Humanized monoclonal antibody targeting BCMA with human-macaque cross-reactivity

Abstract

The present invention relates to an isolated monoclonal antibody targeting B cell maturation antigen (BCMA), wherein the antibody binds to both human BCMA and monkey BCMA. The present invention also relates to a nucleic acid encoding the antibody, a method for producing the monoclonal antibody, and a pharmaceutical composition comprising the antibody of the present invention.

Description

Technical Field

[0001] The present invention relates to monoclonal antibodies and fragments thereof that specifically bind to B cell maturation antigen (BCMA), including monoclonal antibodies that specifically bind to human BCMA and monkey BCMA antigens at the same time.

[0002] The present invention relates to monoclonal antibodies and fragments thereof that specifically bind to BCMA and inhibit the binding of BAFF and APRIL to the BCMA receptor.

[0003] The present invention also relates to monoclonal antibodies and fragments thereof that specifically bind to BCMA and have excellent endocytosis effects. Background Art

[0004] B cells mature in the bone marrow and become plasma cells, which can secrete antibodies to fight foreign viruses or bacteria. When plasma cells become cancerous and become myeloma cells, they will continue to proliferate more malignant myeloma cells and secrete a large amount of useless antibodies. Myeloma usually grows in the spine, skull, pelvis, chest cavity and other locations, and manifests as a tumor or osteolytic lesion. The disease of myeloma is usually progressive, from monoclonal gammopathy of undetermined significance (MGUS) to low-risk smoldering multiple myeloma to high-risk smoldering multiple myeloma (SMM), and finally progresses to multiple myeloma (Nature Reviews Disease Primers, 2017, 3, 17046.). The main symptoms of multiple myeloma (MM) include hypercalcemia, renal dysfunction, anemia, bone dysfunction, etc., accompanied by severe bone pain and easy recurrent fractures (Nature Reviews Clinical Oncology, 2012, 9 (3), 135–143.). According to statistics from the International Myeloma Foundation, as of August 2017, the number of people suffering from the disease worldwide reached approximately 750,000, with approximately 114,000 new cases each year and nearly 90,000 deaths from the disease.

[0005] B cell maturation antigen (BCMA), a member of the tumor necrosis factor superfamily, is primarily expressed on the surface of memory cells, plasmablasts, and plasma cells, but is rarely expressed on other cell surfaces. BCMA is a transmembrane receptor on the cell surface, and its gene is located at the TNFRSF17 locus on chromosome 16. A literature report (Blood Cancer Journal, 2015, 5(2), e282–e282.) shows that BCMA-deficient mice have relatively normal appearance and B cell numbers, but their plasma cell survival is extremely poor.

[0006] BCMA ligands include B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). BAFF receptors also include BAFF-R and TACI, while APRIL receptors also include TACI. The BCMA signaling pathway primarily promotes B cell survival and differentiation and the activation of regulatory T cells. Conversely, the TACI signaling pathway inhibits B cell maturation (Nature reviews immunology, 2009, 9(7):491). Regarding these three receptors (BAFF-R, TACI, and BCMA), during B cell development, immature B cells, migrating B cells, and naive B cells only express BAFF-R on their surfaces; GC B cells all express BAFF-R and BCMA on their surfaces; memory cells all express BAFF-R, TACI, and BCMA on their surfaces; plasmablasts or plasma cells all express TACI and BCMA on their surfaces; and when plasma cells become cancerous and become multiple myeloma cells, their surfaces highly express BCMA, and may express TACI but not BAFF-R (Nature reviews immunology, 2009, 9(7):491.). It can be seen from this that most B cells do not express BCMA. In addition, studies have shown that cells in other organs also rarely express BCMA. Clinically, the levels of BCMA, BAFF, and APRIL in the serum of multiple myeloma patients are higher and their overall survival and prognosis are worse. Therefore, in the treatment of multiple myeloma, BCMA is a new target that is superior to CD19 and other targets, with high specificity and fewer side effects. Therefore, the development of antibody drugs with blocking or endocytic effects against BCMA targets can not only improve the therapeutic effect of multiple myeloma, but also greatly reduce the side effects of treatment and generate huge economic and social value.

[0007] Currently, in terms of ADC (antibody-drug conjugate, ADC) drugs, belantamab mafodotin (abbreviated as GSK2857916), jointly developed by GlaxoGroup and Seattle Genetics, has a significant effect. In 35 patients with R / R MM who were overly pretreated (most patients had received at least 5 therapies and failed treatment), the ORR reached 60%, and the median PFS (Progression-Free-Survival) was 12 months (NCT03848845). In terms of CAR-T cells, Celgene and Bluebird Bio's CAR-T cell therapy Idecabtagenevicleucel (abbreviated as bb2121) achieved an overall response rate of 85% and a median PFS of 11.8 months in 33 patients with R / R MM who had previously failed at least 3 therapies (NCT02658929). In terms of bispecific antibodies, Amgen's AMG 420 is the fastest-progressing therapy. This type of antibody is smaller than traditional antibodies and consists of two antibody domain fragments linked together. It has good activity but a shorter half-life than full-length antibodies (NCT02514239). In terms of clinical results, drugs targeting the BMCA target, whether monoclonal antibodies, bispecific antibodies, ADCs, or CAR-T cell therapies, have achieved impressive results. Clinical results also show that the side effects of the BCMA target are far fewer than those of other targets. Summary of the Invention

[0008] Based on the above background, the present invention aims to develop novel antibodies targeting BCMA.

