WO2021068761A1 - 靶向bcma的具有人猴交叉的人源化单克隆抗体 - Google Patents

靶向bcma的具有人猴交叉的人源化单克隆抗体 Download PDF

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WO2021068761A1
WO2021068761A1 PCT/CN2020/117757 CN2020117757W WO2021068761A1 WO 2021068761 A1 WO2021068761 A1 WO 2021068761A1 CN 2020117757 W CN2020117757 W CN 2020117757W WO 2021068761 A1 WO2021068761 A1 WO 2021068761A1
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antibody
seq
cell
bcma
cdr
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李宁
曹国庆
郎国竣
刘婵娟
胡宇豪
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Suzhou Qin Pharmaceuticals Co Ltd
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Suzhou Qin Pharmaceuticals Co Ltd
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Priority to JP2022521754A priority patent/JP2022551662A/ja
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to a monoclonal antibody that specifically binds to B cell maturation antigen (BCMA), including monoclonal antibodies and fragments thereof that specifically bind to human BCMA and monkey BCMA antigens at the same time.
  • BCMA B cell maturation antigen
  • the present invention relates to monoclonal antibodies and fragments thereof that specifically bind to BCMA and inhibit the binding of BAFF and APRIL to BCMA receptors.
  • the present invention also relates to monoclonal antibodies and fragments thereof that specifically bind to BCMA and have excellent endocytosis effects.
  • B cells mature in the bone marrow and become plasma cells, which can secrete antibodies against foreign viruses or bacteria.
  • plasma cells become cancerous and become myeloma cells, they will continue to proliferate more malignant myeloma cells and secrete a lot of useless antibodies.
  • Myeloma usually grows on the spine, skull, pelvis, chest, etc., and manifests as a tumor or osteolytic lesions.
  • myeloma The condition of myeloma is usually gradual, from monoclonal gammaglobulinemia (MGUS) of unknown significance to low-risk smoky multiple myeloma to high-risk smoky multiple myeloma (SMM), eventually progressing to Multiple myeloma (Nature Reviews Disease Primers, 2017, 3, 17046.).
  • Multiple myeloma (Multiple myeloma, MM) main features include hypercalcemia, renal dysfunction, anemia, bone dysfunction, etc., accompanied by severe bone pain and prone to repeated fractures (Nature Reviews Clinical Oncology, 2012, 9 (3), 135–143.).
  • the International Myeloma Foundation According to statistics from the International Myeloma Foundation, as of August 2017 alone, the number of patients worldwide had reached about 750,000, with approximately 114,400 new cases each year, and nearly 90,000 deaths from the disease.
  • BCMA B cell maturation antigen
  • BCMA belongs to the cell surface Transmembrane receptor, its gene is located at the TNFRSF17 locus of chromosome 16. It has been reported in the literature (Blood Cancer Journal, 2015, 5(2), e282–e282.) that BCMA-deficient mice are relatively normal in appearance and number of B cells, but their plasma cell survival ability is extremely poor.
  • the ligands of BCMA include B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL).
  • BAFF B cell activating factor
  • APRIL proliferation-inducing ligand
  • the receptors of BAFF also include BAFF-R and TACI
  • the receptors of APRIL also include TACI.
  • the main role of the BCMA signaling pathway is to promote the survival, differentiation, and activation of regulatory T cells.
  • the TACI signaling pathway inhibits the maturation of B cells (Nature reviews immunology, 2009, 9(7): 491.).
  • BAFF-R For these three receptors (BAFF-R, TACI and BCMA), during the development of B cells, only BAFF-R is expressed on the surface of immature B cells, migrating B cells and initial B cells; GC B cell surface Both BAFF-R and BCMA are expressed; BAFF-R, TACI and BCMA are expressed on the surface of memory cells; TACI and BCMA are expressed on the surface of plasmablasts or plasma cells; and when plasma cells become cancerous and become multiple myeloma cells, BCMA is highly expressed on its surface, and it is possible to express TACI but not BAFF-R (Nature reviews immunology, 2009, 9(7): 491.). It can be seen that most B cells do not express BCMA.
  • BCMA is a new target that is superior to CD19, etc., with high specificity and fewer side effects of the target. Therefore, the development of antibody drugs with blocking effect or endocytosis effect against the BCMA target can not only improve the treatment effect of multiple myeloma, but also greatly reduce the side effects of the treatment, and can produce huge economic and social value.
  • ADC antibody-drug conjugate
  • CAR-T cell therapy Idecabtagene vicleucel (abbreviated as bb2121), among 33 R/R MM patients who had failed at least 3 therapies in the past, the total remission rate reached The median PFS was 11.8 months (NCT02658929).
  • Amgen's AMG 420 is the fastest-advanced therapy. This type of antibody is smaller than traditional antibodies and is composed of two antibody domain fragments. It has good activity, but has a shorter half-life than full-length antibodies. (NCT02514239).
  • drugs targeting BMCA targets whether it is monoclonal antibody, double antibody, ADC or CAR-T cell therapy, have achieved impressive results, and clinical results show that the side effects of BCMA target It is also much lower than other targets.
  • the present invention intends to develop a novel antibody targeting BCMA.
  • BCMA is used as an immunogen to immunize mice, and an antibody library is constructed and screened through phage display technology to obtain monoclonal antibodies that simultaneously bind to human and monkey BCMA antigens. Subsequent humanization of antibodies was used to transform murine monoclonal antibodies into humanized antibodies, and functional experiments such as affinity, blocking, and endocytosis confirmed that the candidate humanized antibodies exhibited excellent functions, which are at the cellular level.
  • the swallowing function is better than the competing product GSK2857916.
  • the present invention relates to an isolated monoclonal antibody targeting B cell maturation antigen (BCMA), wherein the antibody can bind to both human BCMA and monkey BCMA.
  • BCMA B cell maturation antigen
  • the antibody of the present invention comprises a heavy chain variable region comprising the heavy chain complementarity determining region 1 (CDR-H1) shown in SEQ ID NO: 1 or 2, and/or It includes the heavy chain complementarity determining region 2 (CDR-H2) shown in SEQ ID NO: 3 or 4, and/or includes the heavy chain complementarity determining region 3 (CDR-H3) shown in SEQ ID NO: 5 or 6.
  • CDR-H1 heavy chain complementarity determining region 1
  • CDR-H2 heavy chain complementarity determining region 2
  • CDR-H3 heavy chain complementarity determining region 3
  • the antibody of the present invention comprises a light chain variable region comprising the light chain complementarity determining region 1 (CDR-L1) shown in SEQ ID NO: 7 or 8, and/or It includes the light chain complementarity determining region 2 (CDR-L2) shown in SEQ ID NO: 9 or 10, and/or includes the light chain complementarity determining region 3 (CDR-L3) shown in SEQ ID NO: 11 or 12.
  • CDR-L1 light chain complementarity determining region 1
  • CDR-L2 light chain complementarity determining region 2
  • CDR-L3 light chain complementarity determining region 3
  • the antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the heavy chain complementarity determining region 1 shown in SEQ ID NO: 1 or 2.
  • CDR-H1 and/or comprising the heavy chain complementarity determining region 2 (CDR-H2) shown in SEQ ID NO: 3 or 4, and/or comprising the heavy chain complementarity determining region shown in SEQ ID NO: 5 or 6 3 (CDR-H3)
  • the light chain variable region includes the light chain complementarity determining region 1 (CDR-L1) shown in SEQ ID NO: 7 or 8, and/or includes the light chain complementarity determining region 1 (CDR-L1) shown in SEQ ID NO: 9 or 10
  • the antibody of the present invention comprises a variant of the above-mentioned antibody, and possesses the same or similar activity as the above-mentioned antibody of the present invention.
  • the antibody of the present invention comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 13 or 14, or a variant of the above sequence.
  • the antibody of the present invention comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15 or 16, or a variant of the above sequence.
  • the antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13 or 14, or the sequence of the above sequence.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 15 or 16, or a variant of the above sequence.
  • the heavy chain variable region of the antibody of the present invention includes CDR-H1 shown in SEQ ID NO: 1, CDR-H2 shown in SEQ ID NO: 3, and CDR shown in SEQ ID NO: 5 -H3.
  • the heavy chain variable region of the antibody of the present invention comprises CDR-H1 shown in SEQ ID NO: 2, CDR-H2 shown in SEQ ID NO: 4, and CDR shown in SEQ ID NO: 6 -H3.
  • the light chain variable region of the antibody of the present invention comprises CDR-L1 shown in SEQ ID NO: 7, CDR-L2 shown in SEQ ID NO: 9 and CDR shown in SEQ ID NO: 11 -L3.
  • the light chain variable region of the antibody of the present invention includes CDR-L1 shown in SEQ ID NO: 8, CDR-L2 shown in SEQ ID NO: 10, and CDR shown in SEQ ID NO: 12 -L3.
  • the light chain variable region of the antibody of the present invention has the sequence shown in SEQ ID NO: 13 or 14, or has a sequence that is at least 80% different from any of the above sequences, for example, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity sequence.
  • the heavy chain variable region of the antibody of the present invention has the sequence shown in SEQ ID NO: 15 or 16, or has at least 80% of any of the above sequences, for example, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity sequence.
  • the antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 shown in SEQ ID NO:1, shown in SEQ ID NO:3 CDR-H2 and CDR-H3 shown in SEQ ID NO: 5; the light chain variable region includes CDR-L1 shown in SEQ ID NO: 7, CDR-L2 shown in SEQ ID NO: 9 and SEQ ID CDR-L3 shown in NO:11.
  • the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 13, or has at least 80% of any of the above sequences, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity sequence;
  • the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 15, or has at least the same sequence as any of the above 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similar sequences.
  • the antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 shown in SEQ ID NO: 2, shown in SEQ ID NO: 4 CDR-H2 and CDR-H3 shown in SEQ ID NO: 6; the light chain variable region includes CDR-L1 shown in SEQ ID NO: 8, CDR-L2 shown in SEQ ID NO: 10 and SEQ ID CDR-L3 shown in NO:12.
  • the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 14, or has at least 80% of any of the above sequences, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity sequence;
  • the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 16, or has at least the same sequence as any of the above 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similar sequences.
  • the present invention also relates to a monoclonal antibody targeting B cell maturation antigen (BCMA), which recognizes the same epitope as the antibody described in the present invention.
  • BCMA B cell maturation antigen
  • the present invention also relates to a monoclonal antibody targeting B cell maturation antigen (BCMA), which competitively binds to B cell maturation antigen (BCMA) with the above-mentioned antibody of the present invention.
  • BCMA B cell maturation antigen
  • the present invention also relates to a nucleic acid encoding the above-mentioned antibody of the present invention.
  • the present invention also relates to an expression vector, which contains the above-mentioned nucleic acid of the present invention.
  • the present invention also relates to a host cell, which comprises the expression vector of the present invention or the genome of the present invention integrated with the nucleic acid of the present invention.
  • the present invention also relates to a method for producing a monoclonal antibody, the method comprising culturing the host cell of the present invention to produce the above-mentioned monoclonal antibody of the present invention.
  • the present invention also relates to a pharmaceutical composition, which comprises the monoclonal antibody of the present invention and a pharmaceutically acceptable carrier.
  • the present invention also relates to a kit or product, which includes the monoclonal antibody of the present invention or the pharmaceutical composition of the present invention.
  • the present invention also relates to a method for treating diseases related to the expression of BCMA, which comprises: administering the monoclonal antibody of the present invention or the pharmaceutical composition of the present invention or the present invention to a subject in need thereof The medicine box or product.
  • the aforementioned diseases are selected from B-cell acute lymphocytic leukemia, T-cell acute lymphocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell young lymphocytic leukemia, and blastic plasma cells
  • Dendritic cell tumor Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoma, malignant lymphoid proliferation status , MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor , Waldenstrom macroglobulinemia, myeloma, MG
  • the aforementioned disease is multiple myeloma.
  • the present invention also relates to the use of the monoclonal antibody of the present invention in the preparation of drugs for treating diseases related to the expression of BCMA.
  • the disease is selected from B-cell acute lymphocytic leukemia, T-cell acute lymphocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell young lymphocytic leukemia, and blastic plasma cells
  • Dendritic cell tumor Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoma, malignant lymphoid proliferation status , MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor , Waldenstrom macroglobulinemia, myeloma, MGUS, plasma
  • the aforementioned disease is multiple myeloma.
  • the present invention relates to the following aspects:
  • BCMA B cell maturation antigen
  • An antibody which comprises a heavy chain variable region comprising the heavy chain complementarity determining region 1 (CDR-H1) shown in SEQ ID NO: 1 or 2, and/or comprising SEQ ID NO :
  • An antibody which comprises a light chain variable region comprising the light chain complementarity determining region 1 (CDR-L1) shown in SEQ ID NO: 7 or 8, and/or comprising SEQ ID NO :
  • An antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody;
  • An antibody comprising a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 13 or 14, or a variant of the above sequence;
  • An antibody comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15 or 16, or a variant of the above sequence;
  • An antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody.
  • the antibody according to any one of items 1 to 3, wherein the heavy chain variable region of the antibody comprises CDR-H1 shown in SEQ ID NO: 2, shown in SEQ ID NO: 4 CDR-H2 and CDR-H3 shown in SEQ ID NO: 6.
  • BCMA B cell maturation antigen
  • a monoclonal antibody targeting B cell maturation antigen characterized in that the antibody competitively binds to B cell maturation antigen (BCMA) with the antibody of any one of items 1-9.
  • An expression vector comprising the nucleic acid according to item 12.
  • a host cell comprising the expression vector of item 13 or the nucleic acid of item 12 integrated in the genome.
  • a method of producing a monoclonal antibody comprising culturing the host cell according to item 14 to produce the monoclonal antibody according to any one of items 1 to 11.
  • a pharmaceutical composition comprising the monoclonal antibody according to any one of items 1 to 11 and a pharmaceutically acceptable carrier.
  • kits or product comprising the monoclonal antibody according to any one of items 1 to 11 or the pharmaceutical composition according to item 16.
  • a method of treating diseases related to the expression of BCMA comprising:
  • the monoclonal antibody according to any one of items 1 to 11 or the pharmaceutical composition according to item 16 or the kit or product according to item 17 is administered to a subject in need thereof.
  • the disease is selected from B-cell acute lymphocytic leukemia, T-cell acute lymphocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and B-cell juvenile lymphoid Cell leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, Malignant lymphoma, malignant lymphoproliferative status, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma , Plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia, myeloma,
  • the disease is selected from B-cell acute lymphocytic leukemia, T-cell acute lymphocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and B-cell juvenile lymphoma Cell leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, Malignant lymphoma, malignant lymphoproliferative status, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma , Plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia, myeloma,
  • the antibody of the present invention can bind to both human BCMA and monkey BCMA, and is close to or even better in terms of affinity.
  • the antibody of the present invention is close to or even better than GSK2857916 in blocking the effect of BCMA from binding its ligand BAFF or APRIL.
  • the endocytosis effect of the antibody involved in the present invention it has a better endocytosis effect than GSK2857916.
  • thermal stability the antibody involved in the present invention has better thermal stability than GSK2857916.
  • immunogenicity the antibody involved in the present invention is a humanized antibody with lower immunogenicity.
  • Figure 1 shows the process of antibody production, showing the process of antibody production with human monkey crossover targeting BCMA.
  • Figure 2 shows the activity determination of recombinant proteins BCMA, BAFF and APRIL, and the results show that the activity of each protein is normal.
  • Figure 2(A) shows the binding results of human and monkey BCMA antigens with different tags (Fc and His) and the positive control antibody GSK2857916.
  • Figure 2(B) shows the binding results of human BCMA antigen and BAFF coated with different concentrations.
  • Figure 2(C) shows the binding results of human BCMA antigen and APRIL coated with different concentrations.
  • Figure 3 shows the binding assay of the candidate antibody to cells overexpressing human or monkey BCMA, and the results show that the candidate antibody has human-monkey crossover characteristics.
  • Figure 3(A) shows the binding of some candidate antibodies to HEK293 cells overexpressing human BCMA determined by FACS.
  • Figure 3(B) shows the binding of some candidate antibodies to CHO cells overexpressing monkey BCMA determined by FACS.
  • Figure 4 shows the test results of cross-affinity of some candidate antibodies with human and monkey BCMA.
  • Figure 4(A) shows the affinity effect of some candidate antibodies with human BCMA at the Elisa level. The results show that some candidate antibodies have close or even better affinity than the GSK2857916 antibody.
  • Figure 4(B) shows the affinity effect of some candidate antibodies with monkey BCMA at the Elisa level. The results show that some candidate antibodies have close or even better affinity than the GSK2857916 antibody.
  • Figure 5 shows the results of the blocking effect of some candidate antibodies.
  • Figure 5(A) shows that some candidate antibodies block the binding of BCMA and BAFF at the Elisa level. The results show that some candidate antibodies have close or even better blocking effects than the GSK2857916 antibody.
  • Figure 5(B) shows that some candidate antibodies block the binding of BCMA and APRIL at the Elisa level. The results show that some candidate antibodies have close or even better blocking effects than the GSK2857916 antibody.
  • Figure 6 shows the result of endocytosis effect measurement.
  • Figure 6 (A) and (B) show the endocytosis effect of some candidate antibodies on the human myeloma cell line H929 cells. The results show that some candidate antibodies have better endocytosis effects than the GSK2857916 antibody.
  • Figure 7 shows a summary of the functions of the candidate antibodies.
  • Figure 7 shows the affinity effects of some candidate antibodies on HEK293 cells expressing human BCMA, the affinity effects on CHO cells expressing monkey BCMA, and the level of Elisa compared with human and monkey BCMA.
  • Affinity effect the effect of blocking the binding of BAFF and BCMA at the level of Elisa, the effect of blocking the binding of APRIL and BCMA at the level of Elisa, and the endocytosis effect of the antibody on human myeloma cell H929; the number of'+' increases from At least, it means that the affinity, blocking and endocytosis effects of the antibody are from strong to weak.
  • the results show that the antibodies SY14-3rd-5-6-7 and SY14-3rd-5-6-32 show a more excellent comprehensive effect. .
  • Figure 8 shows the detection results of the cross-affinity activity of the humanized antibody (5-6-7-hu-2) with human and monkey BCMA.
  • Figure 8 (A) shows the detection of SY14-3rd-5-6-7 (also known as 5-6-7 or 5-6-7-WT) antibody before and after humanization, at the level of Elisa affinity with human BCMA The results show that the affinity of the antibody to human BCMA after humanization remains the same as that before humanization.
  • Figure 8(B) shows the detection of the affinity of the SY14-3rd-5-6-7 antibody to monkey BCMA at the Elisa level before and after humanization. The results show that the affinity of the antibody to monkey BCMA after humanization and human origin Be consistent before transformation.
  • Figure 9 shows the test results of the cross-affinity activity of humanized antibody (5-6-32-hu-2) with human and monkey BCMA
  • Figure 9(A) shows the detection of SY14-3rd-5-6-32 (also known as 5-6-32 or 5-6-32-WT) before and after humanization, the affinity of the antibody to human BCMA at the Elisa level. The results show that the affinity of the antibody to human BCMA after humanization is still better than that of the positive antibody (GSK2857916).
  • Figure 9(B) detects the affinity of the SY14-3rd-5-6-32 antibody to monkey BCMA at the Elisa level before and after humanization. The results show that the affinity of the antibody to monkey BCMA after humanization is still better than positive Antibody GSK2857916.
  • Figure 10 shows the detection results of the blocking effect of the antibody (5-6-7-hu-2) after humanization
  • Figure 10(A) shows the detection of SY14-3rd-5-6-7 (also known as 5-6-7 or 5-6-7).
  • 5-6-7-WT After the antibody is humanized, it blocks the ability of BCMA to bind to BAFF at the Elisa level. The results show that the antibody is humanized to block BCMA from binding to BAFF. The effect of blocking the binding of BCMA to BAFF is slightly worse than that of the positive antibody (GSK2857916) .
  • Figure 10(B) shows the detection of the ability of SY14-3rd-5-6-7 antibody to block the binding of BCMA to APRIL at the Elisa level after humanization of the SY14-3rd-5-6-7 antibody. The results show that the humanized antibody blocks the effect of blocking BCMA from binding to APRIL. Slightly worse than the positive antibody (GSK2857916).
  • Figure 11 shows the detection results of the blocking effect of the antibody (5-6-32-hu-2) after humanization
  • Figure 11(A) shows the detection of SY14-3rd-5-6-32 (also known as 5-6-32 or 5-6-32).
