BCMA-TARGETED ANTIBODY AND CHIMERIC ANTIGEN RECEPTOR

Abstract

An antibody specifically binding to BCMA and an antigen binding fragment thereof, a chimeric antigen receptor (CAR) containing the antigen binding fragment, a nucleic acid molecule encoding the CAR, an immune effect cell expressing the CAR, a method for preparing the immune effect cell, use of the CAR and the immune effect cell for preventing and/or treating a B cell-related disease (for example, a B-cell malignancy and an autoimmune disease), and a method for preventing and/or treating the B cell-related disease.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to BCMA, the antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) a sequence set forth in SEQ ID NO:1 or 3; (ii) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) compared to the sequence set forth in SEQ ID NO: 1 or 3; or (iii) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared to the sequence set forth in SEQ ID NO: 1 or 3; and/or, (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) a sequence set forth in SEQ ID NO: 2 or 4; (v) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) compared to the sequence set forth in SEQ ID NO: 2 or 4; or (vi) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared to the sequence set forth in SEQ ID NO: 2 or 4; preferably, the substitution described in (ii) or (v) is a conservative substitution.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) a sequence set forth in SEQ ID NO: 1; (ii) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) compared to the sequence set forth in SEQ ID NO: 1; or (iii) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared to the sequence set forth in SEQ ID NO: 1; and, (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) a sequence set forth in SEQ ID NO: 2; (v) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) compared to the sequence set forth in SEQ ID NO: 2; or (vi) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared to the sequence set forth in SEQ ID NO: 2; preferably, the substitution described in (ii) or (v) is a conservative substitution; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising a sequence set forth in SEQ ID NO: 1 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared thereto, the VL comprising a sequence set forth in SEQ ID NO: 2 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% compared to the %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared thereto.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) a sequence set forth in SEQ ID NO: 3; (ii) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) compared to the sequence set forth in SEQ ID NO: 3; or (iii) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared to the sequence set forth in SEQ ID NO: 3; and, (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) a sequence set forth in SEQ ID NO: 4; (v) a sequence having a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) compared to the sequence set forth in SEQ ID NO: 4; or (vi) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared to the sequence set forth in SEQ ID NO: 4; preferably, the substitution described in (ii) or (v) is a conservative substitution; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising a sequence set forth in SEQ ID NO: 3 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared thereto, the VL comprising a sequence set forth in SEQ ID NO: 4 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% compared to the %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared thereto.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region (CH) and a light chain constant region (CL); preferably, the heavy chain constant region is selected from the group consisting of IgG, IgM, IgE, IgD or IgA; preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; preferably, the light chain constant region is selected from κ or λ; preferably, the light chain constant region is a κ light chain constant region.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of scFv, di-scFv, (scFv).sub.2, Fab, Fab′, (Fab′).sub.2, Fv, disulfide-linked Fv.

6. A chimeric antigen receptor (CAR), which comprises an extracellular antigen-binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises the antibody or antigen-binding fragments thereof according to any one of claims 1 to 5; preferably, the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab fragment, Fab′ fragment, F(ab)′.sub.2 fragment, Fv, disulfide-stabilized Fv protein (“dsFv”), scFv, di-scFv, (scFv).sub.2; preferably, the antibody or antigen-binding fragment thereof is selected from the group consisting of scFv, di-scFv, (scFv).sub.2.

7. The chimeric antigen receptor according to claim 6, wherein the extracellular antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising a sequence set forth in SEQ ID NO: 1 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared thereto, the VL comprising a sequence set forth in SEQ ID NO: 2 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% compared to the %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared thereto; preferably, the VH and VL are connected by a linker; preferably, the linker has a sequence set forth in SEQ ID NO:5.

8. The chimeric antigen receptor according to claim 6, wherein the extracellular antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising a sequence set forth in SEQ ID NO: 3 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared thereto, the VL comprising a sequence set forth in SEQ ID NO: 4 or a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% compared to the %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared thereto; preferably, the VH and VL are connected by a linker; preferably, the linker has a sequence set forth in SEQ ID NO:5.

9. The chimeric antigen receptor according to any one of claims 6 to 8, wherein the spacer domain is selected from the group consisting of a hinge domain and/or CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4); preferably, the spacer domain comprises a hinge region of CD8α; preferably, the spacer domain comprises a sequence set forth in SEQ ID NO:6.