[0009] The present invention used BCMA as an immunogen to immunize mice. Using phage display technology, an antibody library was constructed and screened to obtain monoclonal antibodies that bind to both human and monkey BCMA antigens. Subsequently, through humanization, the murine monoclonal antibody was transformed into a humanized antibody. Functional experiments, including affinity, blocking, and endocytosis, confirmed that the humanized candidate antibody exhibited excellent functionality, with superior endocytosis at the cellular level compared to a competing product, GSK2857916.

[0010] All patents and references disclosed within this specification are expressly and entirely incorporated herein by reference.

[0011] 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.

[0012] In a specific aspect, the antibody of the present invention comprises a heavy chain variable region comprising a heavy chain complementary determining region 1 (CDR-H1) as shown in SEQ ID NO: 1 or 2, and / or comprising a heavy chain complementary determining region 2 (CDR-H2) as shown in SEQ ID NO: 3 or 4, and / or comprising a heavy chain complementary determining region 3 (CDR-H3) as shown in SEQ ID NO: 5 or 6.

[0013] In a specific aspect, the antibody of the present invention comprises a light chain variable region comprising a light chain complementary determining region 1 (CDR-L1) as shown in SEQ ID NO: 7 or 8, and / or a light chain complementary determining region 2 (CDR-L2) as shown in SEQ ID NO: 9 or 10, and / or a light chain complementary determining region 3 (CDR-L3) as shown in SEQ ID NO: 11 or 12.

[0014] In a specific aspect, 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 complementary determining region 1 (CDR-H1) shown in SEQ ID NO: 1 or 2, and / or comprises the heavy chain complementary determining region 2 (CDR-H2) shown in SEQ ID NO: 3 or 4, and / or comprises the heavy chain complementary determining region 3 (CDR-H3) shown in SEQ ID NO: 5 or 6; the light chain variable region comprises the light chain complementary determining region 1 (CDR-L1) shown in SEQ ID NO: 7 or 8, and / or comprises the light chain complementary determining region 2 (CDR-L2) shown in SEQ ID NO: 9 or 10, and / or comprises the light chain complementary determining region 3 (CDR-L3) shown in SEQ ID NO: 11 or 12.

[0015] In one specific aspect, the antibodies of the present invention include variants of the above antibodies, and have the same or similar activities as the above antibodies of the present invention.

[0016] In a specific aspect, 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.

[0017] In a specific aspect, 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 thereof.

[0018] In a specific aspect, 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, and 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.

[0019] In a specific aspect, the heavy chain variable region of the antibody of the present invention comprises CDR-H1 shown in SEQ ID NO: 1, CDR-H2 shown in SEQ ID NO: 3, and CDR-H3 shown in SEQ ID NO: 5.

[0020] In a specific aspect, 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-H3 shown in SEQ ID NO: 6.

[0021] In a specific aspect, the light chain variable region of an antibody of the present invention comprises CDR-L1 shown in SEQ ID NO:7, CDR-L2 shown in SEQ ID NO:9, and CDR-L3 shown in SEQ ID NO:11.

[0022] In a specific aspect, the light chain variable region of the 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.

[0023] In a specific aspect, the light chain variable region of an antibody of the present invention has a sequence as shown in SEQ ID NO: 13 or 14, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences.

[0024] In a specific aspect, the heavy chain variable region of an antibody of the present invention has a sequence as shown in SEQ ID NO: 15 or 16, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences.

[0025] In a specific aspect, 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, CDR-H2 shown in SEQ ID NO: 3, and CDR-H3 shown in SEQ ID NO: 5; and the light chain variable region comprises CDR-L1 shown in SEQ ID NO: 7, CDR-L2 shown in SEQ ID NO: 9, and CDR-L3 shown in SEQ ID NO: 11.

[0026] Further, the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 13, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences; the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 15, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences.

[0027] In a specific aspect, 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, CDR-H2 shown in SEQ ID NO: 4, and CDR-H3 shown in SEQ ID NO: 6; and the light chain variable region 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.

[0028] Further, the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 14, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences; the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 16, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences.

[0029] The present invention also relates to a monoclonal antibody targeting B cell maturation antigen (BCMA), which recognizes the same antigenic determinant as the antibody described above.

[0030] The present invention also relates to a monoclonal antibody targeting B cell maturation antigen (BCMA), which competes with the antibody of the present invention for binding to B cell maturation antigen (BCMA).

[0031] The present invention also relates to nucleic acids encoding the above-mentioned antibodies of the present invention.

[0032] The present invention also relates to an expression vector comprising the nucleic acid described above.

[0033] The present invention also relates to a host cell comprising the expression vector of the present invention or a host cell in which the nucleic acid of the present invention is integrated into its genome.

[0034] The present invention also relates to a method for producing a monoclonal antibody, which comprises culturing the host cell of the present invention to thereby produce the monoclonal antibody of the present invention.

[0035] The present invention also relates to a pharmaceutical composition comprising the monoclonal antibody of the present invention and a pharmaceutically acceptable carrier.

[0036] The present invention also relates to a drug kit or product comprising the monoclonal antibody or the pharmaceutical composition of the present invention.

[0037] The present invention also relates to a method for treating a disease associated with BCMA expression, comprising: administering the monoclonal antibody, the pharmaceutical composition, the kit or the product of the present invention to a subject in need thereof.

[0038] In a specific aspect, the disease is selected from B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis and POEMS syndrome.

[0039] In a specific aspect, the disease is multiple myeloma.

[0040] The present invention also relates to the use of the monoclonal antibody of the present invention in the preparation of a medicament for treating diseases associated with BCMA expression.