  • 5-6-32-WT After the antibody is humanized, it blocks the ability of BCMA to bind to BAFF at the Elisa level. The results show that the humanized antibody is slightly less effective in blocking the binding of BCMA to BAFF than the positive antibody (GSK2857916) .
  • Figure 11(B) shows the detection of the ability of SY14-3rd-5-6-32 antibody to block the binding of BCMA and APRIL at the Elisa level after humanization of the SY14-3rd-5-6-32 antibody. The results show that the antibody blocks the effect of blocking BCMA and APRIL after humanization. Slightly worse than the positive antibody (GSK2857916).
  • Figure 12 shows the results of measuring the binding activity of antibodies before and after humanization in highly expressing human and monkey BCMA cells.
  • Figure 12(A) shows the detection of 5-6-7 and 5-6-32 antibodies before and after humanization and overexpressing human BCMA The results show that the binding ability of the antibody to HEK293 cells after humanization is consistent with that before humanization and is better than that of the GSK2857916 antibody.
  • Figure 12(B) shows the detection of the binding ability of 5-6-7 and 5-6-32 antibodies to CHO cells overexpressing monkey BCMA before and after humanization. The results show that the antibodies have the same binding ability and human origin after humanization. It remains consistent before chemistry and is better than the GSK2857916 antibody.
  • Figure 12 (C) shows the detection of the binding ability of the 5-6-7 and 5-6-32 antibodies to the myeloma cell line H929 cells before and after humanization. The results indicate the binding ability and humanization of the antibody after humanization. The previous remains consistent and superior to the GSK2857916 antibody.
  • Figure 13 shows the results of measuring the endocytosis effect on H929 cells before and after antibody humanization.
  • Figure 13(A) shows the detection of 5-6-7 antibody before and after humanization, on human myeloma cell line H929 cells expressing BCMA. The results show that the endocytosis effect after humanization of the antibody remains the same as that before humanization and is better than that of the GSK2857916 antibody.
  • Figure 13(B) shows the endocytosis effect on the human myeloma cell line H929 cells expressing BCMA before and after humanization of the 5-6-32 antibody. The results show that the endocytosis effect after antibody humanization and before humanization It is consistent and superior to GSK2857916 antibody.
  • Figure 14 shows the summary results of various functions of the humanized antibody.
  • Figure 14 shows the degree of humanization of the humanized antibody, its affinity on HEK293 cells expressing human BCMA, and its affinity on CHO cells expressing monkey BCMA. Effect, the affinity effect with human and monkey BCMA at the level of Elisa, blocking the binding effect of BAFF and BCMA, blocking the binding effect of APRIL and BCMA, and the endocytosis effect of antibody on human myeloma cell H929; where'+' The number is from more to less, representing the effect of antibody affinity, blocking and endocytosis from strong to weak.
  • the results show that the functions of the antibody after humanization remain the same as those before humanization, and some of the functions of the humanized antibody are close to or better than those of the GSK2857916 antibody.
  • an “isolated” antibody is an antibody that has been separated from a component of its natural environment.
  • the antibody is purified to greater than 95% or 99% purity by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse Phase HPLC) to determine.
  • electrophoresis e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reverse Phase HPLC
  • BCMA B cell maturation antigen
  • CD269 a member of the tumor necrosis factor receptor superfamily, namely TNFRSF17
  • TNFRSF17 tumor necrosis factor receptor superfamily 17
  • Human BCMA is almost exclusively expressed in plasma cells and multiple myeloma cells (see, for example, Novak et al., Blood, 103(2):689-694, 2004; Neri et al., Clinical Cancer Research, 73(19):5903 -5909; Felix et al., Mol. Oncology, 9(7):1348-58, 2015).
  • BCMA can bind B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) (for example, Mackay et al., 2003 and Kalled et al., Immunological Review, 204:43-54, 2005).
  • BAFF B cell activating factor
  • APRIL proliferation-inducing ligand
  • BCMA may be a suitable tumor antigen target for immunotherapeutic agents for multiple myeloma.
  • Antigen (Ag) refers to a compound, composition, or substance that can stimulate antibody production or T cell response in an animal, and includes a composition injected or absorbed into an animal (for example, a composition including a cancer-specific protein).
  • the antigen reacts with products of specific humoral or cellular immunity, including products induced by heterologous antigens (such as the disclosed antigens).
  • the target antigen is an epitope of a BCMA polypeptide.
  • Epitopes refers to the region of an antigen that is bound by a binding agent.
  • Epitopes can be formed by contiguous amino acids or discrete amino acids joined by tertiary folding of the protein. Epitopes formed by consecutive amino acids usually remain when exposed to a denaturing solvent, while epitopes formed by tertiary folding usually disappear when treated with a denaturing solvent. Epitopes usually include at least 3, and more usually at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.
  • Antibodies include their antigen-binding fragments, such as camel Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single-chain Fv protein ("scFv”), double -scFv, (scFv)2, mini-antibodies, bifunctional antibodies, tri-functional antibodies, tetra-functional antibodies, disulfide bond stabilized Fv protein (“dsFv”) and single domain antibodies (sdAb, Nanobody) and responsible for antigen binding Part of the full-length antibody.
  • scFv single-chain Fv protein
  • scFv single-chain Fv protein
  • scFv single-chain Fv protein
  • scFv single-chain Fv protein
  • scFv single-chain Fv protein
  • scFv single-chain Fv protein
  • scFv single-chain Fv protein
  • scFv single-chain F
  • the term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), hybrid antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Illinois (IL)); Kuby, Journal of Immunology, 3rd edition, WH Freeman & Co. (WH Freeman & Co.), New York, 1997.
  • chimeric antibodies e.g., humanized murine antibodies
  • hybrid antibodies e.g., bispecific antibodies
  • antigen-binding fragments thereof See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Illinois (IL)); Kuby, Journal of Immunology, 3rd edition, WH Freeman & Co. (WH Freeman & Co.), New York, 1997.
  • a complete antibody contains two heavy chains and two light chains. Each heavy chain is composed of a variable region and first, second, and third constant regions, and each light chain is composed of a variable region and a constant region. Mammalian heavy chains are classified into ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ . Mammalian light chains are classified as lambda or kappa. Immunoglobulins containing alpha, delta, epsilon, gamma, and mu heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. The complete antibody forms a "Y" shape.
  • the stem of Y is composed of the second and third constant regions of the two heavy chains (and for IgE and IgM, the fourth constant region) joined together, and disulfide bonds (interchains) are formed in the hinge.
  • the heavy chains ⁇ , ⁇ , and ⁇ have a constant region composed of three tandem (in a row) Ig domains, and a hinge region for increasing flexibility; the heavy chains ⁇ and ⁇ have a constant region composed of four immunoglobulin domains Area.
  • the second and third constant regions are called "CH2 domain" and "CH3 domain", respectively.
  • Each arm of Y includes a variable region and a first constant region of a single heavy chain that are bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.
  • the light and heavy chain variable regions contain "framework” regions interspersed with three hypervariable regions (also called “complementarity determining regions” or “CDRs”).
  • CDR can be defined or identified by conventional methods, for example by following the sequence of Kabat et al. (Wu, TT and Kabat, EA, Journal of Experimental Medicine 132(2):211-50, (1970); Borden, P. and Kabat EA, PNAS, 84: 2440-2443 (1987); see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (USDepartment of Health and Human Services), 1991, which Incorporated herein by reference), or by following the structure of Chothia et al. (Choithia, C. and Lesk, AM, J Mol. Biol., 196(4): 901-917 (1987) ); Choithia, C. et al., Nature, 342:877-883 (1989)).
  • the sequences of the framework regions of different light chains or heavy chains have relative preservation in species (such as humans).
  • the framework region of the antibody (which is the combined framework region of the light chain and the heavy chain) is used to locate and align the CDRs in a three-dimensional space.
  • CDR is mainly responsible for binding to the epitope of the antigen.
  • the CDRs of each chain are usually called CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are usually identified by the chain in which the specific CDR is located.
  • the CDRs located in the variable domain of the heavy chain of the antibody are called CDR-H1, CDR-H2, and CDR-H3, and the CDRs located in the variable domain of the light chain of the antibody are called CDR-L1, CDR -L2 and CDR-L3.
  • Antibodies with different specificities that is, different combination sites for different antigens have different CDRs.
  • the CDRs are different between antibodies and antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDR are called specificity determining residues (SDR).
  • Illustrative examples of light chain CDRs suitable for construction of the humanized BCMA CAR covered herein include, but are not limited to, the CDR sequences set forth in SEQ ID NO: 1-3.
  • Illustrative examples of heavy chain CDRs suitable for constructing the humanized BCMA CAR covered herein include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 4-6.
  • VH refers to the variable region of an immunoglobulin heavy chain, including the heavy chain variable region of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragments as disclosed herein.
  • V L refers to the variable region of an immunoglobulin light chain comprising a light chain variable region of antibody other antibody fragments, Fv, scFv, dsFv, Fab, or as disclosed herein.
  • a “monoclonal antibody” is an antibody produced by a single clone of B lymphocytes or by cells in which the light chain and heavy chain genes of a single antibody have been transfected.
  • Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by preparing hybrid antibody-forming cells from a fusion of myeloma cells and immune spleen cells.
  • Monoclonal antibodies include humanized monoclonal antibodies.
  • the double-chain Fv species is composed of a dimer of a heavy chain variable domain and a light chain variable domain in a tight non-covalent association.
  • a heavy-chain variable domain and a light-chain variable domain can be covalently linked through a flexible peptide linker, so that the light chain and the heavy chain can be similar to the double-chain Fv category
  • the "dimeric" structure is associated.
  • the three hypervariable regions (HVR) of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer.
  • the six HVRs collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific to the antigen) has the ability to recognize and bind to the antigen, but the affinity is lower than the complete binding site.
  • the Fab fragment contains the variable domain of the heavy chain and the variable domain of the light chain and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments in that several residues are added to the carboxyl end of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of an antibody.
  • Fab'-SH is the name for Fab' herein, in which the cysteine residue of the constant domain carries a free thiol group.
  • F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.
  • the present invention relates to an isolated monoclonal antibody targeting B cell maturation antigen (BCMA), wherein the antibody can bind to both human BCMA and monkey BCMA.
  • BCMA B cell maturation antigen
  • FACS method flow cytometer operation method
  • the monoclonal antibody obtained by the present invention has better affinity for human BCMA-HEK293 cells than the positive control antibody (GSK2857916).
  • the monoclonal antibody obtained in the present invention has an affinity effect on monkey BCMA-CHO cells close to or better than the positive control antibody (GSK2857916).
  • the monoclonal antibody obtained by the present invention can simultaneously bind to the BCMA antigen of human and monkey with high affinity and bind to the BCMA antigen of monkey. It is convenient for the antibody to use monkey (cynomolgus monkey) as a model before entering clinical research. To carry out toxicological evaluation and pharmacokinetic evaluation. At the same time, the monoclonal antibody of the present invention has high affinity, and the high-affinity antibody has more advantages in pharmacodynamics. For example, the antibody dissociates more slowly after binding to the target antigen molecule, which makes the antibody endocytosis more effective in the cell. Good, and to achieve the same cell or animal efficacy, the required antibody dose may also be lower.
  • the terms “specific binding”, “specific recognition” or “specific to" refer to a measurable and reproducible interaction, such as the interaction between a target and an antigen binding protein Combine.
  • an antigen binding protein that specifically binds to a target (which can be an epitope) has a greater affinity, affinity, easier, and/or longer duration than the binding of other targets.
  • the antigen binding protein has a greater affinity, affinity, easier, and/or longer duration than the binding of other targets.
  • the antigen binding protein has a greater affinity, affinity, easier, and/or longer duration than the binding of other targets.
  • the antigen binding protein has a greater affinity, affinity, easier, and/or longer duration than the binding of other targets.
  • the antigen binding protein has a greater affinity, affinity, easier, and/or longer duration than the binding of other targets.
  • the antigen binding protein has a greater affinity, affinity, easier, and/or longer duration than the binding of other targets.
  • the antigen binding protein has a greater affinity, affinity, easier, and/or
  • the antigen binding protein that specifically binds to the target has a dissociation constant (Kd) ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
  • Kd dissociation constant
  • the antibody of the present invention comprises a heavy chain variable region comprising the heavy chain complementarity determining region 1 (CDR-H1) shown in SEQ ID NO: 1 (GHIFTNFHFH) or 2 (GYIFTNYHMH) , And/or include the heavy chain complementarity determining region 2 (CDR-H2) shown in SEQ ID NO: 3 (GIYPGNGDTF) or 4 (GIYPGNGDIF), and/or include SEQ ID NO: 5 (GSYYGYIDAMDY) or 6 (GSYYGYIDAMDY)
  • CDR-H3 The heavy chain complementarity determining region 3 (CDR-H3) is shown.
  • the antibody of the present invention may comprise a light chain variable region comprising the light chain complementarity determining region 1 (CDR-L1) shown in SEQ ID NO: 7 (RASQDISNYLN) or 8 (RASQDISNDLN) ), and/or include the light chain complementarity determining region 2 (CDR-L2) shown in SEQ ID NO: 9 (YTSRLHS) or 10 (YTSRLPS), and/or include SEQ ID NO: 11 (QQGNTLPWT) or 12 (QQGHTLPWT) ) Shown in the light chain complementarity determining region 3 (CDR-L3).
  • the antibody of the present invention may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the heavy chain complementarity determining region 1 (CDR) shown in SEQ ID NO: 1 or 2. -H1), and/or comprising the heavy chain complementarity determining region 2 (CDR-H2) shown in SEQ ID NO: 3 or 4, and/or comprising the heavy chain complementarity determining region 3 shown in SEQ ID NO: 5 or 6 (CDR-H3); the light chain variable region includes the light chain complementarity determining region 1 (CDR-L1) shown in SEQ ID NO: 7 or 8, and/or includes the light chain complementarity determining region 1 (CDR-L1) shown in SEQ ID NO: 9 or 10
  • the antibody of the present invention comprises a light chain variable region comprising
  • the antibody of the present invention includes a heavy chain variable region having SEQ ID NO: 15:
  • the antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13 or 14, or a variant of the above sequence
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 15 or 16, or a variant of the above sequence.
  • the heavy chain variable region of an antibody of the present invention includes CDR-H1 shown in SEQ ID NO: 1, CDR-H2 shown in SEQ ID NO: 3, and CDR shown in SEQ ID NO: 5 -H3.
  • the heavy chain variable region of another antibody of the present invention comprises CDR-H1 shown in SEQ ID NO: 2, CDR-H2 shown in SEQ ID NO: 4, and CDR-H3 shown in SEQ ID NO: 6.
  • the light chain variable region of an antibody of the present invention includes CDR-L1 shown in SEQ ID NO: 7, CDR-L2 shown in SEQ ID NO: 9 and CDR shown in SEQ ID NO: 11 -L3.
  • the light chain variable region of another antibody of the present invention comprises CDR-L1 shown in SEQ ID NO: 8, CDR-L2 shown in SEQ ID NO: 10, and CDR-L3 shown in SEQ ID NO: 12.
  • the light chain variable region of the antibody of the present invention has a sequence shown in SEQ ID NO: 13 or 14, or has a sequence that is at least 80% different from any of the above sequences, for example, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity sequence.
  • the heavy chain variable region of the antibody of the present invention has a sequence shown in SEQ ID NO: 15 or 16, or has a sequence that is at least 80% different from any of the above sequences, for example, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity sequence.
  • an antibody of the present invention includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes CDR-H1 shown in SEQ ID NO: 1, shown in SEQ ID NO: 3. CDR-H2 and CDR-H3 shown in SEQ ID NO: 5; the light chain variable region includes CDR-L1 shown in SEQ ID NO: 7, CDR-L2 shown in SEQ ID NO: 9 and CDR-H3 shown in SEQ ID NO CDR-L3 shown in :11.
  • the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 13, or has at least 80% of any of the above sequences, for example, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% similarity;
  • the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 15, or has a sequence similar to any of the above The sequence is at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similar sequence.
  • another antibody of the present invention includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the CDR-H1 shown in SEQ ID NO: 2, which is shown in SEQ ID NO: 4.
  • the light chain variable region includes CDR-L1 shown in SEQ ID NO: 8, CDR-L2 shown in SEQ ID NO: 10 and SEQ ID CDR-L3 shown in NO:12.
  • the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 14, or has at least 80% of any of the above sequences, for example, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% similarity;
  • the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 16, or has the same sequence as any of the above The sequence is at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similar sequence.
  • variable refers to a heavy chain variable region or a light chain variable region that has been modified with at least one, such as 1, 2 or 3 amino acid substitutions, deletions or additions, including heavy or light chain variants.
  • the modified antigen-binding protein of the body basically retains the biological characteristics of the pre-modified antigen-binding protein.
  • the antigen binding protein containing the sequence of the variable heavy chain variable region or the variable region of the light chain retains 60%, 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein before modification. It should be understood that each heavy chain variable region or light chain variable region can be modified alone or in combination with another heavy chain variable region or light chain variable region.
  • the antigen binding protein of the present disclosure contains 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence of the heavy chain variable region described herein The amino acid sequence of the variable region of the heavy chain.
  • the antigen binding protein of the present disclosure includes 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence of the light chain variable region described herein The amino acid sequence of the variable region of the light chain.
  • the percent homology can be in the entire heavy chain variable region and/or the entire light chain variable region, or the percent homology can be limited to the framework region, and the sequence corresponding to the CDR is the same as the heavy chain variable region and/or light chain.
  • the CDRs disclosed herein have 100% identity within the variable region.
  • the term "CDR variant” refers to a CDR that has been modified with at least one, such as 1, 2 or 3 amino acid substitutions, deletions or additions, wherein the modified antigen binding protein comprising the CDR variant substantially retains the pre-modification The biological characteristics of antigen binding proteins.
  • the antigen binding protein containing the variant CDR retains 60%, 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein before modification. It should be understood that each CDR that can be modified can be modified alone or in combination with another CDR.
  • the modification is a substitution, especially a conservative substitution.
  • the antibody of the present invention has excellent endocytosis effect, which is better than the positive control antibody (GSK2857916).
  • Endocytosis also known as endocytosis or endocytosis, is the process of transporting extracellular material into the cell through the deformation movement of the plasma membrane. According to the different size of the material entering the cell and the different mechanism of the entry, endocytosis can be divided into three types: phagocytosis, phagocytosis, and receptor-mediated endocytosis.
  • the endocytosis of the antibody in the present invention refers to receptor-mediated endocytosis.
  • the BCMA-antibody complex is mediated to form an endocytic body and lysis Enzyme fusion, the BCMA-antibody complex in the lysosome is mediated and degraded, and part of the BCMA or BCMA-antibody complex can also be transported back to the cell membrane.
  • the monoclonal antibody of the present invention is coupled to small toxin molecules to form ADC drugs, they are transported into the cell through endocytosis, and the released toxin molecules can kill target cells (such as multiple myeloma cells). Therefore, when the monoclonal antibody of the present invention is developed as an ADC drug, the better endocytosis effect is very important to help mediate the drug into the target cell.
  • the antibody of the present invention is a humanized antibody.
  • the binding activity of the antibody before and after the humanization of the present invention is basically the same as that of human and monkey BCMA, and the antibody before and after the humanization of the present invention blocks the binding of BCMA and BAFF.
  • the effect is basically the same.
  • the binding effect of the antibody and monkey BCMA-CHO cells before and after the humanization of the present invention is basically the same, and both are better than the positive control antibody (GSK2857916), and the binding effect of the antibody and H929 cells before and after the humanization is basically the same, and both are better than the positive Control antibody (GSK2857916).
  • the endocytosis effect of the antibody before and after humanization on the human myeloma cell line H929 is basically the same, and both are better than the positive control antibody (GSK2857916).
  • Humanized antibody refers to a type of engineered antibody that has its CDRs derived from a non-human donor immunoglobulin, and the remaining immunoglobulin-derived portion of the molecule is derived from one (or more) human immunoglobulins protein.
  • framework support residues can be changed to retain binding affinity (see, for example, Queen et al., Proc. Natl Acad Sci USA, 86: 10029-10032 (1989), Hodgson et al., Bio/Technology, 9: 421 (1991)).
  • Suitable human acceptor antibodies may be antibodies selected from conventional databases such as databases, Los Alamos database, and Swiss protein database by homology with the nucleotide and amino acid sequences of the donor antibody.