10. The chimeric antigen receptor according to any one of claims 6 to 9, wherein the transmembrane domain is a transmembrane region of a protein selected from the group consisting of α, β or ζ chains of T cell receptor, CD8α, CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, DAP10; preferably, the transmembrane domain comprises a transmembrane region of CD8α; preferably, the transmembrane domain comprises a sequence set forth in SEQ ID NO:7.

11. The chimeric antigen receptor according to any one of claims 6 to 10, wherein the intracellular signaling domain comprises a primary signaling domain and optionally a costimulatory signaling domain; preferably, the intracellular signaling domain comprises a costimulatory signaling domain and a primary signaling domain in order from the N-terminal to the C-terminal; preferably, the intracellular signaling domain comprises a primary signaling domain and at least one costimulatory signaling domain; preferably, the primary signaling domain comprises an immunoreceptor tyrosine activation motif (ITAM); preferably, the primary signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD22, CD79a, DAP10, CD79b or CD66d; preferably, the primary signaling domain comprises an intracellular signaling domain of CD3ζ; preferably, the primary signaling domain comprises a sequence set forth in SEQ ID NO: 9; preferably, the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) or DAP10; preferably, the costimulatory signaling domain is selected from an intracellular signaling domain of CD28 or an intracellular signaling domain of CD137 (4-1BB) or a combination thereof; preferably, the costimulatory signaling domain comprises a sequence set forth in SEQ ID NO: 8.

12. The chimeric antigen receptor according to any one of claims 6 to 11, wherein the chimeric antigen receptor comprises an extracellular antigen-binding domain, a spacer domain, a transmembrane domain, an intracellular signaling domain in order from the N-terminal to the C-terminal; preferably, the spacer domain comprises a hinge region of CD8α (e.g., a sequence set forth in SEQ ID NO: 6); preferably, the transmembrane domain comprises a transmembrane region of CD8α (e.g., a sequence set forth in SEQ ID NO: 7); preferably, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises an intracellular signaling domain of CD3ζ (e.g., a sequence set forth in SEQ ID NO: 9), the costimulatory signaling domain comprises an intracellular signaling domain of CD137 (e.g., a sequence set forth in SEQ ID NO: 8); preferably, the chimeric antigen receptor has an amino acid sequence selected from the group consisting of: (1) an amino acid sequence set forth in SEQ ID NO: 10 or 12; (2) a sequence having a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared to the amino acid sequence set forth in SEQ ID NO: 10 or 12.

13. An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 6 to 12; preferably, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 6 to 12, and the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (1) a nucleotide sequence set forth in SEQ ID NO: 11 or 13; (2) a sequence having a sequence identity of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% %, at least 98%, at least 99%, or 100% compared to the nucleotide sequence set forth in SEQ ID NO: 11 or 13.

14. A vector, which comprises the isolated nucleic acid molecule according to claim 13; preferably, the vector comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; preferably, the vector comprises a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 6 to 12; preferably, the vector is selected from the group consisting of DNA vector, RNA vector, plasmid, transposon vector, CRISPR/Cas9 vector, or viral vector; preferably, the vector is an expression vector; preferably, the vector is an episomal vector; preferably, the vector is a viral vector, such as a lentiviral vector, adenoviral vector or retroviral vector.

15. A host cell, which comprises the isolated nucleic acid molecule according to claim 13, or the vector according to claim 14; preferably, the host cell comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or a vector comprising the nucleotide sequence; preferably, the host cell comprises E. coli, yeast, insect cell, or mammalian cell; preferably, the host cell comprises a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 6 to 12 or a vector comprising the nucleotide sequence; preferably, the host cell comprises an immune cell (e.g., a human immune cell); more preferably, the immune cell is selected from T lymphocyte, NK cell, monocyte, macrophage or dendritic cell, and any combination thereof

16. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which comprises, culturing the host cell according to claim 15 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from a culture of the cultured host cell; wherein the host cell comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or a vector comprising the nucleotide sequence.

17. An immune effector cell, which expresses the chimeric antigen receptor according to any one of claims 6 to 12; preferably, the immune effector cell comprises a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 6 to 12 or a vector comprising the nucleotide sequence; preferably, the immune effector cell is selected from the group consisting of T lymphocyte, NK cell, monocyte, macrophage or dendritic cell and any combination thereof; preferably, the immune effector cell is selected from T lymphocyte and/or NK cell.