[0041] In a specific aspect, the disease is selected from B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis and POEMS syndrome.

[0042] In a specific aspect, the disease is multiple myeloma.

[0043] Specifically, the present invention relates to the following aspects:

[0044] 1. An isolated monoclonal antibody targeting B-cell maturation antigen (BCMA), wherein the antibody binds to both human BCMA and monkey BCMA.

[0045] 2. The monoclonal antibody according to item 1, wherein the antibody is selected from any one of the following:

[0046] (1) An antibody comprising a heavy chain variable region comprising a heavy chain complementary determining region 1 (CDR-H1) as set forth in SEQ ID NO: 1 or 2, and / or a heavy chain complementary determining region 2 (CDR-H2) as set forth in SEQ ID NO: 3 or 4, and / or a heavy chain complementary determining region 3 (CDR-H3) as set forth in SEQ ID NO: 5 or 6;

[0047] (2) an antibody comprising a light chain variable region comprising a light chain complementary determining region 1 (CDR-L1) as set forth in SEQ ID NO: 7 or 8, and / or a light chain complementary determining region 2 (CDR-L2) as set forth in SEQ ID NO: 9 or 10, and / or a light chain complementary determining region 3 (CDR-L3) as set forth in SEQ ID NO: 11 or 12;

[0048] (3) an antibody comprising (1) a heavy chain variable region of the antibody and (2) a light chain variable region of the antibody;

[0049] (4) An antibody, which is a variant of the antibody according to any one of (1) to (3), and has the same or similar activity as the antibody according to any one of (1) to (3).

[0050] 3. The antibody according to item 1 or 2, characterized in that the antibody is selected from any one of the following:

[0051] (1) An antibody comprising a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 13 or 14, or a variant thereof;

[0052] (2) an antibody comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15 or 16, or a variant thereof;

[0053] (3) An antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody.

[0054] 4. 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: 1, CDR-H2 shown in SEQ ID NO: 3, and CDR-H3 shown in SEQ ID NO: 5.

[0055] 5. 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, CDR-H2 shown in SEQ ID NO: 4, and CDR-H3 shown in SEQ ID NO: 6.

[0056] 6. The antibody according to any one of items 1 to 3, wherein the light chain variable region of the antibody comprises CDR-L1 shown in SEQ ID NO: 7, CDR-L2 shown in SEQ ID NO: 9, and CDR-L3 shown in SEQ ID NO: 11.

[0057] 7. The antibody according to any one of items 1 to 3, wherein the light chain variable region of the antibody 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.

[0058] 8. The antibody according to any one of items 1 to 3, characterized in that the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 13 or 14, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences.

[0059] 9. The antibody according to any one of items 1 to 3, characterized in that the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 15 or 16, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences.

[0060] 10. A monoclonal antibody targeting B cell maturation antigen (BCMA), characterized in that the antibody recognizes the same antigenic determinant as the antibody of any one of items 1 to 9.

[0061] 11. A monoclonal antibody targeting B cell maturation antigen (BCMA), characterized in that the antibody competes with the antibody of any one of items 1 to 9 for binding to B cell maturation antigen (BCMA).

[0062] 12. A nucleic acid encoding the antibody according to any one of items 1 to 11.

[0063] 13. An expression vector comprising the nucleic acid according to item 12.

[0064] 14. A host cell comprising the expression vector of item 13 or the nucleic acid of item 12 integrated into its genome.

[0065] 15. A method for producing a monoclonal antibody, the method comprising culturing the host cell according to item 14, thereby producing the monoclonal antibody according to any one of items 1 to 11.

[0066] 16. A pharmaceutical composition comprising the monoclonal antibody according to any one of items 1 to 11 and a pharmaceutically acceptable carrier.

[0067] 17. A pharmaceutical kit or article of manufacture comprising the monoclonal antibody according to any one of items 1 to 11 or the pharmaceutical composition according to item 16.

[0068] 18. A method for treating a disease associated with BCMA expression, comprising:

[0069] The monoclonal antibody according to any one of items 1 to 11, the pharmaceutical composition according to item 16, or the kit or preparation according to item 17 is administered to a subject in need thereof.

[0070] 19. The method of claim 18, wherein the disease is selected from the group consisting of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis and POEMS syndrome.

[0071] 20. The method according to item 18 or 19, wherein the disease is multiple myeloma.

[0072] 21. Use of the monoclonal antibody according to any one of items 1 to 11 for the preparation of a medicament for treating a disease associated with BCMA expression.

[0073] 22. The method of claim 21, wherein the disease is selected from the group consisting of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis and POEMS syndrome.

[0074] 23. The use according to item 21 or 22, wherein the disease is multiple myeloma.

[0075] Effects of the present invention

[0076] Compared to the Belantamab mafodotin antibody (abbreviated as GSK2857916), the antibody of the present invention can bind to both human BCMA and monkey BCMA, and its affinity level is close to or even better than that of GSK2857916. The antibody of the present invention is close to or even better than GSK2857916 in terms of blocking the binding of BCMA to its ligands BAFF or APRIL. The antibody of the present invention has a better endocytosis effect than GSK2857916. The antibody of the present invention has a better thermal stability than GSK2857916. In terms of immunogenicity, the antibody of the present invention has lower immunogenicity than the humanized antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The process of antibody production is shown, showing the process of antibody production targeting BCMA with human-monkey cross-talk.