  • Human antibodies characterized by homology (based on amino acids) to the framework regions of the donor antibody may be suitable for providing heavy chain constant regions and/or heavy chain variable framework regions for insertion of donor CDRs.
  • a suitable acceptor antibody capable of providing constant or variable framework regions of the light chain can be selected in a similar manner. It should be noted that the acceptor antibody heavy chain and light chain need not be derived from the same acceptor antibody.
  • the thermal stability of the humanized antibody of the present invention is slightly better than that of the positive control antibody (GSK2857916), and meets the thermal stability conditions of antibody preparation. Poor thermal stability may lead to antibody drugability problems, such as low antibody expression and antibody Gather.
  • the present invention also relates to a monoclonal antibody targeting B cell maturation antigen (BCMA), which recognizes the same epitope as the antibody described in the present invention.
  • the present invention also relates to a monoclonal antibody targeting B cell maturation antigen (BCMA), which competitively binds to B cell maturation antigen (BCMA) with the above-mentioned antibody of the present invention.
  • the present invention also relates to a nucleic acid encoding the above-mentioned antibody of the present invention.
  • the present invention also relates to an expression vector, which contains the above-mentioned nucleic acid of the present invention.
  • the present invention also relates to a host cell, which comprises the expression vector of the present invention or the genome of the present invention integrated with the nucleic acid of the present invention.
  • polynucleotide or “nucleic acid” used interchangeably herein refers to a chain of nucleotides of any length, and includes DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.
  • vector refers to a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors related to cationic flocculants, DNA or RNA expression encapsulated in liposomes Vectors and certain eukaryotic cells, such as producer cells.
  • the terms "host cell”, “host cell line” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of these cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages.
  • the offspring may not be exactly the same as the parent cell in terms of nucleic acid content, but they may contain mutations. This document includes mutant progeny that have the same function or biological activity as the cells screened or selected in the initially transformed cells.
  • the present invention also relates to a method for producing a monoclonal antibody, the method comprising culturing the host cell of the present invention to produce the above-mentioned monoclonal antibody of the present invention.
  • the present invention also relates to a pharmaceutical composition, which comprises the monoclonal antibody of the present invention and a pharmaceutically acceptable carrier.
  • the present invention also relates to a kit or product, which includes the monoclonal antibody of the present invention or the pharmaceutical composition of the present invention.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to maintain biological activity and does not interact with the subject's immune system. reaction. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsion (e.g., oil/water emulsion), and various types of wetting agents.
  • the preferred diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological saline (0.9%).
  • PBS phosphate buffered saline
  • a composition containing such a carrier is formulated by a well-known conventional method (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 21st Ed.MackPublishing,2005).
  • the present invention also relates to a method for treating diseases related to the expression of BCMA, which comprises: administering the monoclonal antibody of the present invention or the pharmaceutical composition of the present invention or the present invention to a subject in need thereof The medicine box or product.
  • diseases are selected from B-cell acute lymphocytic leukemia, T-cell acute lymphocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell young lymphocytic leukemia, blastic plasma cell-like Dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoma, malignant lymphoid proliferation status, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myel
  • MM multiple myeloma
  • Kahler's disease is a type of malignant plasma B cells in close contact with stromal cells in the bone marrow.
  • MM is a progressive disease, especially caused by a variety of genetic damage to precursor plasma B cells, that is, mainly by translocations, such as t(11;14), t(4;14), t(8 14) Chromosomal translocations, or deletions, such as del(13) and del(17), which cause tumor cells to proliferate significantly and become apoptosis-tolerant.
  • B lymphocytes start in the bone marrow and move to the lymph nodes. Due to their development, B lymphocytes mature on their cell surface and display different proteins. When they are activated to secrete antibodies, they are called plasma cells. After they leave part of the lymph nodes called the germinal center, multiple myeloma develops in B cells. The immune system maintains the proliferation of B cells and the secretion of antibodies under strict control. When chromosomes and bases are damaged (usually through rearrangement), this control is lost. Normally, when the promoter gene moves (or translocates) to the chromosome, it stimulates the overproduction of antibody genes.
  • immunoglobulin heavy chain genes on chromosome 14, locus 14q32
  • oncogenes often 11q13, 4p16.3, 6p21, 16q23 and 20q11 [10]
  • the result is the proliferation of plasma cell clones and genomic instability, leading to further mutations and translocations.
  • Chromosome 14 abnormalities are observed in about 50% of all myeloma cases.
  • the deletion of chromosome 13 is also observed in about 50% of cases.
  • the "subject”, “individual” or “subject” is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, race animals, pets, primates, horses, dogs, cats, mice, and rats.
  • administering the monoclonal antibody of the present invention or the pharmaceutical composition of the present invention or the kit or product of the present invention to a subject in need thereof means administering an effective amount of the pharmaceutical combination
  • the term "effective amount” refers to the amount of a drug or agent that elicits a biological or pharmaceutical response of a tissue, system, animal, or human being pursued by, for example, a researcher or clinician.
  • terapéuticaally effective amount refers to an amount that causes an improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or reduces the rate of progression of the disease or condition, compared to a corresponding subject who did not receive the amount. ⁇ The amount. The term also includes within its scope an amount effective to enhance normal physiological functions.
  • the present invention also relates to the use of the monoclonal antibody of the present invention in the preparation of drugs for treating diseases related to the expression of BCMA.
  • diseases are selected from B-cell acute lymphocytic leukemia, T-cell acute lymphocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell young lymphocytic leukemia, blastic plasma cell-like Dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoma, malignant lymphoid proliferation status, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablast
  • the above-mentioned disease is multiple myeloma.
  • cancer new organism
  • tumor tumor-derived cell changes that have undergone malignant transformation or lead to abnormal or unregulated growth or excessive proliferation Cell.
  • Such changes or malignant transformation usually make such cells pathogenic to the host organism, and therefore it is also intended to include primary or precancerous cells that become or can become pathogenic and require intervention or can benefit from intervention.
  • Primary cancer cells i.e., cells taken from near the site of malignant transformation
  • cancer cells includes not only primary cancer cells, but also any cells derived from cancer cell ancestors (ancestors). This includes metastatic cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • "clinically detectable" tumors are those that are detectable on the basis of tumor mass; for example, by e.g. CAT scan, MR imaging, X-ray, ultrasound or palpation And/or tumors that are detectable due to the expression of one or more cancer-specific antigens in samples available from the patient.
  • the term of the present invention includes cells, neoplasms, cancers, and tumors of any stage, including what clinicians call primary cancer, tumor, in situ growth, and late metastatic growth.
  • the tumor may be a hematopoietic tumor, such as a blood cell tumor, which is a liquid tumor.
  • specific examples of clinical conditions based on such tumors include leukemia such as chronic myeloid leukemia or acute myeloid leukemia; myeloma such as multiple myeloma; lymphoma and the like.
  • the antibody of the present invention can bind to both human BCMA and monkey BCMA, and is close to or even better in terms of affinity.
  • the antibody involved in the present invention is close to or better than GSK2857916 in blocking the effect of BCMA from binding its ligand BAFF or APRIL. Blocking the binding of BCMA with BAFF and APRIL can inhibit the activation of intracellular signaling pathways caused by ligand binding (such as the NF ⁇ B signaling pathway), thereby inhibiting the survival of plasma cells.
  • the antibody of the present invention has better endocytosis effect than GSK2857916.
  • the endocytosis effect is the key function of antibody-drug conjugate (ADC) to mediate cell killing.
  • ADC antibody-drug conjugate
  • Antibodies with better endocytosis effect Corresponding to the better advantage of ADC drug killing effect.
  • thermal stability the antibody involved in the present invention has better thermal stability than GSK2857916. Thermal stability is a key indicator of antibody druggability. It has better thermally stable antibodies and may have more advantages in antibody expression, and produces The possibility of aggregates is reduced.
  • the antibody involved in the present invention is a humanized antibody, which has a lower risk of immunogenicity. The immunogenicity can stimulate the immune response in the human body. On the one hand, it produces anti-antibodies and reduces the risk of antibody drugs.
  • the BCMA antigen and its ligand were purchased from ACRO Biosystems.
  • the GSK2857916 positive antibody of GlaxoSmithKline (GSK) was prepared according to the US patent application (US20140105915A). It is worth noting that the positive antibody GSK2857916 was prepared according to the US patent application (US20140105915A). The antibody is not coupled to the toxin molecule but does not affect other possible functions.
  • the positive control antibody GSK2857916 is expressed using the ExpiCHO system, where the main materials used include: Gibco medium (Cat. No.: A29100-01), Gibco Transfection Kit (Cat. No.: A29129).
  • Gibco medium Cat. No.: A29100-01
  • Gibco Transfection Kit Cat. No.: A29129
  • the light chain sequence of GSK2857916 (shown in the sequence of SEQ ID NO: 17) and the heavy chain sequence (shown in the sequence of SEQ ID NO.: 18) of GSK2857916 are synthesized according to the sequence disclosed in the US patent application US20140105915A, A plasmid containing the light chain and heavy chain genes of the complete GSK2857916 antibody was constructed by molecular cloning.
  • the plasmid containing the light chain of GSK2857916 antibody and the plasmid of the heavy chain were mixed at a mass ratio of 2:1.
  • the above plasmid mixture (25 ⁇ g) and transfection reagent were mixed according to the standard procedure and added dropwise to 25mL.
  • ExpiCHO cell expression system After mixing well, express in a cell culture incubator at 37°C for 18-22 hours. Subsequently, feed medium was added to the transfection mixture and placed in a 32°C cell incubator to continue culturing. On the 5th day after transfection, add a second feed, and place the cells in a 32°C cell incubator to continue culturing for 10-12 days.
  • the expressed cell suspension was centrifuged at a high speed and the supernatant was taken, and the obtained supernatant was filtered through a 0.22 ⁇ m filter membrane, and then purified using the Protein A/G affinity chromatography column affinity method. After purification, the target protein was eluted with 100 mM glycinate (pH 3.0), concentrated, replaced, aliquoted, identified by SDS-PAGE and qualified for activity, and then stored and frozen.
  • the human myeloma cell line H929 cells expressing BCMA were purchased, and the cell line BCMA-HEK293 cells overexpressing human BCMA and the cell line BCMA-CHO cells overexpressing monkey BCMA were constructed.
  • H929 was purchased from Peking Union Medical College Cell Bank, the product number is 3111C0001CCC000360, and it is a myeloma cell line that naturally highly expresses human BCMA.
  • a DNA fragment containing the BCMA protein of cynomolgus monkey was synthesized by gene synthesis technology and cloned into an expression vector. Introduce E. coli by chemical transformation method, pick out E. coli single clone and sequence to obtain the correct plasmid clone, carry out plasmid extraction and re-sequence confirmation.
  • Gibco's CD-CHO serum-free medium (Cat. No. 10743029) was used to culture CHO-s cells. The day before electroporation, the cells were passaged to 5 ⁇ 10 6 /mL. The next day, the Invitrogen electroporation kit (Cat. No.: MPK10096) and electroporation instrument (Cat. No.: MP922947) were used to introduce the constructed plasmid into CHO-s cells. Transfer the electroporated cells to CD-CHO medium, and place them in a 37°C cell incubator for 48 hours.
  • the single cell clones grown in the 96-well plate were picked and transferred to a 24-well culture plate for continued expansion. Afterwards, FACS was used to identify cell lines that successfully transformed monkey BCMA.
  • a DNA fragment containing human BCMA protein is synthesized by gene synthesis technology and cloned into an expression vector. Introduce E. coli by chemical transformation method, pick out E. coli single clone and sequence to obtain the correct plasmid clone, carry out plasmid extraction and re-sequence confirmation.
  • Gibco's DMEM serum-free medium (Cat. No. 12634010) was used to culture HEK293 cells. The day before the electroporation, the cells were passaged to 2 ⁇ 10 5 /mL. The next day, the Invitrogen electroporation kit (Cat. No.: MPK10096) and electroporation instrument (Cat. No.: MP922947) were used to introduce the constructed plasmid into HEK293 cells. Transfer the electroporated cells to DMEM medium and place them in a 37°C cell incubator for 48 hours.
  • animal immunization includes several protocols and is carried out at the same time.
  • mouse serum is taken for titer detection and the effect of immunization is evaluated, and finally the number of the mouse to be built into the library is determined based on the serum titer.
  • the materials include the aforementioned human BCMA-Fc and monkey BCMA-Fc Antigen protein (ACRO Biosystems) adopts cross-immunization method.
  • the first immunization dose was 100 ⁇ g/mouse, supplemented with CFA (Complete Freund's Adjuvant), and then the immunization dose was reduced to 50 ⁇ g/mouse, supplemented with IFA (Freund's Incomplete Adjuvant), both of which were cross-immunized for two weeks.
  • the corresponding mouse numbers are Mouse 1 and Mouse 2.
  • the materials include the aforementioned human BCMA-Fc and monkey BCMA-Fc antigen proteins (ACRO Biosystems ), HEK293 cells with high expression of human BCMA and CHO cells with high expression of monkey BCMA, adopt a cross-immunization method.
  • the protein immunization dose is 20 ⁇ g/mouse
  • the cellular immunization dose is 1 ⁇ 10 7 /mouse
  • intraperitoneal injection is a cross-immunization once a week.
  • the corresponding mouse numbers are Mouse 3 and Mouse 4.
  • human BCMA and monkey BCMA were respectively coated one day in advance at a coating concentration of 2 ⁇ g/mL, 30 ⁇ L/well, and overnight at 4°C.
  • the plate was washed three times with PBST, blocked with 5% skim milk at room temperature for two hours, and then washed three times with PBST.
  • Each serum sample was diluted 500 times on the basis of the original solution, and then diluted in a 3-fold gradient, and then added to the ELISA plate as the primary antibody. After incubating for 1 hour at room temperature, the plate was washed three times with PBST, and then the secondary antibody (Goat-anti- mouse-IgG-Fab-HRP, Sigma, M4115), incubate for 1h at room temperature.
  • the secondary antibody Goat-anti- mouse-IgG-Fab-HRP, Sigma, M4115
  • Table 1-1 Use human BCMA to detect the titer of mouse serum
  • the mouse with the best titer in Example 3 was selected.
  • the antibody gene of the B cell in the mouse spleen was cloned into a phage display vector to construct an antibody library.
  • human BCMA and monkey BCMA as screening antigens, through library screening, monoclonal preliminary screening and sequencing, 17 antibody molecules with binding activity against human and monkey BCMA antigens were finally obtained.
  • mice were treated according to the standard procedure of euthanasia. Take the mouse spleen, after grinding and filtering, collect the spleen cells, add 1mL of TRIzol TM Reagent (Thermo Fisher, 15596026) to lyse the spleen cells, extract the total RNA by the phenol-chloroform method, and pass the reverse transcription kit (TaKaRa, 6210A) The extracted RNA is reverse transcribed into cDNA. Afterwards, using cDNA as a PCR template, specific primers amplified from murine antibody sequences were used to amplify the light chain and heavy chain genes of the antibody, respectively.
  • TRIzol TM Reagent Thermo Fisher, 15596026
  • TaKaRa reverse transcription kit
  • the antibody gene fragment was inserted into the vector for phage display, the ligation product was recovered by the DNA recovery kit (Omega, D6492-02), and finally by the electroporation instrument (Bio- Rad, MicroPulser) was transformed into competent E. coli SS320 (Lucigen, MC1061 F), and spread on 2-YT (C+/K+2-YT) solid plates containing ampicillin and tetracycline to amplify the correctly transformed antibody The SS320 strain of the plasmid was finally constructed into a library containing Fab antibody sequences.
  • Magnetic bead screening is based on the combination of Biotin-labeled antigen protein and Avidin-coupled magnetic beads.
  • the antigen-bound magnetic beads and library are incubated, washed and eluted. After 2-4 rounds of sea selection. Selection, the process of finally enriching the specific monoclonal antibodies against the antigen.
  • Biotin-labeled human BCMA antigens and monkey BCMA antigens are used to cross audition principles.
  • the first and third rounds use human BCMA audition, and the second round uses monkey BCMA audition. A total of 3 rounds of audition are used, and then The enriched antibody sequence mixture was subjected to a monoclonal preliminary screening of human and monkey BCMA.
  • the human BCMA protein labeled with Biotin is incubated with the magnetic beads coupled with Avidin, so that the human BCMA protein is bound to the magnetic beads.
  • the magnetic beads with BCMA antigen and the constructed phage library were incubated for 2h at room temperature. After washing 6-8 times with PBST, the non-specifically adsorbed phages were removed, Trypsin (Gibco, 25200072) was added and mixed gently and reacted for 20 minutes to elute the specifically bound antibody display phages.
  • the eluted phage was used to infect logarithmic SS320 bacteria (Lucigen, MC1061 F) and left to stand for 30 minutes, then cultured at 220 rpm for 1 hour, and then added VSCM13 helper phage and allowed to stand for 30 minutes, and continued at 220 rpm Cultivate for 1 hour under the conditions, centrifuge and replace in C+/K+2-YT medium, and the finally obtained phage will continue to be used in the next round of audition.
  • the principle of immune tube screening is to coat the BCMA protein on the surface of the immune tube with high adsorption capacity.
  • the phage display antibody library is added to the immune tube and incubated, washed and eluted with the antigen protein adsorbed on the surface of the immune tube.
  • the process of finally enriching the specific monoclonal antibodies against the antigen after 2-4 rounds of sea selection, the process of finally enriching the specific monoclonal antibodies against the antigen.
  • the purpose of the immunotube method and the magnetic bead method are both to enrich specific antibodies against the antigen, and are two complementary experimental methods.
  • human BCMA antigens and monkey BCMA antigens are used to cross audition.
  • the first and third rounds adopt human BCMA audition, and the second round adopts monkey BCMA audition.
  • a total of 3 rounds of audition are then enriched.
  • the antibody sequence mixture of BCMA was screened for monoclonal preliminary screening of human and monkey BCMA.
  • the specific implementation method is similar to the magnetic bead method.
  • the 17 human monkey crossed Fab antibodies obtained in Example 4 were constructed as a human IgG1 type, in which the light chains are all Kappa, and the antibody type is a human-mouse chimeric antibody.
  • the heavy chain sequence of the sequence obtained from the screening was constructed by fusion with the human IgG1 Fc section, and the light chain was constructed by fusion with the human Kappa constant region.
  • the heavy chain and light chain plasmids were transformed into ExpiCHO cells to induce expression and obtain the full-length antibody.
  • the antibody uses the ExpiCHO transient expression system, the medium is (Gibco, A29100-01), and the transfection kit is (Gibco, A29129).
  • the specific method is as follows: the day before transfection, the ExpiCHO cells are passaged. In a 25mL system, 25 ⁇ g of the constructed plasmid is mixed with the transfection reagent and then added dropwise to 25mL ExpiCHO cells. After mixing well, place in a cell incubator at 37°C. Expression 18-22h. Subsequently, feed medium was added to the transfection mixture and placed in a 32°C cell incubator to continue culturing.
  • the expressed cell suspension is centrifuged at high speed and the supernatant is taken.
  • the obtained supernatant is filtered through a 0.22 ⁇ m filter membrane and purified by the affinity method of a Protein A/G affinity chromatography column. After purification, the target protein was eluted with 100mM glycinate (pH3.0), concentrated, replaced, aliquoted, identified by SDS-PAGE, SEC purity detection, activity identification, and stored and frozen.
  • the ELISA-based method verifies the affinity effect of the candidate antibody on human and monkey BCMA, and the ELISA-based method verifies the candidate antibody's ability to block the binding of BCMA and BAFF or APRIL.
  • the blocking system includes a pre-development process. According to the development results, the actual blocking system parameters with both sensitivity and stability are determined.
  • BAFF blocking plate human BAFF protein, 1 ⁇ g/mL, 30 ⁇ L/well, 4°C overnight. The next day, the well plate was washed 3 times with PBST and then blocked with 5% skimmed milk for 2h. Then, the candidate antibody or the positive control antibody (GSK2857916) was serially diluted, and premixed with Biotin-labeled human BCMA (0.6 ⁇ g/mL) for 0.5h in advance, and added to the 96-well ELISA plate after the completion of blocking and washing. Incubate for 1h. After washing with PBST for 3 times, the secondary antibody (NeutrAvidin-HRP, Therofisher, 31001) was added and incubated for 1 h.