18. The method for preparing the immune effector cell according to claim 17, which comprises: (1) providing an immune effector cell; (2) introducing the isolated nucleic acid molecule according to claim 13 or the vector according to claim 14 into the immune effector cell; wherein the isolated nucleic acid molecule or vector comprises a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 6 to 12; preferably, the immune effector cell is selected from the group consisting of T lymphocyte, NK cell, monocyte, dendritic cell, macrophage and any combination thereof; preferably, the immune effector cell is selected from T lymphocyte and/or NK cell; preferably, in step (1), the immune effector cell is subjected to a pretreatment, and the pretreatment comprises sorting, activation and/or proliferation of the immune effector cell; preferably, the pretreatment comprises contacting the immune effector cell with an anti-CD3 antibody and an anti-CD28 antibody; preferably, in step (2), the nucleic acid molecule or vector is introduced into the immune effector cell by viral infection; preferably, in step (2), the nucleic acid molecule or vector is introduced into the immune effector cell by means of non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method; preferably, a step of expanding the immune effector cell obtained in step (2) is further comprised after step (2).

19. A test kit, which comprises the isolated nucleic acid molecule according to claim 13 or the vector according to claim 14; preferably, the isolated nucleic acid molecule or vector comprises a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 6 to 12.

20. Use of the kit according to claim 19 in the manufacture of a chimeric antigen receptor that specifically binds to BCMA or a cell expressing the chimeric antigen receptor; preferably, the cell is an immune effector cell, such as T lymphocyte and/or NK cell.

21. A conjugate, which comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and a modification moiety linked to the antibody or antigen-binding fragment thereof; preferably, the modification moiety is selected from a detectable label, such as enzyme, radionuclide, fluorescent dye, luminescent substance (e.g., chemiluminescent substance) or biotin; preferably, the modification moiety is selected from a therapeutic agent, such as an anti-tumor activity drug or a cytotoxic agent.

22. A pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the chimeric antigen receptor according to any one of claims 6 to 12, or the isolated nucleic acid molecule according to claim 13, or the vector according to claim 14, or the host cell according to claim 15, or the immune effector cell according to claim 17, or the conjugate according to claim 21, and a pharmaceutically acceptable carrier and/or excipient; preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as alkylating agent, mitotic inhibitor, anti-tumor antibiotic, anti-metabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, anti-angiogenic agent, cytokine, antibody that specifically targets a tumor cell, or immune checkpoint inhibitor.

23. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the chimeric antigen receptor according to any one of claims 6 to 12, or the isolated nucleic acid molecule according to claim 13, or the vector according to claim 14, or the host cell according to claim 15, or the immune effector cell according to claim 17, or the conjugate according to claim 21, or the pharmaceutical combination according to claim 22, in the manufacture of a medicament for the prevention and/or treatment of a B cell-related disease in a subject (e.g., a human); preferably, the B-related disease is a B-cell malignancy, for example, multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL); preferably, the B cell-related disease is an autoimmune disease, such as systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura or myasthenia gravis or autoimmune hemolytic anemia; preferably, the B cell-related disease is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, B cell proliferation of uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disease, immunomodulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas' disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated small vessel vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolysis anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis, or monoclonal gammopathy of undetermined significance.

24. A method for preventing and/or treating a B cell-related disease in a subject (e.g., a human), the method comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the immune effector cell according to claim 17, or the conjugate according to claim 21, or the pharmaceutical combination according to claim 22, preferably, the B-related condition is a B-cell malignancy, for example, multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL); preferably, the B cell-related disease is an autoimmune disease, such as systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura or myasthenia gravis or autoimmune hemolytic anemia; preferably, the B cell-related disease is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, B cell proliferation of uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disease, immunomodulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas' disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated small vessel vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolysis anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immune cell-associated amyloidosis, or monoclonal gammopathy of undetermined significance.

25. A method for preventing and/or treating a B cell-related disease in a subject (e.g., a human), the method comprising the steps of: (1) providing an immune effector cell; (2) introducing an isolated nucleic acid molecule or vector comprising a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 6 to 12 into the immune effector cell described in step (1) to obtain an immune effector cell expressing the chimeric antigen receptor; (3) administering the immune effector cell obtained in step (2) to the subject for treatment; preferably, the immune effector cell is selected from the group consisting of T lymphocyte, NK cell, monocyte, macrophage, dendritic cell, or any combination of these cells; preferably, prior to step (1), the method further comprises a step of obtaining the immune effector cell from the subject.