[0078] Figure 2 shows the activity assays of recombinant proteins BCMA, BAFF, and APRIL, demonstrating normal activity for each protein. Figure 2(A) shows the binding results of human and monkey BCMA antigens with different Fc and His tags and the positive control antibody GSK2857916. Figure 2(B) shows the binding results of human BCMA antigen and BAFF coated at different concentrations. Figure 2(C) shows the binding results of human BCMA antigen and APRIL coated at different concentrations.

[0079] Figure 3 shows the binding assay of candidate antibodies to cells overexpressing human or monkey BCMA. The results indicate that the candidate antibodies have cross-human and monkey properties. Figure 3 (A) shows the binding of some candidate antibodies to HEK293 cells overexpressing human BCMA as determined by FACS. Figure 3 (B) shows the binding of some candidate antibodies to CHO cells overexpressing monkey BCMA as determined by FACS.

[0080] Figure 4 shows the cross-affinity activity test results of some candidate antibodies with human and monkey BCMA. Figure 4(A) shows the affinity of some candidate antibodies for human BCMA at the ELISA level. The results show that some candidate antibodies have similar or even better affinity than GSK2857916 antibody. Figure 4(B) shows the affinity of some candidate antibodies for monkey BCMA at the ELISA level. The results show that some candidate antibodies have similar or even better affinity than GSK2857916 antibody.

[0081] Figure 5 shows the blocking effect test results of some candidate antibodies. Figure 5 (A) shows that some candidate antibodies blocked the binding of BCMA to BAFF at the ELISA level. The results show that some candidate antibodies have similar or even better blocking effects than GSK2857916 antibody. Figure 5 (B) and Figure 5 (B) show that some candidate antibodies blocked the binding of BCMA to APRIL at the ELISA level. The results show that some candidate antibodies have similar or even better blocking effects than GSK2857916 antibody.

[0082] Figure 6 shows the results of the endocytosis assay. Figures 6 (A) and (B) show the endocytosis of some candidate antibodies in the human myeloma cell line H929 cells. The results show that some candidate antibodies have better endocytosis than GSK2857916 antibody.

[0083] Figure 7 Displays a summary of the functions of candidate antibodies, including Figure 7 The data show the affinity effects of some candidate antibodies on HEK293 cells expressing human BCMA, the affinity effects on CHO cells expressing monkey BCMA, the affinity effects on human and monkey BCMA at the ELISA level, the effect of blocking the binding of BAFF to BCMA at the ELISA level, the effect of blocking the binding of APRIL to BCMA at the ELISA level, and the internalization effect of the antibodies on human myeloma cells H929. The number of '+'s from large to small represents the strong to weak antibody affinity, blocking and internalization effects. The results show that antibodies SY14-3rd-5-6-7 and SY14-3rd-5-6-32 exhibited better comprehensive effects.

[0084] Figure 8 shows the results of cross-affinity activity testing of the humanized antibody (5-6-7-hu-2) with human and monkey BCMA. Figure 8(A) shows the affinity of the SY14-3rd-5-6-7 (also known as 5-6-7 or 5-6-7-WT) antibody for human BCMA before and after humanization, as measured by ELISA. The results show that the affinity of the antibody for human BCMA remains the same after humanization. Figure 8(B) shows the affinity of the SY14-3rd-5-6-7 antibody for monkey BCMA before and after humanization, as measured by ELISA. The results show that the affinity of the antibody for monkey BCMA remains the same after humanization.

[0085] Figure 9 shows the results of cross-affinity activity testing of the humanized antibody (5-6-32-hu-2) with human and monkey BCMA. Figure 9(A) shows the affinity of the SY14-3rd-5-6-32 (also known as 5-6-32 or 5-6-32-WT) antibody for human BCMA before and after humanization at the ELISA level. The results show that the affinity of the humanized antibody for human BCMA is still superior to that of the positive antibody (GSK2857916). Figure 9(B) shows the affinity of the SY14-3rd-5-6-32 antibody for monkey BCMA before and after humanization at the ELISA level. The results show that the affinity of the humanized antibody for monkey BCMA is still superior to that of the positive antibody GSK2857916.

[0086] Figure 10 shows the results of the blocking effect test of the humanized antibody (5-6-7-hu-2). Figure 10 (A) shows the ability of the humanized SY14-3rd-5-6-7 (also known as 5-6-7 or 5-6-7-WT) antibody to block BCMA binding to BAFF at the ELISA level. The results show that the humanized antibody is slightly less effective in blocking BCMA binding to BAFF than the positive antibody (GSK2857916). Figure 10 (B) shows the ability of the humanized SY14-3rd-5-6-7 antibody to block BCMA binding to APRIL at the ELISA level. The results show that the humanized antibody is slightly less effective in blocking BCMA binding to APRIL than the positive antibody (GSK2857916).

[0087] Figure 11 shows the results of the blocking effect test of the humanized antibody (5-6-32-hu-2). Figure 11 (A) shows the ability of the humanized SY14-3rd-5-6-32 (also known as 5-6-32 or 5-6-32-WT) antibody to block BCMA binding to BAFF at the ELISA level. The results show that the humanized antibody is slightly less effective in blocking BCMA binding to BAFF than the positive antibody (GSK2857916). Figure 11 (B) shows the ability of the humanized SY14-3rd-5-6-32 antibody to block BCMA binding to APRIL at the ELISA level. The results show that the humanized antibody is slightly less effective in blocking BCMA binding to APRIL than the positive antibody (GSK2857916).