  • the candidate antibody or the positive control antibody GSK2857916
  • Biotin-labeled human BCMA 0.6 ⁇ g/mL
  • the blocking system includes a pre-development process. According to the development results, the actual blocking system parameters with both sensitivity and stability are determined.
  • APRIL blocking plate human APRIL protein, 2 ⁇ g/mL, 30 ⁇ L/well, 4°C overnight. The next day, the well plates were washed 3 times with PBST and then blocked with 5% skimmed milk for 1 hour. Then, the candidate antibody or the positive control antibody (GSK2857916) was serially diluted, and premixed with Biotin-labeled human BCMA (0.6 ⁇ g/mL) for half an hour in advance, and added to the 96-well flat bottom plate after the completion of blocking and washing. Incubate for 1h. After washing with PBST 3 times, the secondary antibody NeutrAvidin-HRP (Therofisher, 31001) was added and incubated for 1 h.
  • PE-labeled anti-human-IgG- Fc flow cytometry antibody Abcam, 98596
  • the operating procedure is completely the same as 7.1.
  • the difference is that the cells are monkey BCMA-CHO cells.
  • the results are shown in Figure 3(B).
  • the results show that the antibody SY14-3rd-5-6-7
  • the affinity effect for monkey BCMA-CHO cells is better than the positive control antibody (GSK2857916).
  • the signal difference between human and monkey may be caused by the difference in BCMA expression on BCMA-HEK293 or BCMA-CHO.
  • Fab-ZAP (Atsbio, IT-51-100) is a Fab fragment connected with saporin, which is a ribosome inhibitor that can inhibit protein synthesis and cause cell death.
  • Fab-ZAP is a Fab fragment that can bind to human antibody Fc.
  • the specific detection method is as follows: H929 cells are resuscitated one week in advance and passaged every 3 days after resuscitation.
  • the cell seeding density is 2 ⁇ 10 5 /mL each time, and the passage of cells does not exceed 3 weeks.
  • Aspirate the cells in the logarithmic growth phase fully mix the cells, count and determine their viability. Take a 96-well flat bottom plate, adjust the cell density to 4 ⁇ 10 5 /mL, add 50 ⁇ L of cells to each well, gently tap to mix, and place the cell culture plate in a 37°C cell incubator for 16 hours.
  • Fab-ZAP As a 27nM (2.16 ⁇ g/ml) diluent, and add it to the serially diluted antibody to make the incubated Fab-ZAP
  • the final concentration of ZAP is 13.5nM (1.08 ⁇ g/ml).
  • the modified candidate antibodies include SY14-3rd-5-6-7 (also known as 5-6-7 or 5-6-7-WT) and SY14-3rd-5-6-32 (also known as 5-6- 32 or 5-6-32-WT), wherein the preferred candidate antibodies after modification are referred to as 5-6-7-hu-2 and 5-6-32-hu-2, respectively.
  • Example 7 For specific operations, see Example 7. The results are shown in Figure 12(B). The results show that the binding effect of the antibody and monkey BCMA-CHO cells before and after humanization is basically the same, and both are better than the positive control antibody (GSK2857916).
  • the thermal stability data of the two candidate antibodies before and after humanization and the positive control antibody were tested.
  • the specific process is as follows: prepare antibody solution, 0.25mg/mL, 19 ⁇ L/well, set three parallel wells for each test product, and use PBS and IPI as references. Then add 1 ⁇ L of SYPRO orange dye at a concentration of 100 ⁇ to each well, and prepare for the machine.
  • Use ABI 7500 FAST RT-PCR instrument to test select melting curve for test type, adopt continuous mode, scan temperature range of 25 ⁇ 95°C, heating rate of 1%, 25°C equilibrium for 5min, collect data during heating process, report base Select ROX for the group, None for the quenching group, and a reaction volume of 20 ⁇ L.
  • the temperature corresponding to the first peak and valley of the first derivative of the melting curve is determined as the denaturation temperature of the candidate antibody.
  • the results are shown in Table 1-3.
  • the Fortebio Octet RED96 instrument was used to detect the affinity of the candidate antibodies before and after humanization of 5-6-7 and 5-6-32 with human BCMA and monkey BCMA.
  • Pre-wet the sensor Anti-Human Fba-CH1 2nd Generation, FAB2G
  • FAB2G Anti-Human Fba-CH1 2nd Generation
  • Add 200 ⁇ L/well of KB buffer to columns 1, 10, and 12 of sample plate 1 add 0.01M glycine solution with pH 2.0 to column 11, and add the prepared sample solution to columns 2-8 (one sample adds 4
  • Add 2 samples to each well, in the ninth column add human BCMA-Fc in order from high to low, that is, add 200nM antigen solution to the first and fifth wells, and add 50nM antigen to the second and sixth wells.
  • 5-6-7-hu-2 and 5-6-32-hu-2 were selected, analyzed and sequenced.
  • the variable region of the human antibody sequence is defined, and the light chain and heavy chain variable regions of the antibody of the present invention are determined (SEQ ID NO: 13-16),
  • the variable region sequence was analyzed and AbM was used to define the CDR to determine the complementarity determining region sequence (SEQ ID NO: 1-12) of the antibody heavy chain and light chain.

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Abstract

提供了一种分离的靶向B细胞成熟抗原(BCMA)的单克隆抗体，该抗体既能结合人BCMA也能结合猴BCMA。还提供了编码该抗体的核酸、生产该抗体的方法，以及包含该抗体的药物组合物。

Description

靶向BCMA的具有人猴交叉的人源化单克隆抗体 技术领域
本发明涉及特异性结合B细胞成熟抗原(BCMA)，包括同时特异性结合人BCMA和猴BCMA抗原的单克隆抗体及其片段。
本发明涉及特异性结合BCMA并且抑制BAFF和APRIL对BCMA受体结合的单克隆抗体及其片段。
本发明还涉及特异性结合BCMA并且具有优异内吞效应的单克隆抗体及其片段。
背景技术
B细胞于骨髓中成熟并成为浆细胞，能够分泌抗体对抗外来的病毒或细菌。当浆细胞发生癌变成为骨髓瘤细胞后，会不断增殖出更多恶性的骨髓瘤细胞并分泌大量无用的抗体。骨髓瘤通常生长在脊柱、颅骨、骨盆、胸腔等位置，表现为一种肿瘤或者是溶骨性病变。骨髓瘤的病情通常是渐进式，从意义未明的单克隆丙种球蛋白血症(MGUS)到低风险冒烟型多发性骨髓瘤再到高危冒烟型多发性骨髓瘤(SMM)，最终进展成多发性骨髓瘤(Nature Reviews Disease Primers,2017,3,17046.)。多发性骨髓瘤(Multiple myeloma，MM)主要的表征包括高血钙症、肾功能减退、贫血、骨骼功能障碍等，伴随剧烈的骨疼痛和容易发生反复性骨折(Nature Reviews Clinical Oncology,2012,9(3),135–143.)。据国际骨髓瘤基金会统计，仅截止2017年8月，全球患病人数达75万人左右，每年新发病例约为11.40万人，并有近9万人死于该疾病。
B细胞成熟抗原(BCMA)，属于肿瘤坏死因子超家族的一员，主要表达于记忆细胞、浆母细胞和浆细胞表面，而在其他细胞表面几乎不表达，另一方面，BCMA属于细胞表面的跨膜受体，其基因定位于16号染色体的TNFRSF17位点。有文献报道(Blood Cancer Journal,2015,5(2),e282–e282.)，BCMA缺陷的小鼠在表观和B细胞数量上都较为正常，但其浆细胞的存活 能力极差。
BCMA的配体包括B细胞活化因子(BAFF)和诱导增殖配体(APRIL)。其中，BAFF的受体还包括BAFF-R和TACI，APRIL的受体还包括TACI。BCMA信号通路主要的作用是促进B细胞的生存、分化和调节性T细胞的激活等，相反的，TACI信号通路抑制B细胞的成熟(Nature reviews immunology,2009,9(7):491.)。对于这三种受体而言(BAFF-R、TACI和BCMA)，在B细胞发育过程中，未成熟B细胞、迁移中的B细胞和初始B细胞表面只表达BAFF-R；GC B细胞表面均有表达BAFF-R和BCMA；记忆细胞表面均有表达BAFF-R、TACI和BCMA；浆母细胞或浆细胞表面均表达TACI和BCMA；而当浆细胞发生癌变成为多发性骨髓瘤细胞后，其表面高度表达BCMA，有可能表达TACI而不表达BAFF-R(Nature reviews immunology,2009,9(7):491.)。由此可知，大部分的B细胞都不表达BCMA，另外，研究表明其他器官的细胞也几乎不表达BCMA。临床上，多发性骨髓瘤病人血清中BCMA、BAFF和APRIL的含量更高且总生存期和预后更差。因此，在治疗多发性骨髓瘤方面，BCMA是一种优于CD19等的新靶点，其特异性高且靶点副作用更小。因此，开发针对BCMA靶点的具有阻断效应或内吞效应的抗体药物不仅能够提高多发性骨髓瘤的治疗效果，还能极大降低治疗的副作用，并且可以产生巨大的经济和社会价值。
目前，在ADC(抗体药物偶联物(antibody-drug conjugate，ADC))药物方面，Glaxo Group公司和Seattle Genetics公司共同开发的Belantamab mafodotin(简称为GSK2857916)效果显著，在35例过度预治疗(大多数患者至少接受了5种疗法且治疗失败)R/R MM患者中，ORR达到了60％，中位PFS(Progression-Free-Survival(无进展生存期))为12个月(NCT03848845)。在CAR-T细胞方面，新基和蓝鸟生物的CAR-T细胞疗法Idecabtagene vicleucel(简称为bb2121)，在33例既往已接受至少3种疗法失败的R/R MM患者中，总缓解率达到了85％，中位PFS为11.8个月(NCT02658929)。在双特异性抗体方面，安进的AMG 420是进展最快的疗法，这类抗体较传统抗体小，由两个抗体域片段连接而成，具有很好的活性，但半衰期比全长抗体短(NCT02514239)。就临床结果而言，针对BMCA靶点的药物，无论是单抗、双抗、ADC还是CAR-T细胞治疗，都取得了令人瞩目的结果，并且临床结果表明，BCMA靶点带来的副作用也远低于其他靶点。
发明内容
基于上述背景，本发明意在开发出新型的靶向BCMA的抗体。
本发明以BCMA作为免疫原免疫小鼠，通过噬菌体展示技术，构建并筛选抗体文库，获得同时结合人和猴BCMA抗原的单克隆抗体。后续通过抗体人源化改造，将鼠源单克隆抗体改造为人源化抗体，通过亲和、阻断和内吞等功能实验证实人源化候选抗体表现出了优异的功能，在细胞水平的内吞功能优于竞品GSK2857916。
本说明书内公开的所有专利和参考文献通过引用清楚和完整地并入本文。
在本发明涉及一种分离的靶向B细胞成熟抗原(BCMA)的单克隆抗体，其中，所述抗体既能结合人BCMA也能结合猴BCMA。
在一具体方面中，本发明的抗体包含重链可变区，所述重链可变区包含SEQ ID NO:1或2所示的重链互补决定区1(CDR-H1)，和/或包含SEQ ID NO:3或4所示的重链互补决定区2(CDR-H2)，和/或包含SEQ ID NO:5或6所示的重链互补决定区3(CDR-H3)。
在一具体方面中，本发明的抗体包含轻链可变区，所述轻链可变区包含SEQ ID NO:7或8所示的轻链互补决定区1(CDR-L1)，和/或包含SEQ ID NO:9或10所示的轻链互补决定区2(CDR-L2)，和/或包含SEQ ID NO:11或12所示的轻链互补决定区3(CDR-L3)。
在一具体方面中，本发明的抗体包含重链可变区和轻链可变区，其中，所述重链可变区包含SEQ ID NO:1或2所示的重链互补决定区1(CDR-H1)，和/或包含SEQ ID NO:3或4所示的重链互补决定区2(CDR-H2)，和/或包含SEQ ID NO:5或6所示的重链互补决定区3(CDR-H3)；所述轻链可变区包含SEQ ID NO:7或8所示的轻链互补决定区1(CDR-L1)，和/或包含SEQ ID NO:9或10所示的轻链互补决定区2(CDR-L2)，和/或包含SEQ ID NO:11或12所示的轻链互补决定区3(CDR-L3)。
在一具体方面中，本发明的抗体包含上述抗体的变体，且具备与上述本发明所述的抗体相同或相似的活性。
在一具体方面中，本发明的抗体包含轻链可变区，所述轻链可变区包含SEQ ID NO:13或14所示的氨基酸序列、或上述序列的变体。
在一具体方面中，本发明的抗体包含重链可变区，所述重链可变区具有SEQ ID NO:15或16所示的氨基酸序列、或上述序列的变体。
在一具体方面中，本发明的抗体包含轻链可变区和重链可变区，其中，所述轻链可变区包含SEQ ID NO:13或14所示的氨基酸序列、或上述序列的变体，所述重链可变区具有SEQ ID NO:15或16所示的氨基酸序列、或上述序列的变体。
在一具体方面中，本发明的抗体的重链可变区包含SEQ ID NO:1所示的CDR-H1，SEQ ID NO:3所示的CDR-H2和SEQ ID NO:5所示的CDR-H3。
在一具体方面中，本发明的抗体的重链可变区包含SEQ ID NO:2所示的CDR-H1，SEQ ID NO:4所示的CDR-H2和SEQ ID NO:6所示的CDR-H3。
在一具体方面中，本发明的抗体的轻链可变区包含SEQ ID NO:7所示的CDR-L1，SEQ ID NO:9所示的CDR-L2和SEQ ID NO:11所示的CDR-L3。
在一具体方面中，本发明的抗体的轻链可变区包含SEQ ID NO:8所示的CDR-L1，SEQ ID NO:10所示的CDR-L2和SEQ ID NO:12所示的CDR-L3。
在一具体方面中，本发明的抗体的轻链可变区具有SEQ ID NO:13或14所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
在一具体方面中，本发明的抗体的重链可变区具有SEQ ID NO:15或16所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
在一具体方面中，本发明的抗体包含重链可变区和轻链可变区，其中，重链可变区包含SEQ ID NO:1所示的CDR-H1，SEQ ID NO:3所示的CDR-H2和SEQ ID NO:5所示的CDR-H3；轻链可变区包含包含SEQ ID NO:7所示的CDR-L1，SEQ ID NO:9所示的CDR-L2和SEQ ID NO:11所示的CDR-L3。
进一步，所述抗体的轻链可变区具有SEQ ID NO:13所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列；所述抗体的重链可变区具有SEQ ID NO:15所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序 列。
在一具体方面中，本发明的抗体包含重链可变区和轻链可变区，其中，重链可变区包含SEQ ID NO:2所示的CDR-H1，SEQ ID NO:4所示的CDR-H2和SEQ ID NO:6所示的CDR-H3；轻链可变区包含包含SEQ ID NO:8所示的CDR-L1，SEQ ID NO:10所示的CDR-L2和SEQ ID NO:12所示的CDR-L3。
进一步，所述抗体的轻链可变区具有SEQ ID NO:14所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列；所述抗体的重链可变区具有SEQ ID NO:16所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
本发明还涉及一种靶向B细胞成熟抗原(BCMA)的单克隆抗体，该抗体与上述本发明所述的抗体识别相同的抗原决定部位。
本发明还涉及一种靶向B细胞成熟抗原(BCMA)的单克隆抗体，该抗体与上述本发明所述的抗体竞争性结合B细胞成熟抗原(BCMA)。
本发明还涉及编码上述本发明的抗体的核酸。
本发明还涉及一种表达载体，其包含上述本发明所述的核酸。
本发明还涉及一种宿主细胞，其包含上述本发明所述的表达载体或基因组中整合有本发明所述的核酸。
本发明还涉及一种生产单克隆抗体的方法，所述方法包括培养本发明所述的宿主细胞从而生产上述本发明所述的单克隆抗体。
本发明还涉及一种药物组合物，其包含本发明所述的单克隆抗体和药学上可接受的载体。