26. A method for diagnosing whether a subject (e.g., a human) suffers from a BCMA-expressing tumor, which comprises detecting an amount of BCMA in a sample from the subject by using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; preferably, the method further comprises a step of comparing the amount of BCMA in the sample from the subject to a reference value; preferably, the amount of BCMA in the sample from the subject is detected by: (1) contacting the sample from the subject with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; (2) detecting an amount of a complex formed by the antibody or antigen-binding fragment thereof and BCMA; preferably, in step (1), the antibody or antigen-binding fragment thereof further bears a detectable label; preferably, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, ascites, circulating cell, circulating tumor cell, non-tissue-associated cell, tissue (e.g., surgically resected tumor tissue, biopsy or fine needle aspiration tissue), histological preparation; preferably, the BCMA-expressing tumor is selected from B-cell malignancies, such as multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL); preferably, the method further comprises administering a BCMA-targeted immunotherapy to the subject diagnosed with a BCMA-expressing tumor; preferably, the BCMA-targeted immunotherapy is the method according to claim 24 or 25.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0260] FIG. 1 shows the binding curves of humanized antibodies C11D5.3-03, C11D5.3-04 and parental murine antibody C11D5.3 to recombinant human BCMA protein.

[0261] FIG. 2 shows the dose-dependent binding curves of humanized antibodies C11D5.3-03, C11D5.3-04 and parental murine antibody C11D5.3 to human multiple myeloma cell line U266.

[0262] FIG. 3 shows the dose-dependent binding curves of humanized antibodies C11D5.3-03, C11D5.3-04 and parental murine antibody C11D5.3 to human multiple myeloma cell line MM.1S.

[0263] FIG. 4 shows the dose-dependent binding curves of humanized antibodies C11D5.3-03, C11D5.3-04 and parental murine antibody C11D5.3 to human multiple myeloma cell line RPMI8226.

[0264] FIG. 5 shows the dose-dependent binding curves of humanized antibodies C11D5.3-03, C11D5.3-04 and parental murine antibody C11D5.3 to human Burkitt's lymphoma cell Daudi that does not express BCMA.

[0265] FIG. 6 shows the binding curves of humanized antibodies C11D5.3-03, C11D5.3-04 and parental murine antibody C11D5.3 to human immortalized keratinocyte cell line Hacat.

[0266] FIG. 7 shows the binding curves of humanized antibodies C11D5.3-03, C11D5.3-04 and parental murine antibody C11D5.3 to human gastric mucosa epithelial cell line GES.

[0267] FIG. 8 shows the expression of BCMA-CAR on T cells transduced with C11D5.3-CAR, or humanized C11D5.3-03-CAR and C11D5.3-04-CAR.

[0268] FIG. 9 shows the IFNy release assay results of C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T, C11D5.3-04 CAR-T.

[0269] FIG. 10 shows the IL2 release assay results of C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T, C11D5.3-04 CAR-T.

[0270] FIG. 11 shows the LDH release assay results of U266 killing caused by C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T and C11D5.3-04 CAR-T.

[0271] FIG. 12 shows the LDH release assay results of Daudi killing caused by C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T and C11D5.3-04 CAR-T.

[0272] FIGS. 13-17 show respectively the detection results of the release levels of IL4 (FIG. 13), IL6 (FIG. 14), IL10 (FIG. 15), TNFα (FIG. 16) and IFNγ (FIG. 17) of C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T, C11D5.3-04 CAR-T and untransfected PBMC cells in the serum-free culture system in the absence of antigen stimulation.

[0273] FIG. 18 shows the detection results of the release levels of IL4, IL6, IL10, TNFα and IFNγ of C11D5.3 CAR-T or humanized C11D5.3-03 CAR-T, C11D5.3-04 CAR-T and untransfected PBMC cells in the culture system containing 5% human serum in the absence of antigen stimulation.

[0274] FIG. 19 shows the detection results of the expression level of HLA-DR on the surface of CAR-T cells.

[0275] FIG. 20 shows the detection results of the expression level of CD25 on the surface of CAR-T cells.

[0276] FIG. 21 shows the detection results of the expression levels of activated Caspase-3 in C11D5.3-03 CAR-T, C11D5.3-04 CAR-T, C11D5.3 CAR-T and the control PBMC.

SEQUENCE INFORMATION

[0277] Information on the partial sequences involved in the present invention is provided in Table 1 below.