[0088] Figure 12 shows the results of humanized antibody binding activity assays on cells overexpressing human and monkey BCMA. Figure 12(A) shows the binding ability of 5-6-7 and 5-6-32 antibodies to HEK293 cells overexpressing human BCMA before and after humanization. The results show that the binding ability of the humanized antibodies remains the same as before humanization and is superior to that of GSK2857916. Figure 12(B) shows 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 binding ability of the humanized antibodies remains the same as before humanization and is superior to that of GSK2857916. Figure 12(C) shows the binding ability of 5-6-7 and 5-6-32 antibodies to the myeloma cell line H929 cells before and after humanization. The results show that the binding ability of the humanized antibodies remains the same as before humanization and is superior to that of GSK2857916.

[0089] Figure 13 shows the results of the endocytosis assay on H929 cells before and after humanization of antibodies. Figure 13(A) shows the endocytosis of the 5-6-7 antibody before and after humanization on H929 cells, a human myeloma cell line expressing BCMA. The results show that the endocytosis of the humanized antibody is consistent with that before humanization and is superior to that of the GSK2857916 antibody. Figure 13(B) shows the endocytosis of the 5-6-32 antibody before and after humanization on H929 cells, a human myeloma cell line expressing BCMA. The results show that the endocytosis of the humanized antibody is consistent with that before humanization and is superior to that of the GSK2857916 antibody.

[0090] Figure 14 Displays the summary results of each function of humanized antibodies. Figure 14 The humanized antibody is shown in Figure 1. The humanization degree of the humanized antibody is demonstrated, along with its affinity on human BCMA-expressing HEK293 cells, its affinity on monkey BCMA-expressing CHO cells, its affinity for human and monkey BCMA at the ELISA level, its ability to block BAFF binding to BCMA, its ability to block APRIL binding to BCMA, and its internalization in human myeloma H929 cells. The number of "+"s in the humanized antibody indicates a decrease in affinity, blocking, and internalization. The results indicate that the functions of the humanized antibody remain consistent with those before humanization, with some functions approaching or exceeding those of the GSK2857916 antibody. DETAILED DESCRIPTION

[0091] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0092] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0093] Generally speaking, the terms used in this specification have the following meanings.

[0094] In this specification, an "isolated" antibody is one that has been separated from the components of its natural environment. In certain embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0095] The term "BCMA" herein is also B cell maturation antigen, also known as CD269, a member of the tumor necrosis factor receptor superfamily, namely TNFRSF17 (Thompson et al., J. Exp. Medicine, 192(1):129-135, 2000). 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 to B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) (e.g., Mackay et al., 2003 and Kalled et al., Immunological Review, 204:43-54, 2005). BCMA may be a suitable tumor antigen target for immunotherapeutic agents against multiple myeloma.

[0096] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T cell response in an animal, including compositions injected or absorbed into an animal (e.g., compositions comprising a cancer-specific protein). Antigens react with products of specific humoral or cellular immunity, including those induced by heterologous antigens (e.g., the disclosed antigens). In certain embodiments, the target antigen is an epitope of a BCMA polypeptide.

[0097] "Epitope" or "antigenic determinant" refers to the region of an antigen that is bound by a binding agent. An epitope can be formed by contiguous amino acids or discontinuous amino acids joined by tertiary folding of the protein. Epitopes formed by contiguous amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope typically comprises at least 3, and more typically at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

[0098] Antibodies include antigen-binding fragments thereof, such as camelid Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv protein ("scFv"), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv"), and single domain antibodies (sdAb, nanobodies), as well as the portion of the full-length antibody responsible for antigen binding. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heterojunction antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J. Immunol., 3rd ed., WH Freeman & Co., New York, 1997.

[0099] As understood by those skilled in the art and as described elsewhere herein, a complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins comprising α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y is composed of the second and third constant regions (and for IgE and IgM, the fourth constant region) of the two heavy chains, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have a constant region consisting of three tandem (in a row) Ig domains and a hinge region for increasing flexibility; heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The second and third constant regions are referred to as "CH2 domain" and "CH3 domain," respectively. Each arm of the Y consists of the variable region of a single heavy chain and a first constant region 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.

[0100] The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions (also called "complementarity determining regions" or "CDRs"). CDRs can be defined or identified by conventional methods, for example, by sequences according to Kabat et al. (Wu, TT and Kabat, EA, J. Exp. Med. 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, US Department of Health and Human Services, 1991, which are incorporated herein by reference), or by structures according to 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)).

[0101] The sequences of the framework regions of different light or heavy chains are relatively conserved within species (e.g., humans). The framework region of an antibody (which is the combined framework region of the component light and heavy chains) is used to locate and align the CDRs in three-dimensional space. CDRs are primarily responsible for binding to the epitope of the antigen. The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are typically also identified by the chain in which the specific CDR is located. Therefore, the CDRs located in the variable domain of the heavy chain of an antibody are referred to as CDR-H1, CDR-H2, and CDR-H3, while the CDRs located in the variable domain of the light chain of an antibody are referred to as CDR-L1, CDR-L2, and CDR-L3. Antibodies with different specificities (i.e., having different combination sites for different antigens) have different CDRs. Although the CDRs differ between antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are referred to as specificity determining residues (SDRs). Illustrative examples of light chain CDRs suitable for constructing the humanized BCMA CARs contemplated herein include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 1 to 3. Illustrative examples of heavy chain CDRs suitable for constructing the humanized BCMA CARs contemplated herein include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 4 to 6.

[0102] Mention "V H" or "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 disclosed herein. Reference to "V L " or "VL" refers to the variable region of an immunoglobulin light chain, including the light chain variable region of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragments as disclosed herein.