本发明还涉及一种药盒或制品，其包括本发明所述的单克隆抗体或本发明所述的药物组合物。
本发明还涉及一种治疗与BCMA的表达相关的疾病的方法，其包括：向有此需要的受试者给药本发明所述的单克隆抗体或本发明所述的药物组合物或本发明所述的药盒或制品。
在一具体方面，上述疾病选自B细胞急性淋巴性白血病、T细胞急性淋巴性白血病、急性淋巴性白血病、慢性髓性白血病、慢性淋巴细胞白血病、 B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、毛细胞白血病，小细胞或大细胞滤泡性淋巴瘤、恶性淋巴瘤、恶性淋巴增殖状况、MALT淋巴瘤、外套细胞淋巴瘤、边缘区淋巴瘤、多发性骨髓瘤、脊髓发育不良和骨髓增生异常综合征、非霍奇金淋巴瘤、浆母细胞淋巴瘤、浆细胞样树突细胞瘤、瓦尔登斯特伦巨球蛋白血症、骨髓瘤、MGUS、浆细胞瘤、系统性淀粉样蛋白轻链淀粉样变性和POEMS综合征。
在一具体方面，上述疾病为多发性骨髓瘤。
本发明还涉及本发明的单克隆抗体在制备用于治疗与BCMA的表达相关的疾病的药物中的用途。
在一具体方面，上述疾病选自B细胞急性淋巴性白血病、T细胞急性淋巴性白血病、急性淋巴性白血病、慢性髓性白血病、慢性淋巴细胞白血病、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、毛细胞白血病，小细胞或大细胞滤泡性淋巴瘤、恶性淋巴瘤、恶性淋巴增殖状况、MALT淋巴瘤、外套细胞淋巴瘤、边缘区淋巴瘤、多发性骨髓瘤、脊髓发育不良和骨髓增生异常综合征、非霍奇金淋巴瘤、浆母细胞淋巴瘤、浆细胞样树突细胞瘤、瓦尔登斯特伦巨球蛋白血症、骨髓瘤、MGUS、浆细胞瘤、系统性淀粉样蛋白轻链淀粉样变性和POEMS综合征。
在一具体方面，上述疾病为多发性骨髓瘤。
具体来说，本发明涉及如下方面：
1.一种分离的靶向B细胞成熟抗原(BCMA)的单克隆抗体，其中，所述抗体既能结合人BCMA也能结合猴BCMA。
2.根据项1所述的单克隆抗体，所述抗体选自以下的任一种：
(1)抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO:1或2所示的重链互补决定区1(CDR-H1)，和/或包含SEQ ID NO:3或4所示的重链互补决定区2(CDR-H2)，和/或包含SEQ ID NO:5或6所示的重链互补决定区3(CDR-H3)；
(2)抗体，其包含轻链可变区，所述轻链可变区包含SEQ ID NO:7或8所示的轻链互补决定区1(CDR-L1)，和/或包含SEQ ID NO:9或10所示的轻链互补决定区2(CDR-L2)，和/或包含SEQ ID NO:11或12所示的轻链互补 决定区3(CDR-L3)；
(3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；
(4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
3.如项1或2所述的抗体，其特征在于，所述抗体选自以下的任一种：
(1)抗体，包含轻链可变区，所述轻链可变区包含SEQ ID NO:13或14所示的氨基酸序列、或上述序列的变体；
(2)抗体，包含重链可变区，所述重链可变区具有SEQ ID NO:15或16所示的氨基酸序列、或上述序列的变体；
(3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区。
4.如项1～3中任一项所述的抗体，其特征在于，所述的抗体的重链可变区包含SEQ ID NO:1所示的CDR-H1，SEQ ID NO:3所示的CDR-H2和SEQ ID NO:5所示的CDR-H3。
5.如项1～3中任一项所述的抗体，其特征在于，所述的抗体的重链可变区包含SEQ ID NO:2所示的CDR-H1，SEQ ID NO:4所示的CDR-H2和SEQ ID NO:6所示的CDR-H3。
6.如项1～3中任一项所述的抗体，其特征在于，所述的抗体的轻链可变区包含SEQ ID NO:7所示的CDR-L1，SEQ ID NO:9所示的CDR-L2和SEQ ID NO:11所示的CDR-L3。
7.如项1～3中任一项所述的抗体，其特征在于，所述的抗体的轻链可变区包含SEQ ID NO:8所示的CDR-L1，SEQ ID NO:10所示的CDR-L2和SEQ ID NO:12所示的CDR-L3。
8.根据项1～3中任一项所述的抗体，其特征在于，所述的抗体的轻链可变区具有SEQ ID NO:13或14所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
9.根据项1～3中任一项所述的抗体，其特征在于，所述的抗体的重链可变区具有SEQ ID NO:15或16所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、 98％、99％相似性的序列。
10.一种靶向B细胞成熟抗原(BCMA)的单克隆抗体，其特征在于，该抗体与项1-9中任一项所述的抗体识别相同的抗原决定部位。
11.一种靶向B细胞成熟抗原(BCMA)的单克隆抗体，其特征在于，该抗体与项1-9中任一项所述的抗体竞争性结合B细胞成熟抗原(BCMA)。
12.编码项1-11中任一项所述的抗体的核酸。
13.一种表达载体，其包含项12所述的核酸。
14.一种宿主细胞，其包含项13所述的表达载体或基因组中整合有项12所述的核酸。
15.一种生产单克隆抗体的方法，所述方法包括培养根据项14所述的宿主细胞从而生产根据项1～11中任一项所述的单克隆抗体。
16.一种药物组合物，其包含根据项1～11中任一项所述的单克隆抗体和药学上可接受的载体。
17.一种药盒或制品，其包括根据项1～11中任一项所述的单克隆抗体或根据项16所述的药物组合物。
18.一种治疗与BCMA的表达相关的疾病的方法，其包括：
向有此需要的受试者给药根据项1～11中任一项所述的单克隆抗体或根据项16所述的药物组合物或项17所述的药盒或制品。
19.根据项18所述的方法，其中，所述疾病选自B细胞急性淋巴性白血病、T细胞急性淋巴性白血病、急性淋巴性白血病、慢性髓性白血病、慢性淋巴细胞白血病、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、毛细胞白血病，小细胞或大细胞滤泡性淋巴瘤、恶性淋巴瘤、恶性淋巴增殖状况、MALT淋巴瘤、外套细胞淋巴瘤、边缘区淋巴瘤、多发性骨髓瘤、脊髓发育不良和骨髓增生异常综合征、非霍奇金淋巴瘤、浆母细胞淋巴瘤、浆细胞样树突细胞瘤、瓦尔登斯特伦巨球蛋白血症、骨髓瘤、MGUS、浆细胞瘤、系统性淀粉样蛋白轻链淀粉样变性和POEMS综合征。
20.根据项18或19所述的方法，其中，所述疾病为多发性骨髓瘤。
21.根据项1～11中任一项所述的单克隆抗体在制备用于治疗与BCMA的表达相关的疾病的药物中的用途。
22.根据项21所述的用途，其中，所述疾病选自B细胞急性淋巴性白 血病、T细胞急性淋巴性白血病、急性淋巴性白血病、慢性髓性白血病、慢性淋巴细胞白血病、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、毛细胞白血病，小细胞或大细胞滤泡性淋巴瘤、恶性淋巴瘤、恶性淋巴增殖状况、MALT淋巴瘤、外套细胞淋巴瘤、边缘区淋巴瘤、多发性骨髓瘤、脊髓发育不良和骨髓增生异常综合征、非霍奇金淋巴瘤、浆母细胞淋巴瘤、浆细胞样树突细胞瘤、瓦尔登斯特伦巨球蛋白血症、骨髓瘤、MGUS、浆细胞瘤、系统性淀粉样蛋白轻链淀粉样变性和POEMS综合征。
23.根据项21或22所述的用途，其中，所述疾病为多发性骨髓瘤。
本发明的效果
相比于Belantamab mafodotin抗体(简称为GSK2857916)，本发明涉及的抗体既能结合人BCMA也能结合猴BCMA，在亲和水平方面，与之接近甚至更好。本发明涉及的抗体在阻断BCMA结合其配体BAFF或APRIL效果方面，与GSK2857916接近甚至更好。本发明涉及的抗体在内吞效果方面，具有比GSK2857916更好的内吞效果。本发明涉及的抗体在热稳定性方面，具有比GSK2857916更好的热稳定性。本发明涉及的抗体，在免疫原性方面，为人源化后的抗体，具有更低的免疫原性。
附图说明
图1显示抗体产生的过程，显示靶向BCMA的具有人猴交叉的抗体产生的过程。
图2显示重组蛋白BCMA、BAFF和APRIL活性测定，结果显示各蛋白活性正常。图2(A)显示不同标签(Fc和His)的人和猴BCMA抗原和阳性对照抗体GSK2857916的结合结果。图2(B)显示人BCMA抗原和不同浓度包板的BAFF的结合结果。图2(C)显示人BCMA抗原和不同浓度包板的APRIL的结合结果。
图3显示候选抗体与过表达人或猴BCMA的细胞结合测定，结果表明候选抗体具有人猴交叉的特性。图3(A)显示通过FACS确定部分候选抗体与过表达人BCMA的HEK293细胞的结合。图3(B)显示通过FACS确定部分候选抗体与过表达猴BCMA的CHO细胞的结合。
图4显示部分候选抗体与人、猴BCMA交叉亲和活性检测结果。图4(A)显示部分候选抗体在Elisa水平上与人BCMA的亲和效果，结果表明，部分候选抗体相比于GSK2857916抗体有接近甚至更好的亲和效果。图4(B)显示部分候选抗体在Elisa水平上与猴BCMA的亲和效果，结果表明，部分候选抗体相比于GSK2857916抗体有接近甚至更好的亲和效果。
图5显示部分候选抗体阻断效果检测结果。图5(A)显示部分候选抗体在Elisa水平上阻断BCMA与BAFF的结合，结果表明，部分候选抗体相比于GSK2857916抗体有接近甚至更好的阻断效果。图5(B)和显示部分候选抗体在Elisa水平上阻断BCMA与APRIL的结合，结果表明，部分候选抗体相比于GSK2857916抗体有接近甚至更好的阻断效果。
图6显示内吞效果测定结果。图6(A)和(B)显示部分候选抗体在人骨髓瘤细胞系H929细胞上的内吞效果，结果表明，部分候选抗体相比于GSK2857916抗体有更好的内吞效果。
图7显示候选抗体各个功能汇总，其中图7展示了部分候选抗体在表达人BCMA的HEK293细胞上的亲和效果，表达猴BCMA的CHO细胞上的亲和效果，Elisa水平上和人、猴BCMA的亲和效果，Elisa水平上阻断BAFF与BCMA结合的效果、Elisa水平上阻断APRIL与BCMA结合的效果，以及抗体在人骨髓瘤细胞H929上的内吞效果；其中‘+’数量从多到少，代表抗体亲和、阻断及内吞效果由强到弱，结果表明，抗体SY14-3rd-5-6-7和SY14-3rd-5-6-32表现出了更优异的综合效果。
图8显示人源化后抗体(5-6-7-hu-2)与人、猴BCMA交叉亲和活性检测结果。图8(A)显示检测SY14-3rd-5-6-7(又称5-6-7或5-6-7-WT)抗体人源化前后，在Elisa水平上与人BCMA的亲和效果，结果表明，抗体人源化后与人BCMA的亲和力和人源化前保持一致。图8(B)显示检测SY14-3rd-5-6-7抗体人源化前后，在Elisa水平上与猴BCMA的亲和效果，结果表明，抗体人源化后与猴BCMA的亲和力和人源化前保持一致。
图9显示人源化后抗体(5-6-32-hu-2)与人、猴BCMA交叉亲和活性检测结果，图9(A)显示检测SY14-3rd-5-6-32(又称5-6-32或5-6-32-WT)抗体人源化前后，在Elisa水平上与人BCMA的亲和效果，结果表明，抗体人源化后与人BCMA的亲和力依旧优于阳性抗体(GSK2857916)。图9(B)检测SY14-3rd-5-6-32抗体人源化前后，在Elisa水平上与猴BCMA的亲和效果， 结果表明，抗体人源化后与猴BCMA的亲和力依旧优于阳性抗体GSK2857916。
图10显示人源化后抗体(5-6-7-hu-2)阻断效果检测结果，图10(A)显示检测SY14-3rd-5-6-7(又称5-6-7或5-6-7-WT)抗体人源化后，在Elisa水平上阻断BCMA与BAFF结合的能力，结果表明，抗体人源化后阻断BCMA结合BAFF的效果稍差于阳性抗体(GSK2857916)。图10(B)显示检测SY14-3rd-5-6-7抗体人源化后，在Elisa水平上阻断BCMA与APRIL结合的能力，结果表明，抗体人源化后阻断BCMA结合APRIL的效果稍差于阳性抗体(GSK2857916)。
图11显示人源化后抗体(5-6-32-hu-2)阻断效果检测结果，图11(A)显示检测SY14-3rd-5-6-32(又称5-6-32或5-6-32-WT)抗体人源化后，在Elisa水平上阻断BCMA与BAFF结合的能力，结果表明，抗体人源化后阻断BCMA结合BAFF的效果稍差于阳性抗体(GSK2857916)。图11(B)显示检测SY14-3rd-5-6-32抗体人源化后，在Elisa水平上阻断BCMA与APRIL结合的能力，结果表明，抗体人源化后阻断BCMA与APRIL的效果稍差于阳性抗体(GSK2857916)。
图12显示人源化前后抗体在高表达人、猴BCMA的细胞结合活性测定结果，图12(A)显示检测5-6-7和5-6-32抗体人源化前后与过表达人BCMA的HEK293细胞的结合能力，结果表明抗体人源化后与之的结合能力和人源化前保持一致并且优于GSK2857916抗体。图12(B)显示检测5-6-7和5-6-32抗体人源化前后与过表达猴BCMA的CHO细胞的结合能力，结果表明抗体人源化后与之的结合能力和人源化前保持一致并且优于GSK2857916抗体。图12(C)显示检测5-6-7和5-6-32抗体人源化前后与骨髓瘤细胞系H929细胞的结合能力，结果表明抗体人源化后与之的结合能力和人源化前保持一致并且优于GSK2857916抗体。
图13显示抗体人源化前后在H929细胞上的内吞效果测定结果，图13(A)显示检测5-6-7抗体人源化前后，在表达BCMA的人骨髓瘤细胞系H929细胞上内吞的效果，结果表明抗体人源化后内吞效果和人源化前保持一致并且优于GSK2857916抗体。图13(B)显示检测5-6-32抗体人源化前后，在表达BCMA的人骨髓瘤细胞系H929细胞上内吞的效果，结果表明抗体人源化后内吞效果和人源化前保持一致并且优于GSK2857916抗体。
图14显示人源化抗体各个功能汇总结果，图14展示了人源化抗体的人源化程度，在表达人BCMA的HEK293细胞上的亲和效果，在表达猴BCMA的CHO细胞上的亲和效果，在Elisa水平上和人、猴BCMA的亲和效果，阻断BAFF与BCMA结合效果、阻断APRIL与BCMA结合效果，以及抗体在人骨髓瘤细胞H929上的内吞效果；其中‘+’数量从多到少，代表抗体亲和、阻断及内吞效果由强至弱。结果表明，抗体人源化后各个功能和人源化前保持一致，人源化抗体部分功能接近或优于GSK2857916抗体。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明。
本说明书中提及的科技术语具有与本领域技术人员通常理解的含义相同的含义，如有冲突以本说明书中的定义为准。
一般而言，本说明书中采用的术语具有如下含义。
在本说明书中，“分离的”抗体是已经与它的天然环境的组分分离的抗体。在某些实施方案中，将抗体纯化至大于95％或99％纯度，所述纯度通过例如电泳(例如，SDS-PAGE等电聚焦(IEF)、毛细管电泳)或色谱(例如，离子交换或反相HPLC)来确定。
本文中的术语“BCMA”，也是B细胞成熟抗原，也称为CD269，是肿瘤坏死因子受体超家族成员，即TNFRSF17(Thompson等人,J.Exp.Medicine,192(1):129-135,2000)。人BCMA几乎排他性地在浆细胞和多发性骨髓瘤细胞中表达(参见例如Novak等人,Blood,103(2):689-694,2004；Neri等人,Clinical Cancer Research,73(19):5903-5909；Felix等人,Mol.Oncology,9(7):1348-58,2015)。BCMA可结合B细胞活化因子(BAFF)和增殖诱导配体(APRIL)(例如Mackay等人,2003和Kalled等人,Immunological Review,204:43-54,2005)。BCMA可以是针对多发性骨髓瘤的免疫治疗剂的合适肿瘤抗原靶标。
“抗原(Ag)”是指可以刺激动物中的抗体产生或T细胞应答的化合物、组合物或物质，包括注射或吸收到动物中的组合物(例如包括癌症特异性蛋白的组合物)。抗原与特异性体液或细胞免疫的产物(包括由异源抗原(例如所公开的抗原)诱导的产物)反应。在特定实施例中，靶抗原是BCMA多肽的表位。
“表位”或“抗原决定子”是指抗原的被结合剂结合的区。表位可以由连续氨基酸或经蛋白质的三级折叠并接的不连续氨基酸形成。由连续氨基酸形成的表位在暴露于变性溶剂时通常保留，而通过三级折叠形成的表位在用变性溶剂处理时通常消失。表位通常在独特空间构象中包括至少3个，并且更通常至少5个、约9个或约8-10个氨基酸。
抗体包括其抗原结合片段，例如骆驼Ig、Ig NAR、Fab片段、Fab'片段、F(ab)'2片段、F(ab)'3片段、Fv、单链Fv蛋白(“scFv”)、双-scFv、(scFv)2、微型抗体、双功能抗体、三功能抗体、四功能抗体、二硫键稳定的Fv蛋白(“dsFv”)和单结构域抗体(sdAb，纳米抗体)以及负责抗原结合的全长抗体的部分。所述术语还包括经遗传工程改造的形式，例如嵌合抗体(例如人类化鼠抗体)、杂结合抗体(例如双特异性抗体)和其抗原结合片段。还参看皮尔斯目录与手册(Pierce Catalog and Handbook),1994-1995(皮尔斯化学公司(PierceChemical Co.),罗克福德(Rockford),伊利诺伊州(IL))；Kuby,免疫学杂志,第3版,W.H.弗里曼公司(W.H.Freeman&Co.),纽约,1997。
如技术人员所理解并且如本文别处所描述，完全抗体包含两个重链和两个轻链。每个重链由可变区以及第一、第二和第三恒定区组成，而每个轻链由可变区和恒定区组成。哺乳动物重链分类为α、δ、ε、γ和μ。哺乳动物轻链分类为λ或κ。包含α、δ、ε、γ和μ重链的免疫球蛋白分类为免疫球蛋白(Ig)A、IgD、IgE、IgG和IgM。完全抗体形成“Y”形状。Y的茎由两个重链的第二和第三恒定区(并且对于IgE和IgM，第四恒定区)结合在一起组成，并且二硫键(链间)在铰链中形成。重链γ、α和δ具有由三个串联(成一行)Ig结构域构成的恒定区，和用于增加柔性的铰链区；重链μ和ε具有由四个免疫球蛋白结构域构成的恒定区。第二和第三恒定区分别称为“CH2结构域”和“CH3结构域”。Y的每个臂包括结合到单个轻链的可变和恒定区的单个重链的可变区和第一恒定区。轻链和重链的可变区负责抗原结合。
轻链和重链可变区含有间杂有三个高变区(也称为“互补决定区”或“CDR”)的“构架”区。CDR可以通过常规方法定义或鉴别，例如通过根据Kabat等人的序列(Wu,TT和Kabat,E.A.,实验医学杂志132(2):211-50,(1970)；Borden,P.和Kabat E.A.,PNAS,84:2440-2443(1987)；参看Kabat等人,免疫学感兴趣的蛋白质的序列(Sequences of Proteins ofImmunological Interest),美国卫生和公众服务部(U.S.Department of Health andHuman Services),1991，其在此 以引用的方式并入)，或通过根据Chothia等人的结构(Choithia,C.和Lesk,A.M.,分子生物学杂志(J Mol.Biol.),196(4):901-917(1987)；Choithia,C.等人,自然(Nature),342:877-883(1989))。
不同轻链或重链的构架区的序列在物种(例如人类)内具有相对保存性。抗体的构架区(其是成分轻链和重链的组合构架区)用以在三维空间中定位和比对CDR。CDR主要负责结合到抗原的表位。每个链的CDR通常称为CDR1、CDR2和CDR3，从N末端开始依序编号，并且通常还通过特定CDR所位于的链鉴别。因此，位于抗体的重链的可变结构域中的CDR称为CDR-H1、CDR-H2和CDR-H3，而位于抗体的轻链的可变结构域中的CDR称为CDR-L1、CDR-L2和CDR-L3。具有不同特异性(即针对不同抗原有不同组合位点)的抗体具有不同CDR。尽管抗体与抗体之间的CDR不同，但CDR内仅有限数目的氨基酸位置直接参与抗原结合。CDR内的这些位置称为特异性决定残基(SDR)。适用于构建本文所涵盖的人类化BCMA CAR的轻链CDR的说明性实例包括(但不限于)SEQ ID NO:1-3中所阐述的CDR序列。适用于构建本文所涵盖的人类化BCMA CAR的重链CDR的说明性实例包括(但不限于)SEQ ID NO:4-6中所阐述的CDR序列。
提及“V H”或“VH”是指免疫球蛋白重链的可变区，包括抗体、Fv、scFv、dsFv、Fab或如本文所公开的其它抗体片段的重链可变区。提及“V L”或“VL”是指免疫球蛋白轻链的可变区，包括抗体、Fv、scFv、dsFv、Fab或如本文所公开的其它抗体片段的轻链可变区。
“单克隆抗体”是由B淋巴细胞的单个克隆或由其中已经转染单个抗体的轻链和重链基因的细胞产生的抗体。单克隆抗体通过本领域的技术人员已知的方法产生，例如通过由骨髓瘤细胞与免疫脾细胞的融合体制备杂交抗体形成细胞。单克隆抗体包括人类化单克隆抗体。
“Fv”是含有完全抗原结合位点的最小抗体片段。在一个实施例中，双链Fv种类由一个重链可变结构域和一个轻链可变结构域呈紧密非共价缔合的二聚体组成。在单链Fv(scFv)种类中，一个重链可变结构域与一个轻链可变结构域可以通过柔性肽连接子共价连接，使得轻链和重链可以按类似于双链Fv种类的“二聚”结构缔合。在这一配置中，每个可变结构域的三个高变区(HVR)相互作用以定义VH-VL二聚体的表面上的抗原结合位点。六个HVR共同地赋予对抗体的抗原结合特异性。然而，即使单个可变结构域(或包含 仅三个对抗原具有特异性的HVR的Fv的一半)也具有识别和结合抗原的能力，但亲和力低于完整结合位点。
Fab片断含有重链可变结构域和轻链可变结构域并且还含有轻链的恒定结构域和重链的第一恒定结构域(CH1)。Fab′片段与Fab片段不同之处在于，重链CH1结构域的羧基末端增添了几个残基，包括一个或多个来自抗体铰链区的半胱氨酸。Fab′-SH是本文关于Fab′的名称，其中恒定结构域的半胱氨酸残基携有游离硫醇基。F(ab′)2抗体片段最初是作为其间具有铰链半胱氨酸的Fab′片段对产生。还已知抗体片段的其它化学偶合。
如上所述，本发明涉及一种分离的靶向B细胞成熟抗原(BCMA)的单克隆抗体，其中，所述抗体既能结合人BCMA也能结合猴BCMA。具体来说，在本发明中，利用流式细胞仪操作方法(FACS方法)验证了本发明的抗体对表达人和猴BCMA细胞的亲和效果。本发明所得到的单克隆抗体对于人BCMA-HEK293细胞的亲和效果都优于阳性对照抗体(GSK2857916)。本发明所得到的单克隆抗体对于猴BCMA-CHO细胞的亲和效果都接近或优于阳性对照抗体(GSK2857916)。本发明所得到的单克隆抗体，能够同时高亲和地结合人和猴的BCMA抗原，和猴的BCMA抗原结合，便于抗体在进入临床研究前，以猴(食蟹猴)作为模型，很好地进行毒理评估和药代动力学评估。同时，本发明的单克隆抗体具有很高的亲和力，高亲和力的抗体在药效上更具优势，诸如抗体结合到靶标抗原分子上后解离下来更慢，使得抗体在细胞内吞效果等更好，而且达到同等细胞或动物药效作用情况下，所需的抗体剂量可能也更低。