TABLE-US-00001 TABLE 1 Description of sequences SEQ ID NO Description 1 C11D5.3-03 VH 2 C11D5.3-03 VL 3 C11D5.3-04 VH 4 C11D5.3-04 VL 5 Linker 6 Hinge region 7 Transmembrane region 8 CD137 intracellular signaling domain 9 CD3ζ ITAM 10 C11D5.3-03-CAR full-length amino acid sequence 11 nucleic acid sequence encoding C11D5.3-03-CAR 12 C11D5.3-04-CAR full-length amino acid sequence 13 Nucleic acid sequence encoding C11D5.3-04-CAR 14 C11D5.3-CAR full-length amino acid sequence 15 Nucleic acid sequence encoding C11D5.3-CAR 16 Nucleic acid sequence encoding P2A-tEGFR

EXAMPLES

[0278] The present invention will now be described with reference to the following examples, which are intended to illustrate, but not limit, the present invention.

[0279] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Refined Laboratory Guide for Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes were used according to the conditions recommended by the product manufacturer. Those skilled in the art appreciate that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention as claimed.

Example 1

Humanization of Anti-BCMA Murine Antibody and Preparation of Humanized Antibody

[0280] In order to improve the sequence homology between the antibody and the human antibody and to reduce the immunogenicity of the antibody to human, the mouse antibody C11D5.3 (of which the heavy chain variable region sequence and light chain variable region sequence referred to SEQ ID NO: 3 and SEQ ID NO: 12 in CN 201510142069.2, respectively) were subjected to the design for humanization by using methods known in the art to insert murine CDR regions into human framework sequences (see U.S. Pat. No. 5,225,539 to Winter; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.; and Lo, Benny, K C, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Generally, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FRs and/or constant regions) are derived from human immunoglobulin (receptor antibody). According to the above method and after extensive screening, two humanized antibodies (scFv) were constructed based on the CDRs of the murine antibody C11D5.3, named C11D5.3-03 and C11D5.3-04 respectively. The VH and VL of C11D5.3-03 were set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the VH and VL of C11D5.3-04 were set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. In addition, the murine antibody C11D5.3 (scFv) was prepared as a reference antibody.

[0281] The HEK293-F cells (Invitrogen, R7907) transiently transfected with expression plasmids encoding the above antibodies were cultured in Freestyle 293 expression medium (Invitrogen, 12338018) and incubated in a CO.sub.2 incubator at 37° C. for 5-7 days. Cells and cell debris were removed by centrifugation, and the culture supernatant was collected, filtered through a 0.22 μm membrane. Purification was performed by using a protein A column (GE Life Science, 17127901) according to the manufacturer's instructions, in which after washing with PBS, an acid buffer (pH 3.5) containing 20 mM citrate and 150 mM NaCl was used to elute antibodies, and the eluate was neutralized with 1M Tris (pH 8.0), and dialyzed against PBS. Antibody concentration was determined by measuring absorbance at 280 nm, and aliquoted antibodies were stored in a −80° C. refrigerator.

Example 2

Affinity Assessment of Anti-BCMA Humanized Antibodies to Recombinant Human BCMA Protein

[0282] The binding activity of the antibodies to human BCMA protein was assessed using an ELISA method, and the antibody potency was compared using dose-dependent binding curves in ELISA.

[0283] BCMA protein (Sino Biological, 10620-H08H) was diluted to 1 μg/mL in PBS, 100 μL of the protein solution was added to each well of the ELISA plate and incubated at 4° C. overnight. The plate was washed 3 times in wash buffer (PBS, 0.05% Tween 20), and non-specific sites were blocked by adding 200 μl/w PBS+2% BSA blocking solution. C11D5.3-03, C11D5.3-04 or murine antibody C11D5.3 was added and incubated at 37° C. for 2 h. The plate was washed 3 times in wash buffer, and HRP-conjugated goat anti-human secondary antibody was added and incubated for 1 h at room temperature. The plate was washed 3 times in wash buffer, and the bound secondary antibody was detected by adding TMB (HRP substrate) and incubating the plate in the dark at room temperature for 5 to 10 minutes. The enzymatic reaction was stopped by adding 1M of sulfuric acid solution, and the light absorption at 450 nm was measured.

[0284] The results were shown in FIG. 1. The humanized antibodies C11D5.3-03 and C11D5.3-04 had EC50 values (effective concentration at 50% activity) of 0.011 μg/mL and 0.011 μg/mL, respectively, indicating they had equivalent binding activity compared with the murine antibody C11D5.3 (EC50=0.011 μg/mL).

Example 3

Affinity Assessment of Anti-BCMA Humanized Antibodies to BCMA-Expressing Tumor Cells

[0285] The binding activity of the humanized antibodies obtained in Example 1 to BCMA-expressing cells was evaluated using the FACS method, and the antibody potency was compared using the dose-dependent binding curves in FACS.