[0103] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell into which the light and heavy chain genes for 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 the fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0104] "Fv" is the smallest antibody fragment containing a complete antigen binding site. In one embodiment, the two-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. In the single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker so that the light chain and the heavy chain can be associated in a "dimerization" structure similar to the two-chain Fv species. In this configuration, the three hypervariable regions (HVRs) 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 comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigens, but the affinity is lower than the complete binding site.

[0105] The Fab fragment contains the heavy and light chain variable domains 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 a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0106] As described above, the present invention relates to a separated monoclonal antibody targeting B cell maturation antigen (BCMA), wherein the antibody can bind to both human BCMA and monkey BCMA. Specifically, in the present invention, the affinity effect of the antibody of the present invention on cells expressing human and monkey BCMA was verified using a flow cytometer operation method (FACS method). The affinity effect of the monoclonal antibody obtained by the present invention on human BCMA-HEK293 cells was better than that of the positive control antibody (GSK2857916). The affinity effect of the monoclonal antibody obtained by the present invention on monkey BCMA-CHO cells was close to or better than that of the positive control antibody (GSK2857916). The monoclonal antibody obtained by the present invention can simultaneously bind to human and monkey BCMA antigens with high affinity, and bind to monkey BCMA antigens, which facilitates the toxicological evaluation and pharmacokinetic evaluation of the antibody using monkeys (crab-eating macaques) as a model before entering clinical research. At the same time, the monoclonal antibodies of the present invention have very high affinity. Antibodies with high affinity have more advantages in terms of efficacy. For example, after the antibody binds to the target antigen molecule, it dissociates more slowly, making the antibody more effective in cell endocytosis. Moreover, to achieve the same cellular or animal efficacy, the required antibody dose may also be lower.

[0107] As used herein, the terms "specifically bind," "specifically recognize," or "have specificity for" refer to a measurable and reproducible interaction, such as binding between a target and an antigen binding protein. For example, an antigen binding protein that specifically binds to a target (which may be an epitope) is an antigen binding protein that binds to that target with greater affinity, avidity, more readily, and / or for a longer duration than binding to other targets. In some embodiments, the extent of binding of the antigen binding protein to an unrelated target is about 10% less than the binding of the antigen binding protein to the target, as determined, for example, by radioimmunoassay (RIA). In some embodiments, the antigen binding protein that specifically binds to a target has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM.

[0108] Specifically, the antibody of the present invention comprises a heavy chain variable region comprising a heavy chain complementary determining region 1 (CDR-H1) as shown in SEQ ID NO: 1 (GHIFTNFHFH) or 2 (GYIFTNYHMH), and / or a heavy chain complementary determining region 2 (CDR-H2) as shown in SEQ ID NO: 3 (GIYPGNGDTF) or 4 (GIYPGNGDIF), and / or a heavy chain complementary determining region 3 (CDR-H3) as shown in SEQ ID NO: 5 (GSYYGYIDAMDY) or 6 (GSYYGYIDAMDY).

[0109] Specifically, the antibody of the present invention may comprise a light chain variable region comprising a light chain complementary determining region 1 (CDR-L1) as shown in SEQ ID NO: 7 (RASQDISNYLN) or 8 (RASQDISNDLN), and / or a light chain complementary determining region 2 (CDR-L2) as shown in SEQ ID NO: 9 (YTSRLHS) or 10 (YTSRLPS), and / or a light chain complementary determining region 3 (CDR-L3) as shown in SEQ ID NO: 11 (QQGNTLPWT) or 12 (QQGHTLPWT).

[0110] Specifically, 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 complementary determining region 1 (CDR-H1) shown in SEQ ID NO: 1 or 2, and / or comprises the heavy chain complementary determining region 2 (CDR-H2) shown in SEQ ID NO: 3 or 4, and / or comprises the heavy chain complementary determining region 3 (CDR-H3) shown in SEQ ID NO: 5 or 6; the light chain variable region comprises the light chain complementary determining region 1 (CDR-L1) shown in SEQ ID NO: 7 or 8, and / or comprises the light chain complementary determining region 2 (CDR-L2) shown in SEQ ID NO: 9 or 10, and / or comprises the light chain complementary determining region 3 (CDR-L3) shown in SEQ ID NO: 11 or 12.

[0111] Specifically, the antibody of the present invention comprises a light chain variable region comprising

[0112] SEQ ID NO: 13:

[0113] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGQGTKLEIK

[0114] or

[0115] SEQ ID NO: 14:

[0116] DIQMTQSPSSSLSASVGDRVTITCRASQDISNDLNWYQQKPGKAPKLLIYYTSRLPSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGHTLPWTFGQGTKLEIK

[0117] The amino acid sequence shown, or a variant of the above sequence.

[0118] Specifically, the antibody of the present invention comprises a heavy chain variable region having

[0119] SEQ ID NO: 15:

[0120] QVQLVQSGAEVKKPGSSVKISCKASGHIFTNFHFHWVRQAPGQGLEWIGGIYPGNGDTFYNQKFQGRATITADKSTSTAYMELSSLRSEDTAVYYCVRGSYYGYIDAMDYWGQGTSVTVSS

[0121] or

[0122] SEQ ID NO: 16:

[0123] QVQLVQSGAEVKKPGSSVKISCKASGHIFTNFHFHWVRQAPGQGLEWIGGIYPGNGDTFYNQKFQGRATITADKSTSTAYMELSSLRSEDTAVYYCVRGSYYGYIDAMDYWGQGTSVTVSS

[0124] The amino acid sequence shown, or a variant of the above sequence.