在本文所用，术语“特异性结合”、“特异性识别”或“对......具有特异性”是指可测定的和可再现的相互作用，诸如靶标和抗原结合蛋白之间的结合。例如，特异性结合靶标(可以是表位)的抗原结合蛋白是与其他靶标的结合相比，与该靶标的结合具有更大的亲和力、亲合力、更容易和/或具有更长的持续时间的抗原结合蛋白。在一些实施方案中，如例如通过放射性免疫测定法(RIA)所测定，抗原结合蛋白与不相关靶标的结合程度比抗原结合蛋白与靶标的结合小约10％。在一些实施方案中，特异性结合靶标的抗原结合蛋白具有的解离常数(Kd)≤1μM、≤100nM、≤10nM、≤1nM或≤0.1nM。
具体来说，本发明的抗体包含重链可变区，所述重链可变区包含SEQ ID NO:1(GHIFTNFHFH)或2(GYIFTNYHMH)所示的重链互补决定区 1(CDR-H1)，和/或包含SEQ ID NO:3(GIYPGNGDTF)或4(GIYPGNGDIF)所示的重链互补决定区2(CDR-H2)，和/或包含SEQ ID NO:5(GSYYGYIDAMDY)或6(GSYYGYIDAMDY)所示的重链互补决定区3(CDR-H3)。
具体来说，本发明的抗体可以包含轻链可变区，所述轻链可变区包含SEQ ID NO:7(RASQDISNYLN)或8(RASQDISNDLN)所示的轻链互补决定区1(CDR-L1)，和/或包含SEQ ID NO:9(YTSRLHS)或10(YTSRLPS)所示的轻链互补决定区2(CDR-L2)，和/或包含SEQ ID NO:11(QQGNTLPWT)或12(QQGHTLPWT)所示的轻链互补决定区3(CDR-L3)。
具体来说，本发明的抗体可以包含重链可变区和轻链可变区，其中，所述重链可变区包含SEQ ID NO:1或2所示的重链互补决定区1(CDR-H1)，和/或包含SEQ ID NO:3或4所示的重链互补决定区2(CDR-H2)，和/或包含SEQ ID NO:5或6所示的重链互补决定区3(CDR-H3)；所述轻链可变区包含SEQ ID NO:7或8所示的轻链互补决定区1(CDR-L1)，和/或包含SEQ ID NO:9或10所示的轻链互补决定区2(CDR-L2)，和/或包含SEQ ID NO:11或12所示的轻链互补决定区3(CDR-L3)。
具体来说，本发明的抗体包含轻链可变区，所述轻链可变区包含
SEQ ID NO:13：
Figure PCTCN2020117757-appb-000001
或
SEQ ID NO:14：
Figure PCTCN2020117757-appb-000002
所示的氨基酸序列、或上述序列的变体。
具体来说，本发明的抗体包含重链可变区，所述重链可变区具有SEQ ID NO:15：
Figure PCTCN2020117757-appb-000003
Figure PCTCN2020117757-appb-000004
或
SEQ ID NO:16：
Figure PCTCN2020117757-appb-000005
所示的氨基酸序列、或上述序列的变体。
具体来说，本发明的抗体包含轻链可变区和重链可变区，其中，所述轻链可变区包含SEQ ID NO:13或14所示的氨基酸序列、或上述序列的变体，所述重链可变区具有SEQ ID NO:15或16所示的氨基酸序列、或上述序列的变体。
具体来说，本发明的一种抗体的重链可变区包含SEQ ID NO:1所示的CDR-H1，SEQ ID NO:3所示的CDR-H2和SEQ ID NO:5所示的CDR-H3。本发明的另一种抗体的重链可变区包含SEQ ID NO:2所示的CDR-H1，SEQ ID NO:4所示的CDR-H2和SEQ ID NO:6所示的CDR-H3。
具体来说，本发明的一种抗体的轻链可变区包含SEQ ID NO:7所示的CDR-L1，SEQ ID NO:9所示的CDR-L2和SEQ ID NO:11所示的CDR-L3。本发明的另一种抗体的轻链可变区包含SEQ ID NO:8所示的CDR-L1，SEQ ID NO:10所示的CDR-L2和SEQ ID NO:12所示的CDR-L3。
具体来说，本发明的抗体的轻链可变区具有SEQ ID NO:13或14所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。具体来说，本发明的抗体的重链可变区具有SEQ ID NO:15或16所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
具体来说，本发明的一种抗体包含重链可变区和轻链可变区，其中，重链可变区包含SEQ ID NO:1所示的CDR-H1，SEQ ID NO:3所示的CDR-H2和SEQ ID NO:5所示的CDR-H3；轻链可变区包含SEQ ID NO:7所示的CDR-L1，SEQ ID NO:9所示的CDR-L2和SEQ ID NO:11所示的CDR-L3。具体来说，所述抗体的轻链可变区具有SEQ ID NO:13所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、 96％、97％、98％、99％相似性的序列；所述抗体的重链可变区具有SEQ ID NO:15所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
具体来说，本发明的另一种抗体包含重链可变区和轻链可变区，其中，重链可变区包含SEQ ID NO:2所示的CDR-H1，SEQ ID NO:4所示的CDR-H2和SEQ ID NO:6所示的CDR-H3；轻链可变区包含SEQ ID NO:8所示的CDR-L1，SEQ ID NO:10所示的CDR-L2和SEQ ID NO:12所示的CDR-L3。具体来说，所述抗体的轻链可变区具有SEQ ID NO:14所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列；所述抗体的重链可变区具有SEQ ID NO:16所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
如本文所用，术语“变体”是指已经修饰至少一个，例如1、2或3个氨基酸取代、缺失或添加的重链可变区或轻链可变区，其中包含重链或轻链变体的经修饰的抗原结合蛋白基本上保留修饰前抗原结合蛋白的生物学特征。在一个实施方案中，含有变体重链可变区或轻链可变区序列的抗原结合蛋白保留修饰前抗原结合蛋白的60％、70％、80％、90％、100％生物学特征。应当理解，可以单独或在与另一个重链可变区或轻链可变区组合修饰每个重链可变区或轻链可变区。本公开的抗原结合蛋白包含与本文描述的重链可变区氨基酸序列90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源的重链可变区氨基酸序列。本公开的抗原结合蛋白包括与本文描述的轻链可变区氨基酸序列90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源的轻链可变区氨基酸序列。同源性百分比可以在整个重链可变区和/或整个轻链可变区上，或者百分比同源性可以限于框架区，而对应于CDR的序列与重链可变区和/或轻链可变区内本文中公开的CDR具有100％同一性。如本文所用，术语“CDR变体”是指已经修饰至少一个，例如1、2或3个氨基酸取代、缺失或添加的CDR，其中包含CDR变体的经修饰的抗原结合蛋白基本上保留修饰前抗原结合蛋白的生物学特征。在一个实施方案中，含有变体CDR的抗原结合蛋白保留修饰前抗原结合蛋白的60％、70％、80％、90％、100％生物学特征。应当理解，可以修饰的每个CDR可以单独或与另 一个CDR组合修饰。在一个实施方案中，修饰是取代，特别是保守取代。
如上所述，本发明的抗体具有优异的内吞效果，均优于阳性对照抗体(GSK2857916)。内吞作用(endocytosis)又称入胞作用或胞吞作用，是通过质膜的变形运动将细胞外物质转运入细胞内的过程。根据入胞物质的不同大小，以及入胞机制的不同可将内吞作用分为三种类型：吞噬作用、吞饮作用、受体介导的内吞作用。本发明所述抗体的内吞作用，指的是受体介导的内吞作用，通过靶向BCMA的单克隆抗体和BCMA结合后，介导BCMA-抗体复合物形成内吞小体后和溶酶体融合，在溶酶体内BCMA-抗体复合物被溶酶体介导降解，部分BCMA或BCMA-抗体复合物也可转运回细胞膜上。当本发明所述的单克隆抗体偶联毒素小分子形成ADC药物后，通过内吞转运至胞内，释放的毒素分子能够杀死靶细胞(如多发性骨髓瘤细胞)。因此，当本发明所述单克隆抗体开发为ADC药物，所述更好的内吞效果对于帮助介导药物进入靶细胞非常关键。
此外，本发明的抗体是经人源化改造的抗体，经本发明人源化改造前后的抗体与人和猴BCMA结合活性基本一致，且人源化改造前后的抗体阻断BCMA和BAFF结合的效果基本一致。本发明人源化改造前后抗体和猴BCMA-CHO细胞结合效果基本一致，且均优于阳性对照抗体(GSK2857916)，且人源化改造前后抗体和H929细胞结合效果基本一致，且均优于阳性对照抗体(GSK2857916)。且人源化前后抗体抗体在人骨髓瘤细胞系H929细胞上的内吞效果基本一致，且均优于阳性对照抗体(GSK2857916)。
“人源化抗体”是指一类工程化抗体，其具有源自非人供体免疫球蛋白的其CDR，该分子的剩余免疫球蛋白衍生部分源自一种(或多种)人免疫球蛋白。此外，框架支持残基可以改变以保留结合亲和力(参见例如Queen等,Proc.Natl Acad Sci USA,86:10029-10032(1989),Hodgson等,Bio/Technology,9:421(1991))。合适的人受体抗体可以是通过与供体抗体的核苷酸和氨基酸序列的同源性从常规数据库，例如数据库、Los Alamos数据库和Swiss蛋白质数据库选择的抗体。以与供体抗体的框架区的同源性(基于氨基酸)表征的人抗体可以适合于提供用于插入供体CDR的重链恒定区和/或重链可变框架区。可以以类似的方式选择能够提供轻链恒定或可变框架区的合适受体抗体。应当注意的是，受体抗体重链和轻链不需要来源于相同的受体抗体。
本发明经人源化改造后的抗体的热稳定性略优于阳性对照抗体(GSK2857916)，且达到抗体成药的热稳定性条件，热稳定性差可能导致抗体成药性问题，如抗体低表达和抗体聚集。
另外，本发明还涉及一种靶向B细胞成熟抗原(BCMA)的单克隆抗体，该抗体与上述本发明所述的抗体识别相同的抗原决定部位。本发明还涉及一种靶向B细胞成熟抗原(BCMA)的单克隆抗体，该抗体与上述本发明所述的抗体竞争性结合B细胞成熟抗原(BCMA)。
具体来说，本发明还涉及编码上述本发明的抗体的核酸。本发明还涉及一种表达载体，其包含上述本发明所述的核酸。本发明还涉及一种宿主细胞，其包含上述本发明所述的表达载体或基因组中整合有本发明所述的核酸。
如本领域已知，在本文中可交换使用的“多核苷酸”或“核酸”是指任何长度的核苷酸链，并且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、修饰的核苷酸或碱基、和/或它们的类似物、或者能够通过DNA或RNA聚合酶掺入链的任何底物。如本文所用，“载体(vector)”表示构建体，其能够将一种或多种所关注的基因或序列递送入宿主细胞并且优选在宿主细胞中表达所述基因或序列。载体的实例包括但不限于病毒载体、裸DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子凝聚剂相关的DNA或RNA表达载体、包囊化于脂质体中的DNA或RNA表达载体以及某些真核细胞，例如生产细胞。在本发明中术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，并且是指已经引入外源性核酸的细胞，包括这些细胞的子代。宿主细胞包括“转化子”和“转化的细胞”，其包括原代转化细胞以及由此来源的子代，而不考虑传代次数。子代在核酸含量上与亲代细胞可能不完全相同，但可能含有突变。本文包括与在初始转化的细胞中筛选或选择的细胞具有相同功能或生物学活性的突变子代。
本发明还涉及一种生产单克隆抗体的方法，所述方法包括培养本发明所述的宿主细胞从而生产上述本发明所述的单克隆抗体。
本发明还涉及一种药物组合物，其包含本发明所述的单克隆抗体和药学上可接受的载体。本发明还涉及一种药盒或制品，其包括本发明所述的单克隆抗体或本发明所述的药物组合物。如本文所用，“药学可接受的载体”或“药学可接受的赋形剂"包括任何这样的材料，当与活性成分组合时，其允许所述成分保持生物活性，并且不与对象的免疫系统反应。实例包括但不限于任 何标准药学载体，例如磷酸缓冲盐溶液、水、乳剂(如油/水乳剂)以及各种类型的湿润剂。用于气溶胶或肠胃外施用的优选稀释剂为磷酸缓冲盐水(PBS)或生理盐水(0.9％)。通过公知的常规方法配制包含这类载体的组合物(参见，例如，Remington'sPharmaceutical Sciences,18th edition,A.Gennaro,ed.,Mack Publishing Co.,Easton,PA,1990；和Remington,The Science and Practice of Pharmacy 21st Ed.MackPublishing,2005)。
本发明还涉及一种治疗与BCMA的表达相关的疾病的方法，其包括：向有此需要的受试者给药本发明所述的单克隆抗体或本发明所述的药物组合物或本发明所述的药盒或制品。具体来说，上述疾病选自B细胞急性淋巴性白血病、T细胞急性淋巴性白血病、急性淋巴性白血病、慢性髓性白血病、慢性淋巴细胞白血病、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、毛细胞白血病，小细胞或大细胞滤泡性淋巴瘤、恶性淋巴瘤、恶性淋巴增殖状况、MALT淋巴瘤、外套细胞淋巴瘤、边缘区淋巴瘤、多发性骨髓瘤、脊髓发育不良和骨髓增生异常综合征、非霍奇金淋巴瘤、浆母细胞淋巴瘤、浆细胞样树突细胞瘤、瓦尔登斯特伦巨球蛋白血症、骨髓瘤、MGUS、浆细胞瘤、系统性淀粉样蛋白轻链淀粉样变性和POEMS综合征。具体来说，上述疾病为多发性骨髓瘤。
具体来说，术语“多发性骨髓瘤(MM)”，也称为浆细胞骨髓瘤或Kahler氏病(Otto Kahler之后)，是一种以与基质细胞密切接触的恶性浆B细胞在骨髓内的积累为特征的难治性克隆B细胞肿瘤。MM是一种渐进性疾病，特别是例如由对前体浆B细胞的多种遗传损伤引起的，即主要由易位，例如t(11；14)、t(4；14)、t(8；14)导致的染色体易位，或删除，例如del(13)和del(17)，使得肿瘤细胞大幅增殖并成为凋亡耐受。B淋巴细胞起始于骨髓中并移动到淋巴结。由于它们的发育，B淋巴细胞在其细胞表面上成熟并展示不同的蛋白。当它们被活化而分泌抗体时，将它们称作浆细胞。在它们离开称为生发中心的部分淋巴结后，多发性骨髓瘤在B细胞中发展。免疫系统在严格控制下保持B细胞的增殖和抗体的分泌。当染色体和基受损时(通常通过重排)，失去了这种控制。通常情况下，启动子基因移动(或易位)到染色体，它刺激抗体基因过量产生。
如上所述，在多发性骨髓瘤患者中经常观察到免疫球蛋白重链基因(在 染色体14，基因座14q32上)和癌基因(经常11q13、4p16.3、6p21、16q23和20q11[10])之间的染色体易位。该突变导致所述癌基因的失调，这被认为是骨髓瘤发病机制中的一个重要的起始事件。该结果是浆细胞克隆的增殖和基因组不稳定性，导致进一步的突变和易位。在约50％的所有骨髓瘤病例中观察到了14号染色体异常。在约50％的病例中也观察到了13号染色体(一部分)的缺失。
在本文中，“受试者”、“个体”或“对象”是哺乳动物，更优选是人。哺乳动物还包括但不限于农场动物、竞赛动物、宠物、灵长类、马、犬、猫、小鼠和大鼠。在本发明中，向有此需要的受试者给药本发明所述的单克隆抗体或本发明所述的药物组合物或本发明所述的药盒或制品是指给予有效量的药物组合物或药剂或制品等，如本发明所用，术语“有效量”表示引发例如研究者或临床医师所追求的组织、系统、动物或人的生物学或药学响应的药物或药剂的量。此外，术语“治疗有效量”表示，与没有接受该量的相应受试者相比，引起疾病、病症或副作用的改进治疗、治愈、预防或减轻的量，或者使疾病或病况的进展速率降低的量。该术语在其范围内还包括有效增强正常生理功能的量。
本发明还涉及本发明的单克隆抗体在制备用于治疗与BCMA的表达相关的疾病的药物中的用途。具体来说，上述疾病选自B细胞急性淋巴性白血病、T细胞急性淋巴性白血病、急性淋巴性白血病、慢性髓性白血病、慢性淋巴细胞白血病、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、毛细胞白血病，小细胞或大细胞滤泡性淋巴瘤、恶性淋巴瘤、恶性淋巴增殖状况、MALT淋巴瘤、外套细胞淋巴瘤、边缘区淋巴瘤、多发性骨髓瘤、脊髓发育不良和骨髓增生异常综合征、非霍奇金淋巴瘤、浆母细胞淋巴瘤、浆细胞样树突细胞瘤、瓦尔登斯特伦巨球蛋白血症、骨髓瘤、MGUS、浆细胞瘤、系统性淀粉样蛋白轻链淀粉样变性和POEMS综合征。具体来说，上述疾病为多发性骨髓瘤。如本文所用，术语“癌症”、“新生物”和“肿瘤”可互换使用，并且为单一或复数形式，是指已经经过恶性转化或导致异常或不受调节的生长或过度增殖的细胞变化的细胞。此种变化或恶性转化通常会使此类细胞对宿主生物体有致病性，因此也意图包括变为或可以变成致病性且需要干预或可以受益于干预的初癌或癌前细胞。原发性癌细胞(即，从恶性转化部位附近 取得的细胞)可以通过完善建立的技术，特别是组织学检查而与非癌细胞区分。如本发明所用，癌细胞的定义不仅包括原发性癌细胞，也包括衍生自癌细胞祖先(ancestor)的任何细胞。这包括转移的癌细胞、和体外培养物和衍生自癌细胞的细胞系。当提及通常表现为实体瘤的癌症类型时，“临床上可检测”的肿瘤为在肿瘤块的基础上可检测的那些；例如通过如CAT扫描、MR成像、X-射线、超声或触诊的操作，和/或由于从患者可获得的样品中一种或多种癌症特异性抗原的表达而可检测的肿瘤。换言之，本发明的术语包括细胞、新生物、癌症和任何阶段的肿瘤，包括临床医生称为初癌、肿瘤、原位生长、以及晚期转移性生长的。肿瘤可以为造血肿瘤，例如血细胞肿瘤等，即为液体肿瘤。基于此类肿瘤的临床病况的具体实例包括白血病如慢性髓细胞性白血病或急性髓细胞性白血病；骨髓瘤如多发性骨髓瘤；淋巴瘤等。
如上所述，相比于Belantamab mafodotin抗体(简称为GSK2857916)，本发明涉及的抗体既能结合人BCMA也能结合猴BCMA，在亲和水平方面，与之接近甚至更好。本发明涉及的抗体在阻断BCMA结合其配体BAFF或APRIL效果方面，与GSK2857916接近甚至更好，阻断BCMA和BAFF及APRIL结合，能够抑制受配体的结合导致的胞内信号通路活化(如NFκB信号通路)，从而抑制浆细胞的生存。本发明涉及的抗体在内吞效果方面，具有比GSK2857916更好的内吞效果，内吞效果是抗体药物偶联物(ADC)介导细胞杀伤的关键功能，具有更好的内吞效果的抗体对应ADC药物杀伤效果更优优势。本发明涉及的抗体在热稳定性方面，具有比GSK2857916更好的热稳定性，热稳定是抗体成药性的关键指标，具有更好的热稳定抗体，在抗体表达上可能更具优势，且产生聚体的可能性降低。本发明涉及的抗体，在免疫原性方面，为人源化后的抗体，具有更低的免疫原性风险，免疫原性会激起人体内的免疫反应，一方面产生抗抗体，降低抗体药的效果，另一方面强的免疫原性可能会带来药物更大的副作用风险。而人源化的抗体，将抗体CDR区以外的序列进行突变改造，使之更接近于人的抗体序列，从而降低抗体产生强免疫原性的风险。
实施例
实施例1
原材料的制备
在本实施例中，BCMA抗原及其配体均购自ACRO Biosystems，同时，根据美国专利申请(US20140105915A)制备了葛兰素史克(GSK)的阳性抗体GSK2857916，值得注意的是，制备的该阳性抗体并没有偶连毒素分子但并不影响其他可能的功能。
1.1抗原的准备
本申请中抗原共用到如下4种：human-BCMA-Fc、human-BCMA-His、cyno-BCMA-Fc和cyno-BCMA-His，均购买于ACRO Biosystems，货号分别为BC7-H5254、BCA-H522y、BCA-C5253、BCA-C52H7。另外，其活性已与GSK2857916进行相互验证，并与美国专利申请US20140105915A上披露的相符，结果显示在图2(A)中。
1.2配体的准备
本申请中使用的配体共用到如下2种，human-BAFF-Fc和human-APRIL-Fc，均购买于ACRO Biosystems，货号分别为BAF-H4268、APL-H5267。另外，其活性已与human-BCMA进行受配体结合验证，并与美国专利申请US20140105915A上披露的相符，结果显示在图2(B)和2(C)中。
1.3阳性对照抗体的制备
本申请中，阳性对照抗体GSK2857916采用瞬转系统(ExpiCHO)进行表达，其中，用到的主要材料包括：Gibco培养基(货号：A29100-01)，Gibco转染试剂盒(货号：A29129)。首先，根据美国专利申请US20140105915A披露的序列合成GSK2857916的轻链序列(如序列号SEQ ID NO:17的序列所示)和重链序列(如序列号SEQ ID NO.:18的序列所示)，通过分子克隆方法构建出包含完整GSK2857916抗体轻链和重链基因的质粒。将含有GSK2857916抗体轻链的质粒和重链的质粒按照2:1的质量比进行混合，在25mL表达体系内，按照标准流程将上述质粒混合物(25μg)与转染试剂进行混合并滴加入25mL的ExpiCHO细胞表达体系中。充分混匀后，于37℃细胞培养箱内表达18-22小时。随后，向上述转染混合物中添加补料培养基并置于32℃细胞培养箱内继续培养。转染后第5天，添加第二次补料，并将细胞置于32℃细胞培养箱内继续培养10-12天。接着，将表达好的细胞混悬液进行高速离心并取上清，所得上清经0.22μm滤膜过滤后，采用Protein A/G亲和层 析柱亲和法进行纯化。纯化后，用100mM甘氨酸盐(pH3.0)洗脱目的蛋白，浓缩，置换，分装，经SDS-PAGE鉴定和活性鉴定合格后入库冻存。
实施例2
细胞株的制备
在本实施例中，购买了表达BCMA的人骨髓瘤细胞系H929细胞，构建了过表达人BCMA的细胞株BCMA-HEK293细胞和过表达猴BCMA的细胞株BCMA-CHO细胞。
2.1 H929细胞株的准备
本申请中，H929购于北京协和细胞库，货号为3111C0001CCC000360，为天然高表达人BCMA的骨髓瘤细胞株。