[0286] Cells (human myeloma cells expressing BCMA or tissue cells not expressing BCMA) were digested, and 500,000 cells were seeded in 50 μL of PBS containing 2% FBS (FACS buffer) in a U-bottom 96-well plate. The test samples were subjected to a 3-fold serial dilution by diluting ⅓ volume (100 μL) in 200 μL of FACS buffer, starting at a concentration of 300 μg/mL (final concentration). 50 μL of the diluted antibody was added to each well of the cell plate and incubated at 4° C. for 1 hour. After washing twice with FACS buffer, 100 μL of secondary antibody (Abcam ab97003, 5 μg/mL, diluted in FACS buffer) was added to each well, and incubated at 4° C. for 0.5 hours. After staining and washing twice with FACS buffer, 200 μL of FACS buffer was added to each well, and reading was performed on the machine (BD C6plus).

[0287] FIGS. 2, 3, 4 and 5 showed the dose-dependent binding curves of the antibodies to human multiple myeloma cells U266, MM.1S, RPMI8226, and human Burkitt's lymphoma cell Daudi that did not express BCMA, respectively. The EC50 values were shown in Table 2. The results showed that for the three BCMA-expressing tumor cells, the humanized antibodies C11D5.3-03 and C11D5.3-04 had a high affinity comparable to that of the murine antibody; in the meantime, for the tumor cells not expressing BCMA, the humanized antibodies C11D5.3-03 and C11D5.3-04 also exhibited a low affinity comparable to that of the murine antibody.

TABLE-US-00002 TABLE 2 EC50 values of anti-BCMA antibodies binding to BCMA-expressing tumor cells EC50 (μg/ml) C11D5.3 C11D5.3-03 C11D5.3-04 U266 4.4 7.57 1.99 MM.1S 1.62 0.81 0.47 RPMI8226 3.81 2.25 0.82 Daudi 58.25 36.32 30.71

Example 4

Evaluation of Non-Specific Binding of Anti-BCMA Humanized Antibodies to Non-Tumor Tissue Cells

[0288] The non-specific binding between the purified humanized antibodies obtained in Example 1 and non-tumor tissue cells was evaluated using FACS method, and using tissue cells that did not express BCMA, and the specific method was as described above. FIGS. 6-7 showed the binding curves of the antibodies to human immortalized keratinocyte Hacat and human gastric mucosal epithelial cell GES, respectively. As shown in the figures, the murine antibody C11D5.3 bound to both Hacat and GES cells at high concentration (100 μg/mL), in contrast, the two humanized antibodies C11D5.3-03 and C11D5.3-04 did not bind to either of the two non-tumor tissue cells at the concentrations tested (0.003 to 100 μg/mL). The above results showed that the humanized antibodies of the present invention had reduced non-specific binding compared to the murine antibody C11D5.3, and such a technical effect was significant and unexpected.

Example 5

Construction and Preparation of Chimeric Antigen Receptor (CAR) Lentiviral Expression Vector

[0289] Based on the humanized antibodies obtained in the above example, CAR lentiviral expression vectors were further constructed. In the constructed chimeric antigen receptor lentiviral expression vectors, the amino acid sequences of elements from the N-terminus to the C-terminus were shown in Table 3 below. The CAR comprised the scFv (VH+VL) of the humanized antibody described above, CD8α hinge region, CD8α transmembrane region, CD137 intracellular domain, and CD3ζ ITAM region. The nucleic acid sequence encoding the above CAR was linked with the sequence (SEQ ID NO: 16) encoding P2A-tEGFR and inserted into the GV401 vector (Genechem), with the frame of EF1a Promoter-BCMA CAR-P2A-tEGFR-WPRE, and the BCMA CAR expression was monitored by detecting tEGFR expression.