[0125] Specifically, 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, and 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.

[0126] Specifically, the heavy chain variable region of one antibody of the present invention comprises CDR-H1 set forth in SEQ ID NO: 1, CDR-H2 set forth in SEQ ID NO: 3, and CDR-H3 set forth in SEQ ID NO: 5. The heavy chain variable region of another antibody of the present invention comprises CDR-H1 set forth in SEQ ID NO: 2, CDR-H2 set forth in SEQ ID NO: 4, and CDR-H3 set forth in SEQ ID NO: 6.

[0127] Specifically, the light chain variable region of one antibody of the present invention comprises CDR-L1 set forth in SEQ ID NO: 7, CDR-L2 set forth in SEQ ID NO: 9, and CDR-L3 set forth in SEQ ID NO: 11. The light chain variable region of another antibody of the present invention comprises CDR-L1 set forth in SEQ ID NO: 8, CDR-L2 set forth in SEQ ID NO: 10, and CDR-L3 set forth in SEQ ID NO: 12.

[0128] Specifically, the light chain variable region of the antibody of the present invention has a sequence as shown in SEQ ID NO: 13 or 14, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences. Specifically, the heavy chain variable region of the antibody of the present invention has a sequence as shown in SEQ ID NO: 15 or 16, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences.

[0129] Specifically, an 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, CDR-H2 shown in SEQ ID NO: 3, and CDR-H3 shown in SEQ ID NO: 5; the light chain variable region comprises CDR-L1 shown in SEQ ID NO: 7, CDR-L2 shown in SEQ ID NO: 9, and CDR-L3 shown in SEQ ID NO: 11. Specifically, the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 13, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences; the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 15, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences.

[0130] Specifically, another 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, CDR-H2 shown in SEQ ID NO: 4, and CDR-H3 shown in SEQ ID NO: 6; the light chain variable region 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. Specifically, the light chain variable region of the antibody has the sequence shown in SEQ ID NO: 14, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences; the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO: 16, or a sequence having at least 80%, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to any of the above sequences.

[0131] As used herein, the term "variant" refers to a heavy chain variable region or light chain variable region that has been modified by at least one, e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions, wherein the modified antigen-binding protein comprising the heavy chain or light chain variant substantially retains the biological characteristics of the antigen-binding protein before modification. In one embodiment, the antigen-binding protein containing the variant heavy chain variable region or light chain variable region sequence retains 60%, 70%, 80%, 90%, or 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 proteins of the present disclosure comprise heavy chain variable region amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequences described herein. The antigen-binding proteins of the present disclosure include light chain variable region amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the light chain variable region amino acid sequences described herein. The percentage of homology can be over the entire heavy chain variable region and / or the entire light chain variable region, or the percentage homology can be limited to the framework region, and the sequence corresponding to the CDR has 100% identity with the CDR disclosed herein within the heavy chain variable region and / or the light chain variable region. As used herein, the term "CDR variant" refers to a CDR that has been modified by 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 biological characteristics of the antigen-binding protein before modification. In one embodiment, the antigen-binding protein containing the variant CDR retains 60%, 70%, 80%, 90%, or 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. In one embodiment, the modification is a substitution, particularly a conservative substitution.

[0132] As described above, the antibodies of the present invention have excellent endocytosis effects, outperforming the positive control antibody (GSK2857916). Endocytosis, also known as endocytosis or phagocytosis, is the process of transporting extracellular substances into cells through the deformation movement of the plasma membrane. Based on the size of the endocytic substance and the different mechanisms of entry, endocytosis can be divided into three types: phagocytosis, pinocytosis, and receptor-mediated endocytosis. The endocytosis of the antibodies of the present invention refers to receptor-mediated endocytosis. After the monoclonal antibody targeting BCMA binds to BCMA, it mediates the formation of BCMA-antibody complexes into endosomes, which then fuse with lysosomes. Within the lysosomes, the BCMA-antibody complex is degraded by lysosomes, and some BCMA or BCMA-antibody complexes can also be transported back to the cell membrane. When the monoclonal antibody of the present invention is conjugated to a toxin small molecule to form an ADC drug, it is 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 into an ADC drug, the better endocytosis effect is critical for helping to mediate the drug into target cells.

[0133] In addition, the antibodies of the present invention are humanized antibodies. The antibodies before and after humanization of the present invention have substantially the same binding activity to human and monkey BCMA, and the antibodies before and after humanization have substantially the same effect in blocking the binding of BCMA and BAFF. The antibodies before and after humanization of the present invention have substantially the same binding effect to monkey BCMA-CHO cells, and both are superior to the positive control antibody (GSK2857916). The antibodies before and after humanization have substantially the same binding effect to H929 cells, and both are superior to the positive control antibody (GSK2857916). The endocytosis effect of the antibodies before and after humanization on the human myeloma cell line H929 cells is substantially the same, and both are superior to the positive control antibody (GSK2857916).

[0134] "Humanized antibody" refers to a class of engineered antibodies having CDRs derived from non-human donor immunoglobulins, with the remaining immunoglobulin-derived portion of the molecule derived from one (or more) human immunoglobulins. In addition, framework support residues may 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 databases, and Swiss protein databases, based on the homology to the nucleotide and amino acid sequences of donor antibodies. Human antibodies characterized by the homology (based on amino acids) to the framework regions of donor antibodies may be suitable for providing heavy chain constant regions and / or heavy chain variable framework regions for inserting donor CDRs. Suitable acceptor antibodies capable of providing light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains do not need to be derived from the same acceptor antibody.