2.2 BCMA-CHO细胞株的制备
2.2.1表达猴BCMA全长的质粒的构建
通过基因合成技术合成含有食蟹猴的BCMA蛋白的DNA片段，并将其克隆至表达载体。通过化转的方法导入大肠杆菌，挑取大肠杆菌单克隆后测序得到正确的质粒克隆，进行质粒抽提并再次测序确认。
2.2.2电转
使用Gibco的CD-CHO无血清培养基(货号：10743029)培养CHO-s细胞。电转前一天，将细胞传代至5×10 6/mL，次日使用Invitrogen的电转试剂盒(货号：MPK10096)和电转仪(货号：MP922947)将构建好的质粒导入CHO-s细胞中。将电转后的细胞移至CD-CHO培养基中，放置于37℃细胞培养箱中培养48h。
2.2.3电转后细胞铺板
将电转后的CHO-s细胞按2000个细胞/孔铺到96孔板中，加入终浓度30μM MSX(Millipore，GSS-1015-F)和GS supplement(Sigma，58672C-100ml)，放置于37℃二氧化碳培养箱中培养，10天后补充加入含30μM MSX和1×GS supplement的培养基。
2.2.4克隆挑选、细胞扩培和FACS鉴定
挑取96孔板中长出的单细胞克隆，转移至24孔培养板中继续扩大培养，之后通过FACS鉴定猴BCMA稳转成功的细胞株。
2.3 BCMA-HEK293细胞株的制备
2.3.1表达人BCMA全长的质粒的构建
通过基因合成技术合成含有人的BCMA蛋白的DNA片段，并将其克隆至表达载体。通过化转的方法导入大肠杆菌，挑取大肠杆菌单克隆后测序得到正确的质粒克隆，进行质粒抽提并再次测序确认。
2.3.2电转
使用Gibco的DMEM无血清培养基(货号：12634010)培养HEK293细胞。电转前一天，将细胞传代至2×10 5/mL，次日使用Invitrogen的电转试剂盒(货号：MPK10096)和电转仪(货号：MP922947)将构建好的质粒导入HEK293细胞中。将电转后的细胞移至DMEM培养基中，放置于37℃细胞培养箱中培养48h。
2.3.3电转后细胞铺板
将电转后的HEK293细胞按1000个细胞/孔铺到96孔板中，加入终浓度2μg/mL的puromycin，放置于37℃二氧化碳培养箱中培养，14天后补充加入2μg/mL的puromycin的培养基。
2.3.4克隆挑选、细胞扩培和FACS鉴定
挑取96孔板中长出的单细胞克隆，转移至24孔培养板中继续扩大培养，之后通过FACS鉴定人BCMA稳转成功的细胞株。
实施例3
动物免疫
在本实施例中，动物免疫包括数种方案且同时进行，在免疫过程中，取小鼠血清进行效价检测并评估免疫的效果，最终根据血清效价决定建库的小鼠编号。
3.1动物免疫
3.1.1.免疫方案一
通过皮下注射和腹腔注射的方式免疫Babl/c小鼠(上海灵畅生物科技有限公司，雌性，6-8周，n＝2)，材料包括上述提到的人BCMA-Fc和猴BCMA-Fc抗原蛋白(ACRO Biosystems)，采用交叉免疫的方式。首次免疫剂量为100μg/只，并辅以CFA(完全弗氏佐剂)，之后免疫剂量降至50μg/只，并辅以IFA(弗式不完全佐剂)，均为两周一交叉免疫。对应小鼠编号为Mouse 1和Mouse 2。
3.1.2免疫方案二
通过皮下注射和腹腔注射的方式免疫Babl/c小鼠(小鼠与方案一为同一批次，n＝2)，材料包括上述提到的人BCMA-Fc、猴BCMA-Fc抗原蛋白(ACRO Biosystems)、高表达人BCMA的HEK293细胞和高表达猴BCMA的CHO细胞，采用交叉免疫的方式。蛋白免疫剂量为20μg/只，细胞免疫剂量为1×10 7/只，腹腔注射，为一周一交叉免疫。对应小鼠编号为Mouse 3和Mouse 4。
3.2血清效价ELISA检测
3.2.1抗原包被和封闭
在ELISA板上，提前一天分别包被人BCMA和猴BCMA，包被浓度为2μg/mL，30μL/孔，4℃过夜。在免疫效价测定当日，用PBST洗板三次，并用5％的脱脂牛奶室温封闭两小时，再用PBST洗板三次。
3.2.2一抗和二抗
各个血清样本在原液的基础上先稀释500倍，再按3倍梯度进行稀释后，作为一抗加到ELISA板中，室温下孵育1h，PBST洗板三次后，加入二抗(Goat-anti-mouse-IgG-Fab-HRP，Sigma，M4115),室温下孵育1h。
3.2.3显色、终止和读板
孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色，根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板。血清效价结果表明，免疫后的小鼠对人BCMA和猴BCMA的效价均很高，可用于下一步的抗体库构建，其结果显示在下表1-1和1-2中。
表1-1用人BCMA检测小鼠血清的效价
Figure PCTCN2020117757-appb-000006
表1-2用猴BCMA检测小鼠血清的效价
Figure PCTCN2020117757-appb-000007
实施例4
在本实施例中，优选了实施例3中效价最优的小鼠，采用噬菌体展示技术，将小鼠脾脏中B细胞的抗体基因克隆至噬菌体展示载体，构建成抗体文库。以人BCMA和猴BCMA为筛选抗原，通过文库海选、单克隆初筛和测序等过程，最终获得17个针对人和猴的BCMA抗原具有结合活性的抗体分子。
4.1噬菌体展示抗体基因文库构建
在免疫结束后，按安乐死标准流程处理小鼠。取小鼠脾脏，经研磨和过滤后，收集脾细胞，加入1mL的TRIzol TM Reagent(Thermo Fisher，15596026)裂解脾细胞，通过酚氯仿法提取总RNA，通过反转录试剂盒(TaKaRa,6210A)将提取的RNA反转录成cDNA。之后，以cDNA为PCR模板，采用鼠源抗体序列扩增的特异性引物，分别扩增抗体的轻链和重链基因。最后，通过NcoI+NotI双酶切和T4连接酶连接，将抗体基因片段插入至噬菌体展示用载体上，连接产物通过DNA回收试剂盒(Omega,D6492-02)回收，最后通过电转仪(Bio-Rad，MicroPulser)转化至感受态大肠杆菌SS320中(Lucigen,MC1061 F)，并涂布于含有氨苄青霉素和四环素的2-YT(C+/K+2-YT)固体平板，扩增正确转化的抗体质粒的SS320菌，最终构建成含Fab段抗体序列的文库。
4.2噬菌体展示抗体基因文库筛选
4.2.1磁珠法筛选噬菌体展示抗体基因文库
磁珠法筛选是基于将Biotin标记的抗原蛋白和偶联有Avidin的磁珠结合，通过将结合抗原的磁珠和文库进行孵育、洗涤和洗脱的海选过程，经历2-4轮的海选，最终将针对抗原的特异性单克隆抗体富集下来的过程。本实施例中利用Biotin标记的人BCMA抗原和猴BCMA抗原交叉海选的原则， 其中第一和第三轮采用人BCMA海选，第二轮采用猴BCMA海选，一共海选3轮，然后将富集的抗体序列混合物，进行人和猴BCMA的单克隆初筛。
具体实施方法如下：
首先用Biotin标记的人BCMA蛋白与Avidin偶联的磁珠孵育，使得人BCMA蛋白结合到磁珠上。将结合有BCMA抗原的磁珠和构建的噬菌体库室温下孵育2h。经PBST洗涤6-8次后，去除非特异性吸附的噬菌体，加入Trypsin(Gibco，25200072)轻轻混匀并反应20min，以洗脱特异性结合的抗体展示噬菌体。随后，用洗脱下来的噬菌体侵染对数期的SS320菌体(Lucigen,MC1061 F)并静置30min，然后在220rpm条件下培养1h，再通过加入VSCM13辅助噬菌体并静置30min，继续在220rpm条件下培养1h，离心并置换至C+/K+2-YT培养基中，最终得到的噬菌体继续用于下一轮的海选。
4.4.2免疫管法筛选噬菌体展示抗体基因文库
免疫管筛选的原理是将BCMA蛋白包被在具有高吸附力的免疫管表面，通过将噬菌体展示抗体文库加入免疫管中并和吸附于免疫管表面的抗原蛋白进行孵育、洗涤和洗脱的海选过程，经历2-4轮海选，最终将针对抗原的特异性单克隆抗体富集下来的过程。免疫管法和磁珠法的目的均为富集针对抗原的特异性抗体，为两个互补的实验方法。本实施例中以人BCMA抗原和猴BCMA抗原交叉海选的原则，其中第一和第三轮采用人BCMA海选，第二轮采用猴BCMA海选，一共海选3轮，然后将富集的抗体序列混合物，进行人和猴BCMA的单克隆初筛。具体实施方法与磁珠法筛选类同。
4.5单克隆的挑选
在三轮筛选后，从第三轮的Pool中挑选部分单克隆进行ELISA检测，包括与人BCMA和猴BCMA的结合。最终，在1344个克隆中共挑到93个能够与人和猴BCMA都结合的阳性克隆，经测序分析及与人和猴BCMA亲和力排序结果分析后，最终选取了17个克隆的序列构建全长以进行下一步的实验。
实施例5
全长抗体构建、表达与纯化
在本实施例中，将实施例4中获得的17个人猴交叉的Fab抗体构建为人IgG1型，其中轻链均为Kappa，抗体类型为人鼠嵌合抗体。
5.1质粒的构建
将筛选获得的序列的重链序列与人IgG1Fc段融合构建，轻链与人的Kappa恒定区融合构建，将重链和轻链的质粒转化ExpiCHO细胞，诱导表达并得到全长抗体。
5.2抗体的表达纯化
本申请中，抗体采用的是ExpiCHO瞬转表达系统，培养基为(Gibco,A29100-01)，转染试剂盒为(Gibco，A29129)。具体方法如下：转染前一天将ExpiCHO细胞进行传代，在25mL体系内，将构建好的质粒25μg与转染试剂混合之后滴加入25mL ExpiCHO细胞中，充分混匀后，于37℃细胞培养箱内表达18-22h。随后，向上述转染混合物中添加补料培养基并置于32℃细胞培养箱内继续培养。转染后第5天，添加第二次补料，并将细胞置于32℃细胞培养箱内继续培养10-12天。接着，将表达好的细胞混悬液进行高速离心并取上清，所得上清经0.22μm滤膜过滤后，采用Protein A/G亲和层析柱亲和法进行纯化。纯化后，用100mM甘氨酸盐(pH3.0)洗脱目的蛋白，浓缩，置换，分装，经SDS-PAGE鉴定、SEC纯度检测、活性鉴定后入库冻存。
实施例6
候选抗体ELISA水平亲和阻断效果检测
在本实施例中，基于ELISA的方法验证了候选抗体对人和猴BCMA的亲和效果，基于ELISA的方法验证了候选抗体阻断BCMA和BAFF或APRIL结合的效果。
6.1基于ELISA检测候选抗体对人和猴BCMA的亲和效果
在96孔ELISA板上，分别包被人BCMA和猴BCMA，2μg/mL，30μL/孔，4度过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h，用PBST洗板3次后，加入梯度稀释的候选抗体及阳性对照抗体(GSK2857916)并孵育1h。之后，用PBST清洗3次后加入二抗(anti-human-IgG-Kappa-HRP，abcam，ab79115)并孵育1h。孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色。根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板，结果显示在图4(A)至图4(B)中，结果表明，候选抗体拥有接近甚至优于GSK2857916抗体的亲和人BCMA和猴BCMA的效果。
6.2基于ELISA检测候选抗体阻断BCMA和BAFF的结合
在本实施例中，阻断体系包含前期开发的过程，根据开发结果，确定实际使用的兼具灵敏度及稳定性的阻断体系参数。
对于BAFF的阻断，包板人BAFF蛋白，1μg/mL，30μL/孔，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h。然后分别将候选抗体或阳性对照抗体(GSK2857916)梯度稀释，并与Biotin标记的人BCMA(0.6μg/mL)提前预混0.5h，在封闭完成并洗板结束后加至96孔ELISA板中，孵育1h。之后，用PBST清洗3次后加入二抗(NeutrAvidin-HRP，Therofisher，31001)并孵育1h。孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色，根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板，结果显示在图5(A)中，结果表明，候选抗体拥有接近甚至优于GSK2857916抗体的阻断人BCMA和BAFF结合的效果。
6.3基于ELISA检测候选抗体阻断BCMA和APRIL的结合
在本实施例中，阻断体系包含前期开发的过程，根据开发结果，确定实际使用的兼具灵敏度及稳定性的阻断体系参数。
对于APRIL的阻断，包板人APRIL蛋白，2μg/mL，30μL/孔，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭1h。然后分别将候选抗体或阳性对照抗体(GSK2857916)梯度稀释，并与Biotin标记的人BCMA(0.6μg/mL)提前预混半小时，在封闭完成并洗板结束后加至96孔平底板中，孵育1h。之后，用PBST清洗3次后加入二抗NeutrAvidin-HRP(Therofisher，31001)并孵育1h。孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色，根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)在OD450下读板，结果显示在图5(B)中，结果表明，候选抗体拥有接近甚至优于GSK2857916抗体的阻断人BCMA和APRIL的结合的效果。
实施例7
候选抗体FACS水平与人和猴BCMA表达细胞株亲和效果检测
在本实施例中，基于FACS方法验证候选抗体对表达人和猴BCMA细胞的亲和效果。
7.1基于FACS检测候选抗体对人BCMA-HEK293细胞的亲和效果
收集指数生长期的人BCMA-HEK293细胞，300g离心去上清，将细胞用配制好的FACS缓冲液重悬，计数并将细胞悬液密度调整为2×10 6/mL。随后，将BCMA-HEK293细胞以每孔100μL加入96孔圆底板中，300g离心去上清。向对应孔中加入梯度稀释的候选抗体和阳性对照抗体稀释液，用排枪将细胞吹匀并放置于4℃下孵育30min。将孵育后的细胞混合液300g离心去上清，向对应孔中加入200μL的FACS缓冲液并使用排枪重悬细胞；重复两次，300g离心去上清；加入PE标记的anti-human-IgG-Fc流式抗体(Abcam,98596)，用排枪将细胞吹匀并放置于4℃下孵育30min，300g离心去上清。随后，加入FACS缓冲液并重悬细胞，重复两次后向孔中加入FACS缓冲液，每孔200μL，重悬细胞。最后，通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)上机检测。本实施例中，结果显示在图3(A)中，结果表明，抗体SY14-3rd-5-6-7对于人BCMA-HEK293细胞的亲和效果优于阳性对照抗体(GSK2857916)。
7.2基于FACS检测候选抗体对猴BCMA-CHO细胞的亲和效果
本实施例中，操作流程与7.1完全一致，与之不同的是，细胞为猴BCMA-CHO细胞，其结果显示在图3(B)中，结果表明，抗体SY14-3rd-5-6-7对于猴BCMA-CHO细胞的亲和效果优于阳性对照抗体(GSK2857916)。另外，人、猴间的信号差异猜测可能是BCMA-HEK293或BCMA-CHO上BCMA表达量差异造成的。
实施例8
候选抗体内吞效应检测
在本实施例中，开发了检测抗体在人骨髓瘤细胞系H929细胞上内吞的方法体系并对候选抗体内吞效应进行了相应检测，基本原理为通过细胞毒性来检测抗体的内吞效应。Fab-ZAP(Atsbio,IT-51-100)是一种连接了saporin(皂素)的Fab片段，saporin是一种核糖体抑制剂，能够抑制蛋白质的合成而使细胞死亡。本实施例中，Fab-ZAP是一种能够和人源抗体Fc结合的Fab片段，Fab-ZAP和抗BCMA的抗体共孵育后使抗体带上毒素，当抗BCMA的抗体被H929细胞内吞后，毒素随着抗体进入到细胞内并使细胞死亡，因此，可以通过MTS试剂盒(Promega，G3580)检测细胞的活性来检测抗体的内吞 效应。
具体检测方法如下：H929细胞提前一周复苏，复苏后每3天传代一次，每次细胞接种密度为2×10 5/mL，细胞传代不超过3周。吸取处于对数生长期的细胞,充分混匀细胞后，计数并测定其活率。取1个96孔平底板，将细胞密度调整为4×10 5/mL，每孔中加入50μL细胞，轻轻拍打混匀，将细胞培养板放入37℃细胞培养箱中孵育16小时。然后用1640+10％FBS+1％PS+50μM β-巯基乙醇完全培养基将Fab-ZAP先配制成27nM(2.16μg/ml)的稀释液，加至梯度稀释的抗体内使得孵育的Fab-ZAP终浓度为13.5nM(1.08μg/ml)。按设计好的布板，用300μL排枪从稀释板中取50μL稀释液加入到细胞板中，轻轻拍打混匀，放入细胞培养箱培养3天。之后，提前将MTS置于室温融化，将细胞板从培养箱中取出，用100μL的12道移液枪取20μL的MTS加入到各孔中，轻轻拍打后置于培养箱孵育2h，最后，将细胞板放入酶标仪中，在492nm波长下，读数并保存。
本实施例中，结果显示在图6(A)和6(B)中，结果表明，候选抗体SY14-3rd-5-6-7和SY14-3rd-5-6-32的内吞效果均优于阳性对照抗体(GSK2857916)。
至此，候选抗体所有的检测结果汇总在图7中。
实施例9
抗体人源化改造
在本实施例中，对鼠源抗体重链和轻链V区的框架进行了氨基酸的点突变，使之更接近人的Germline。其中，改造的候选抗体包括SY14-3rd-5-6-7(又称5-6-7或5-6-7-WT)和SY14-3rd-5-6-32(又称5-6-32或5-6-32-WT)，其中改造后的优选候选抗体分别称为5-6-7-hu-2和5-6-32-hu-2。
实施例10
人源化抗体功能验证
10.1基于ELISA检测两个候选抗体在人源化改造前后对人和猴BCMA的亲和效果
具体操作方法参见实施例6，结果显示在图8(A)、图8(B)、图9(A)和图9(B)中，结果表明，人源化改造前后的抗体与人和猴BCMA结合活性基本一 致。
10.2基于ELISA检测两个候选抗体在人源化改造前后阻断BCMA和BAFF的效果
具体操作参见实施例6，结果显示在图10(A)和11(A)中，结果表明，人源化改造后的抗体阻断BCMA和BAFF结合的效果稍差于阳性抗体。
10.3基于ELISA检测两个候选抗体在人源化改造前后阻断BCMA和APRIL的效果
具体操作参见实施例6，结果显示在10(B)和11(B)中，结果表明，人源化改造后的抗体阻断BCMA和APRIL结合的效果稍差于阳性抗体。
10.4基于FACS检测候选抗体对人BCMA-HEK293细胞的亲和效果
具体操作参见实施例7，结果显示在图12(A)中，结果表明，人源化改造前后抗体和人BCMA-HEK293细胞结合效果基本一致，且均优于阳性对照抗体(GSK2857916)。
10.5基于FACS检测候选抗体对猴BCMA-CHO细胞的亲和效果
具体操作参见实施例7，结果显示在图12(B)中，结果表明，人源化改造前后抗体和猴BCMA-CHO细胞结合效果基本一致，且均优于阳性对照抗体(GSK2857916)。
10.6基于FACS检测候选抗体对人骨髓瘤细胞系H929细胞的亲和效果
具体操作参见实施例7，结果显示在图12(C)中，结果表明，人源化改造前后抗体和H929细胞结合效果基本一致，且均优于阳性对照抗体(GSK2857916)。
10.7抗体在人源化改造前后内吞效应检测
在本实施例中，还检测了两个候选抗体人源化改造前后在人骨髓瘤细胞系H929细胞上的内吞效应。具体操作参见实施例8，结果显示在图13-A和13-B中，结果表明，人源化抗体5-6-7-huV2的内吞效果比人源化前(5-6-7-WT)有部分提升，其他人源化前后抗体抗体在人骨髓瘤细胞系H929细胞上的内吞效果基本一致，且均优于阳性对照抗体(GSK2857916)。
至此，两个候选抗体在人源化改造前后所有的检测结果汇总在图14中。
实施例11
人源化前后候选抗体DSF检测
在本实施例中，检测了两个候选抗体在人源化改造前后以及阳性对照抗体(GSK2857916)的热稳定性数据。具体流程如下：制备抗体溶液，0.25mg/mL，19μL/孔，每个供试品设置三个平行孔，并以PBS和IPI作为参比。然后在每个孔中加入1μL浓度为100×的SYPRO orange染料，准备上机。利用ABI 7500 FAST RT-PCR仪器进行测试，试验类型选择熔解曲线，采用连续模式，扫描温度范围为25～95℃，升温速率为1％，25℃平衡5min，在升温过程中采集数据，报告基团选择ROX，淬灭基团选择None，反应体积20μL，以熔解曲线一阶导数的第一个峰谷对应的温度确定为候选抗体的变性温度，结果详见表1-3。
表1-3人源化抗体热稳定性检测结果
Figure PCTCN2020117757-appb-000008
实施例12
人源化前后候选抗体亲和力检测
在本实施例中，采用Fortebio Octet RED96仪器，检测了5-6-7和5-6-32的人源化前后候选抗体，与人BCMA及猴BCMA的亲和力。
12.1材料准备
称取1g的BSA，量取500μL的Tween 20，加入到1000mL的1×PBS，混匀。过滤后分装保存。吸取0.1mL 0.1M pH＝2.0的甘氨酸溶液加入0.9mL的超纯水，混匀。抗体以KB buffer稀释成10μg/mL，抗原以KB buffer稀释成系列浓度梯度，依次为200、50、12.5、0nM。
12.2实验流程
避光预湿sensor(Anti-Human Fba-CH1 2nd Generation，FAB2G)至少10min后开始测试样品板(GreinierBio，PN655209)，测试无误后按预设程序进行。其中，样品板1的第1、10和12列加入200μL/孔的KB buffer，第11列加入0.01M pH2.0的甘氨酸溶液，第2-8列加入制备好的样品溶液(一个样品加4个孔，即一列加2个样品)，第九列按照浓度从高到低依次加入人 BCMA-Fc，即第1，5个孔加入200nM的抗原溶液，第2，6个孔加入50nM的抗原溶液，第3，7个孔加入12.5nM的抗原溶液，第4、8个孔加入0nM的抗原溶液。样品板2的准备除第九列的抗原换成猴BCMA蛋白，其余保持不变。数据结果详见表1-4。
表1-4人源化抗体与人、猴BCMA亲和力检测结果
Figure PCTCN2020117757-appb-000009
实施例13
基于上述实施例选定了5-6-7-hu-2和5-6-32-hu-2，对其进行分析和测序。基于IMGT数据库(http://www.imgt.org/)对人源抗体序列可变区进行定义，确定本发明抗体轻链和重链可变区的序列(SEQ ID NO:13-16)，针对可变区序列进行分析，采用AbM定义CDR的方式，确定了抗体重链和轻链的互补决定区序列(SEQ ID NO:1-12)。
本发明保护的序列具体如下：
SEQ ID NO:1
GHIFTNFHFH
SEQ ID NO:2