TABLE-US-00003 TABLE 3 Amino acid sequences of elements from N-terminal to C-terminal in chimeric antigen receptors N-terminal .fwdarw. C-terminal, amino acid sequence Encoding of each element (SEQ ID NO:) Full- nucleic CAR Hinge Transmembrane length acid name VH Linker VL region region CD137 CD3ζ sequence sequence C11D5.3- 1 5 2 6 7 8 9 10 11 03 C11D5.3- 3 4 12 13 04 C11D5.3 SEQ ID NO: 3 of SEQ ID NO: 12 of 14 15 CN201510142069.2 CN201510142069.2

Example 6

Preparation of CAR-T Cells

[0290] PBMC cells were collected from healthy people (Xuanfeng Bio, SLB-HP010); 1 μg/ml CD3 antibody (Miltenyi, 170-076-124) and 1 μg/ml CD28 antibody (Miltenyi, 170-076-117) were coated in a 6-well plate; PBMC was added to the coated 6-well plate and incubated overnight. The CAR lentiviral vectors described in Example 5 were added to PBMC at MOI=3, and centrifuged at 2000 rpm for 60 minutes for transduction, thereby obtaining the T cells transduced with murine C11D5.3-CAR (C11D5.3 CAR-T), the T cells transduced with humanized Cl1D5.3-03-CAR (C11D5.3-03 CAR-T), and the T cells transduced with humanized C11D5.3-04-CAR (C11D5.3-04 CAR-T). After 48 hours of culture, tEGFR expression was detected with Cetuximab labeled with PE fluorescence.

[0291] FIG. 8 showed the expression of BCMA-CAR on untransfected control PBMC and the above three CAR-T cells. The results showed that all the above three CAR-Ts could express the corresponding anti-BCMA CAR, indicating that the anti-BCMA CAR-T was successfully constructed.

Example 7

Evaluation of In Vitro Anti-tumor Ability of Anti-BCMA CAR-T

[0292] Cytokine release assay: The anti-BCMA CAR-T cells prepared in Example 6 were collected, washed twice with DPBS solution, and resuspended in RPMI1640 medium containing 2% FBS; the tumor cells were collected, and then washed and resuspended in RPMI1640 medium containing 2% FBS, the tumor cells included: tumor cells that did not express BCMA (293T, K562), tumor cells that expressed BCMA (M1ss, U266, RPMI8226, Daudi).

[0293] The anti-BCMA CAR-T cells and the above tumor cell lines were mixed and cultured at E:T (effector cells/target cells)=1:1 for 16 hours, and supernatants were collected and the release of cytokines was determined by BD CBA assay. The IFNγ and IL2 release results of the control PBMC, C11D5.3 CAR-T, C11D5.3-03 CAR-T, and C11D5.3-04 CAR-T were shown in FIG. 9 and FIG. 10, respectively.

[0294] The above results showed that the humanized C11D5.3-03 CAR-T and C11D5.3-04 CAR-T were similar to the murine C11D5.3 CAR-T, which could specifically recognize the BCMA+ cell line and release IFNγ and IL2.

[0295] Tumor cell lysis experiment: BCMA-expressing cell lines (U266, Daudi) and anti-BCMA CAR-T cells were mixed and cultured for 4 hours according to E:T=30:1, 10:1, and 3:1, respectively, and the supernatants were collected to detect LDH release (CytoTox96 Non-Radioactive Cytotoxicity Assay). The LDH release results of U266 and Daudi killed by the control PBMC, C11D5.3 CAR-T, C11D5.3-03 CAR-T, C11D5.3-04 CAR-T were shown in FIG. 11 and FIG. 12, respectively.

[0296] The above results showed that the humanized C11D5.3-03 CAR-T and C11D5.3-04 CAR-T were similar to the murine C11D5.3 CAR-T, and could recognize and kill BCMA+ tumor cells.

Example 8

Cytokine Release of Humanized Anti-BCMA CAR-T Cells

[0297] It was reported that anti-BCMA CAR-T cells could release cytokines in the absence of BCMA antigen, and this release level varied with the structural differences in anti-BCMA CAR molecules; the toxicity caused by enhanced cytokine release was one of the known toxicity problems of anti-BCMA CAR T cell clinical therapy. It was known that CAR-T cell therapy based on murine antibody C11D5.3 had not found significant enhanced cytokine release problems in the current clinical practice. However, the study further found that some humanized anti-BCMA CARs caused antigen-independent cytokine release, which would make humanized anti-BCMA CARs unsuitable for T-cell therapy. To this end, this example evaluated the antigen-independent cytokine release of humanized anti-BCMA CAR-T cells.

