FOR ADOPTIVE T-CELL THERAPY OF MULTIPLE MYELOMA, CLINICAL CANCER RESEARCH, 15 April 2013 (2013-04-15)
CELLULAR IMMUNOLOGY, 14 March 2016 (2016-03-14)
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883
SKERRA ET AL., SCIENCE, vol. 242, 1988, pages 1038 - 1041
WARD ET AL., NATURE, vol. 334, 1989, pages 54454

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