GYIFTNYHMH
SEQ ID NO:3
GIYPGNGDTF
SEQ ID NO:4
GIYPGNGDIF
SEQ ID NO:5
GSYYGYIDAMDY
SEQ ID NO:6
GSYYGYIDAMDY
SEQ ID NO:7
RASQDISNYLN
SEQ ID NO:8
RASQDISNDLN
SEQ ID NO:9
YTSRLHS
SEQ ID NO:10
YTSRLPS
SEQ ID NO:11
QQGNTLPWT
SEQ ID NO:12
QQGHTLPWT
SEQ ID NO:13
Figure PCTCN2020117757-appb-000010
SEQ ID NO:14
Figure PCTCN2020117757-appb-000011
Figure PCTCN2020117757-appb-000012
SEQ ID NO:15
Figure PCTCN2020117757-appb-000013
SEQ ID NO:16
Figure PCTCN2020117757-appb-000014
SEQ ID NO:17
Figure PCTCN2020117757-appb-000015
SEQ ID NO:18
Figure PCTCN2020117757-appb-000016
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (23)

  1. 一种分离的靶向B细胞成熟抗原(BCMA)的单克隆抗体，其中，所述抗体既能结合人BCMA也能结合猴BCMA。
  2. 根据权利要求1所述的单克隆抗体，所述抗体选自以下的任一种：
    (1)抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO:1或2所示的重链互补决定区1(CDR-H1)，和/或包含SEQ ID NO:3或4所示的重链互补决定区2(CDR-H2)，和/或包含SEQ ID NO:5或6所示的重链互补决定区3(CDR-H3)；
    (2)抗体，其包含轻链可变区，所述轻链可变区包含SEQ ID NO:7或8所示的轻链互补决定区1(CDR-L1)，和/或包含SEQ ID NO:9或10所示的轻链互补决定区2(CDR-L2)，和/或包含SEQ ID NO:11或12所示的轻链互补决定区3(CDR-L3)；
    (3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；
    (4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
  3. 根据权利要求1或2所述的抗体，其特征在于，所述抗体选自以下的任一种：
    (1)抗体，包含轻链可变区，所述轻链可变区包含SEQ ID NO:13或14所示的氨基酸序列、或上述序列的变体；
    (2)抗体，包含重链可变区，所述重链可变区具有SEQ ID NO:15或16所示的氨基酸序列、或上述序列的变体；
    (3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区。
  4. 根据权利要求1～3中任一项所述的抗体，其特征在于，所述的抗体的重链可变区包含SEQ ID NO:1所示的CDR-H1，SEQ ID NO:3所示的CDR-H2和SEQ ID NO:5所示的CDR-H3。
  5. 根据权利要求1～3中任一项所述的抗体，其特征在于，所述的抗体 的重链可变区包含SEQ ID NO:2所示的CDR-H1，SEQ ID NO:4所示的CDR-H2和SEQ ID NO:6所示的CDR-H3。
  6. 根据权利要求1～3中任一项所述的抗体，其特征在于，所述的抗体的轻链可变区包含SEQ ID NO:7所示的CDR-L1，SEQ ID NO:9所示的CDR-L2和SEQ ID NO:11所示的CDR-L3。
  7. 根据权利要求1～3中任一项所述的抗体，其特征在于，所述的抗体的轻链可变区包含SEQ ID NO:8所示的CDR-L1，SEQ ID NO:10所示的CDR-L2和SEQ ID NO:12所示的CDR-L3。
  8. 根据权利要求1～3中任一项所述的抗体，其特征在于，所述的抗体的轻链可变区具有SEQ ID NO:13或14所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
  9. 根据权利要求1～3中任一项所述的抗体，其特征在于，所述的抗体的重链可变区具有SEQ ID NO:15或16所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
  10. 一种靶向B细胞成熟抗原(BCMA)的单克隆抗体，其特征在于，该抗体与权利要求1-9中任一项所述的抗体识别相同的抗原决定部位。
  11. 一种靶向B细胞成熟抗原(BCMA)的单克隆抗体，其特征在于，该抗体与权利要求1-9中任一项所述的抗体竞争性结合B细胞成熟抗原(BCMA)。
  12. 编码权利要求1-11中任一项所述的抗体的核酸。
  13. 一种表达载体，其包含权利要求12所述的核酸。
  14. 一种宿主细胞，其包含权利要求13所述的表达载体或基因组中整合有权利要求12所述的核酸。
  15. 一种生产单克隆抗体的方法，所述方法包括培养根据项14所述的宿主细胞从而生产根据权利要求1～11中任一项所述的单克隆抗体。
  16. 一种药物组合物，其包含根据权利要求1～11中任一项所述的单克隆抗体和药学上可接受的载体。
  17. 一种药盒或制品，其包括根据权利要求1～11中任一项所述的单克隆抗体或根据权利要求16所述的药物组合物。
  18. 一种治疗与BCMA的表达相关的疾病的方法，其包括：
    向有此需要的受试者给药权利要求1～11中任一项所述的单克隆抗体或权利要求16所述的药物组合物或权利要求17所述的药盒或制品。
  19. 根据权利要求18所述的方法，其中，所述疾病选自B细胞急性淋巴性白血病、T细胞急性淋巴性白血病、急性淋巴性白血病、慢性髓性白血病、慢性淋巴细胞白血病、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、毛细胞白血病，小细胞或大细胞滤泡性淋巴瘤、恶性淋巴瘤、恶性淋巴增殖状况、MALT淋巴瘤、外套细胞淋巴瘤、边缘区淋巴瘤、多发性骨髓瘤、脊髓发育不良和骨髓增生异常综合征、非霍奇金淋巴瘤、浆母细胞淋巴瘤、浆细胞样树突细胞瘤、瓦尔登斯特伦巨球蛋白血症、骨髓瘤、MGUS、浆细胞瘤、系统性淀粉样蛋白轻链淀粉样变性和POEMS综合征。
  20. 根据权利要求18或19所述的方法，其中，所述疾病为多发性骨髓瘤。
  21. 根据权利要求1～11中任一项所述的单克隆抗体在制备用于治疗与BCMA的表达相关的疾病的药物中的用途。
  22. 根据权利要求21所述的用途，其中，所述疾病选自B细胞急性淋巴性白血病、T细胞急性淋巴性白血病、急性淋巴性白血病、慢性髓性白血病、慢性淋巴细胞白血病、B细胞幼淋巴细胞白血病、母细胞性浆细胞样树突状细胞瘤、伯基特淋巴瘤、弥散性大B细胞淋巴瘤、滤泡性淋巴瘤、毛细胞白血病，小细胞或大细胞滤泡性淋巴瘤、恶性淋巴瘤、恶性淋巴增殖状况、MALT淋巴瘤、外套细胞淋巴瘤、边缘区淋巴瘤、多发性骨髓瘤、脊髓发育不良和骨髓增生异常综合征、非霍奇金淋巴瘤、浆母细胞淋巴瘤、浆细胞样树突细胞瘤、瓦尔登斯特伦巨球蛋白血症、骨髓瘤、MGUS、浆细胞瘤、系统性淀粉样蛋白轻链淀粉样变性和POEMS综合征。
  23. 根据权利要求21或22所述的用途，其中，所述疾病为多发性骨髓瘤。
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115028734A (zh) * 2022-06-29 2022-09-09 广东康盾创新产业集团股份公司 一种靶向bcma的嵌合抗原受体及其用途
WO2023283615A1 (en) * 2021-07-09 2023-01-12 Dynetherapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11771776B2 (en) 2021-07-09 2023-10-03 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
CN117069842A (zh) * 2022-12-06 2023-11-17 成都赛恩吉诺生物科技有限公司 具有特定等电点的抗人bcma纳米抗体及car-t和应用
US11833217B2 (en) 2018-08-02 2023-12-05 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12128109B2 (en) 2021-07-09 2024-10-29 Dyne Therapeutics, Inc. Muscle targeting complexes and formulations for treating dystrophinopathies
US12239717B2 (en) 2021-07-09 2025-03-04 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12329825B1 (en) 2018-08-02 2025-06-17 Dyne Therapeutics, Inc. Muscle targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and method of use thereof to induce exon skipping of exon 44 of dystrophin in a subject

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140105915A1 (en) 2011-05-27 2014-04-17 Glaxo Group Limited Bcma (cd269/tnfrsf17) - binding proteins
WO2017021450A1 (en) * 2015-08-03 2017-02-09 Engmab Ag Monoclonal antibodies against bcma

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
UA112434C2 (uk) 2011-05-27 2016-09-12 Ґлаксо Ґруп Лімітед Антигензв'язувальний білок, який специфічно зв'язується з всма
TWI679212B (zh) * 2011-11-15 2019-12-11 美商安進股份有限公司 針對ｂｃｍａ之e3以及ｃｄ３的結合分子
AR095374A1 (es) 2013-03-15 2015-10-14 Amgen Res Munich Gmbh Moléculas de unión para bcma y cd3
KR102526945B1 (ko) 2014-04-30 2023-04-27 막스-델부뤽-센트럼 퓌어 몰레쿨라레 메디친 인 데어 헬름홀츠-게마인샤프트 Ｃｄ269에 대한 인간화 항체
DE102014107077B3 (de) * 2014-05-20 2015-08-13 Ecs Engineered Control Systems Ag Schaltvorrichtung
CN108778329B (zh) * 2016-02-17 2022-09-16 西雅图基因公司 Bcma抗体和其用以治疗癌症和免疫病症的用途
CN108004259B (zh) * 2016-11-02 2020-06-30 上海恒润达生生物科技有限公司 靶向b细胞成熟抗原的嵌合抗原受体及其用途
CN109942708B (zh) * 2019-03-28 2021-01-05 上海科棋药业科技有限公司 一种抗bcma的单域抗体及其应用
CN110229232B (zh) * 2019-06-19 2020-05-19 北京智仁美博生物科技有限公司 双特异性抗体及其用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140105915A1 (en) 2011-05-27 2014-04-17 Glaxo Group Limited Bcma (cd269/tnfrsf17) - binding proteins
WO2017021450A1 (en) * 2015-08-03 2017-02-09 Engmab Ag Monoclonal antibodies against bcma

Non-Patent Citations (23)

* Cited by examiner, † Cited by third party
Title
BLOOD CANCER JOURNAL, vol. 5, no. 2, 2015, pages e282 - e282
BORDEN, P.KABAT EA, PNAS, vol. 84, 1987, pages 2440 - 2443
CHOITHIA, C. ET AL., NATURE, vol. 342, 1989, pages 877 - 883
CHOITHIA, C.LESK, AM, J MOL. BIOL., vol. 196, no. 4, 1987, pages 901 - 917
ECKHERT ERIK, HEWITT RHONDA, LIEDTKE MICHAELA: "B-cell maturation antigen directed monoclonal antibody therapies for multiple myeloma", IMMUNOTHERAPY, vol. 11, no. 9, 16 May 2019 (2019-05-16), pages 801 - 811, XP009527380, ISSN: 1750-743X, DOI: 10.2217/imt-2018-0199 *
FELIX ET AL., MOL. ONCOLOGY, vol. 9, no. 7, 2015, pages 1348 - 58
HODGSON ET AL., BIO/TECHNOLOGY, vol. 9, 1991, pages 421
KUBY: "Journal of Immunology", 1997, W.H. FREEMAN & CO.
LIU, MING-JIE ET AL.: "Research Progress in B Cell Maturation Antigen-Targeted Immunotherapy in Multiple Myeloma", JOURNAL OF LANZHOU UNIVERSITY (MEDICAL SCIENCES), vol. 45, no. 4, 8 August 2019 (2019-08-08), pages 67 - 73, XP055802010, ISSN: 1000-2812 *
MACKAYKALLED ET AL., IMMUNOLOGICAL REVIEW, vol. 204, 2003, pages 43 - 54
NATURE REVIEWS CLINICAL ONCOLOGY, vol. 9, no. 3, 2012, pages 135 - 143
NATURE REVIEWS DISEASE PRIMERS, vol. 3,, 2017, pages 17046
NATURE REVIEWS IMMUNOLOGY, vol. 9, no. 7, 2009, pages 491
NATURE REVIEWS IMMUNOLOGY,, vol. 9, no. 7, 2009, pages 491
NERI ET AL., CLINICAL CANCER RESEARCH, vol. 73, no. 19, pages 5903 - 5909
NOVAK ET AL., BLOOD, vol. 103, no. 2, 2004, pages 689 - 694
PIERCE: "Catalog and Handbook", 1994, PIERCE CHEMICAL CO.
QUEEN ET AL., PROC. NATL ACAD SCI USA, vol. 86, 1989, pages 10029 - 10032
REMINGTON: "Pharmaceutical Sciences", 1990, MACK PUBLISHING CO.
REMINGTON: "The Science and Practice of Pharmacy", 2005, MACK PUBLISHING
See also references of EP4023673A4
THOMPSON ET AL., J.EXP.MEDICINE, vol. 192, no. 1, 2000, pages 129 - 135
WU, TTKABAT, EA, JOURNAL OF EXPERIMENTAL MEDICINE, vol. 132, no. 2, 1970, pages 211 - 50

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12005124B2 (en) 2018-08-02 2024-06-11 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12478687B2 (en) 2018-08-02 2025-11-25 Dyne Therapeutics, Inc. United states
US12357703B2 (en) 2018-08-02 2025-07-15 Dyne Therapeutics, Inc. Muscle-targeting complexes comprising an anti-transferin receptor antibody linked to an oligonucleotide and method of use thereof to induce exon skipping
US12329825B1 (en) 2018-08-02 2025-06-17 Dyne Therapeutics, Inc. Muscle targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and method of use thereof to induce exon skipping of exon 44 of dystrophin in a subject
US11833217B2 (en) 2018-08-02 2023-12-05 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12263225B2 (en) 2018-08-02 2025-04-01 Dyne Therapeutics, Inc. Muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and methods of use thereof to target dystrophin and to treat Duchenne muscular dystrophy
US12144867B2 (en) 2021-07-09 2024-11-19 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12403203B2 (en) 2021-07-09 2025-09-02 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12128109B2 (en) 2021-07-09 2024-10-29 Dyne Therapeutics, Inc. Muscle targeting complexes and formulations for treating dystrophinopathies
US12144868B2 (en) 2021-07-09 2024-11-19 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11986537B2 (en) 2021-07-09 2024-05-21 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12239717B2 (en) 2021-07-09 2025-03-04 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12239716B2 (en) 2021-07-09 2025-03-04 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
WO2023283615A1 (en) * 2021-07-09 2023-01-12 Dynetherapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12329824B1 (en) 2021-07-09 2025-06-17 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12440575B2 (en) 2021-07-09 2025-10-14 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11771776B2 (en) 2021-07-09 2023-10-03 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12397062B2 (en) 2021-07-09 2025-08-26 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
CN115028734A (zh) * 2022-06-29 2022-09-09 广东康盾创新产业集团股份公司 一种靶向bcma的嵌合抗原受体及其用途
CN117069842A (zh) * 2022-12-06 2023-11-17 成都赛恩吉诺生物科技有限公司 具有特定等电点的抗人bcma纳米抗体及car-t和应用
CN117069842B (zh) * 2022-12-06 2024-03-29 成都赛恩吉诺生物科技有限公司 具有特定等电点的抗人bcma纳米抗体及car-t和应用

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