[0298] The CAR-T obtained in Example 6 or PBMC control was statically cultured in AIM-V culture medium (Gibco, A3021002) containing 60 IU/ml IL2 (Miltenyi, 130-097-748) in an incubator at 5% CO.sub.2 and 37° C., and the cytokine release in the absence of stimulation was determined at various incubation time points. The control PBMC, C11D5.3 CAR-T, C11D5.3-03 CAR-T, or C11D5.3-04 CAR-T was cultured, supernatant was collected on the first day (before the first medium change), the second day, the third day, the fifth day (before the second medium change), the seventh day, and the ninth day, and the multiplexed magnetic bead method (TH1/TH2 cytokine CBA kit, BD, #550749) was used to determine the release levels of 5 cytokines released into the supernatant, including IL4, IL6, IL10, TNFα and IFNγ. FIGS. 13-17 showed the comparison of the release levels of IL4, IL6, IL10, TNFα and IFNγ at different culture time points for the above three CAR-T cells and untransfected PBMC cells in the serum-free culture system without antigen stimulation, respectively. It could be seen that in the absence of stimulation, the release of type 1 inflammatory factors of humanized C11D5.3-03 CAR-T and C11D5.3-04 CAR-T did not change significantly compared with the untransfected PBMC cells.

[0299] The above results indicated that the humanized anti-BCMA CAR-T cells of the present invention did not induce significant antigen-independent cytokine release.

[0300] In AIM-V medium (Gibco, A3021002) containing human serum (Corning, MT35060CI) but without BCMA antigen, 1×10.sup.5 cells of control PBMC, C11D5.3 CAR-T, C11D5.3-03 CAR-T, or C11D5.3-04 CAR-T were cultured for 48 hours, and the multiplexed magnetic bead method was used to determine the levels of 5 cytokines released into the supernatant, including IL4, IL6, IL10, TNFα and IFNγ. The release cytokines of the above three CAR-T cells were compared to assess whether sequence changes in C11D5.3 caused by humanization would introduce a new reactivity to proteins in human serum that was not seen in mouse anti-BCMA CAR-T cells. FIG. 18 showed a comparison of release levels of IL4, IL6, IL10, TNFα and IFNγ for the above three CAR-T cells and the untransfected PBMC cells in the absence of antigen stimulation in a culture system containing 5% human serum. It could be seen that the release of type 1 inflammatory factors of the two humanized anti-BCMA CAR-T cells did not change significantly compared with the murine CAR-T cells, and did not change significantly in terms of the release of type 1 inflammatory factors compared with the untransfected PBMC cells either.

[0301] The above results indicated that the sequence changes to C11D5.3 caused by the humanization of C11D5.3-03 and C11D5.3-04 did not introduce a new reactivity to proteins in human serum that was not seen in mouse anti-BCMA CAR-T cells.

[0302] In sum, the CARs obtained based on the humanized antibodies C11D5.3-03 and C11D5.3-04 of the present invention could reduce the potential immunogenicity caused by the immune response of the mouse sequence, while showing no obvious antigen-independent cytokine release and new reactivity to proteins in human serum, thereby having significantly improved safety. Such technical effects were remarkable and unexpected.

Example 9

Evaluation of Activation Status of Humanized Anti-BCMA CAR T Cells

[0303] The CAR-T or PBMC control prepared in Example 6 were cultured in the culture system described in Example 8, the expression of T cell activation phenotype markers on the surface of the three CAR-T cells were detected on the fifth day and the ninth day (end point) of culturing, to assess whether sequence changes to C11D5.3 caused by humanization would introduce hyperactivation of CAR-T cells that was not seen in mouse anti-BCMA CAR-T cells. The expression levels of HLA-DR (APC anti-human HLA-DR Antibody, Biolegend) and CD25 (FITC anti-human CD25 Antibody, Biolegend) on the surface of CAR T cells were detected by FACS method.

[0304] The results were shown in FIG. 19 and FIG. 20, respectively. The results showed that the humanized C11D5.3-03 CAR-T and C11D5.3-04 CAR-T did not show hyperactivation of CAR-T cells compared with the murine C11D5.3 CAR-T.

Example 10

Apoptosis Evaluation of Humanized Anti-BCMA CAR T Cells

[0305] The exhaustion of T cells is often associated with the activation-induced cell death caused by apoptosis. Levels of activated Caspase-3 (CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit, Invitrogen) were measured to examine whether the introduction of anti-BCMA CAR could lead to higher levels of T cell apoptosis. The expression levels of activated Caspase-3 in C11D5.3-03 CAR-T, C11D5.3-04 CAR-T, C11D5.3 CAR-T and the control PBMC were detected by FACS method, and the results were shown in FIG. 21. The results showed that the introduction of humanized C11D5.3-03 CAR and C11D5.3-04 CAR did not affect the expression level of activated Caspase-3 in T cells, and did not lead to cell death caused by T cell apoptosis.

[0306] Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details in light of all the teachings that have been published, and that these changes are all within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

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