[0135] 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 for antibody druggability. Poor thermal stability may lead to antibody druggability problems, such as low antibody expression and antibody aggregation.

[0136] In addition, the present invention also relates to a monoclonal antibody targeting B cell maturation antigen (BCMA), which recognizes the same antigenic determinant as the antibody described above. The present invention also relates to a monoclonal antibody targeting B cell maturation antigen (BCMA), which competes with the antibody described above for binding to B cell maturation antigen (BCMA).

[0137] Specifically, the present invention also relates to nucleic acids encoding the antibodies of the present invention. The present invention also relates to an expression vector comprising the nucleic acid of the present invention. The present invention also relates to a host cell comprising the expression vector of the present invention or having the nucleic acid of the present invention integrated into its genome.

[0138] As is known in the art, "polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a nucleotide chain of any length and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate capable of being incorporated into a chain by DNA or RNA polymerase. As used herein, "vector" refers to a construct that is 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. Examples of 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 associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells. In the present invention, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of these cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as the cell screened or selected for in the initially transformed cell are included herein.

[0139] The present invention also relates to a method for producing a monoclonal antibody, which comprises culturing the host cell of the present invention to thereby produce the monoclonal antibody of the present invention.

[0140] The present invention also relates to a pharmaceutical composition comprising the monoclonal antibody of the present invention and a pharmaceutically acceptable carrier. The present invention also relates to a kit or article of manufacture comprising the monoclonal antibody of the present invention or the pharmaceutical composition of the present invention. As used herein, a "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 react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline, water, emulsions (such as oil / water emulsions), and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline (PBS) or normal saline (0.9%). Compositions containing such carriers are prepared by conventional methods known in the art (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. Mack Publishing, 2005).

[0141] The present invention also relates to a method for treating a disease associated with BCMA expression, comprising: administering the monoclonal antibody, the pharmaceutical composition, the kit or the product of the present invention to a subject in need thereof. Specifically, the disease is selected from B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis and POEMS syndrome. Specifically, the disease is multiple myeloma.

[0142] Specifically, the term "multiple myeloma (MM)", also known as plasma cell myeloma or Kahler's disease (after Otto Kahler), is a refractory clonal B cell neoplasm characterized by the accumulation of malignant plasma B cells in close contact with stromal cells in the bone marrow. MM is a progressive disease caused, inter alia, by multiple genetic damage to precursor plasma B cells, i.e., chromosomal translocations, primarily caused by translocations such as t(11;14), t(4;14), t(8;14), or deletions such as del(13) and del(17), which allow the tumor cells to proliferate extensively and become resistant to apoptosis. B lymphocytes originate in the bone marrow and migrate to the lymph nodes. As they develop, B lymphocytes mature and display different proteins on their cell surface. When they become activated to secrete antibodies, they are called plasma cells. Multiple myeloma develops in B cells after they leave parts of the lymph nodes called germinal centers. The immune system keeps B cells' proliferation and antibody secretion under strict control. This control is lost when chromosomes and genes are damaged (usually through rearrangements). Normally, the promoter gene moves (or translocates) to a chromosome, where it stimulates overproduction of the antibody gene.

[0143] As mentioned above, chromosomal translocations between immunoglobulin heavy chain genes (on chromosome 14, locus 14q32) and oncogenes (often 11q13, 4p16.3, 6p21, 16q23, and 20q11

[10] ) are frequently observed in patients with multiple myeloma. This mutation leads to deregulation of the oncogenes, which is considered an important initiating event in the pathogenesis of myeloma. The result is the proliferation of plasma cell clones and genomic instability, leading to further mutations and translocations. Chromosome 14 abnormalities are observed in approximately 50% of all myeloma cases. Deletions of (a portion of) chromosome 13 are also observed in approximately 50% of cases.

[0144] As used herein, a "subject," "individual," or "object" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, racing animals, pets, primates, horses, dogs, cats, mice, and rats. In the present invention, 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 refers to administering an effective amount of the pharmaceutical composition or agent or product, etc. As used herein, the term "effective amount" refers to the amount of the drug or agent that elicits a biological or pharmaceutical response of a tissue, system, animal, or human being, for example, that is sought by a researcher or clinician. In addition, the term "therapeutically effective amount" refers to an amount that causes improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received the amount. The term also includes within its scope an amount that effectively enhances normal physiological function.

[0145] The present invention also relates to the use of the monoclonal antibody of the present invention in the preparation of a medicament for treating a disease associated with BCMA expression. Specifically, the above-mentioned disease is selected from B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis and POEMS syndrome. Specifically, the above-mentioned disease is multiple myeloma. As used herein, the terms "cancer", "neoplastic organism" and "tumor" are used interchangeably and are single or plural, referring to cells that have undergone malignant transformation or caused abnormal or unregulated growth or excessive proliferation of cells. Such changes or malignant transformations can usually make such cells pathogenic to the host organism, and are therefore also intended to include becoming or can become pathogenic and need intervention or can benefit from the initial cancer or precancerous cells of intervention. Primary cancer cells (that is, cells obtained near the site of malignant transformation) can be distinguished from non-cancerous cells by perfecting the technology established, particularly histological examination. As used in the present invention, the definition of cancer cells not only includes primary cancer cells, but also includes any cell derived from cancer cell ancestors (ancestors). This includes the cancer cells and in vitro cultures and cell lines derived from cancer cells that have shifted. When referring to cancer types that typically manifest as solid tumors, "clinically detectable" tumors are those that are detectable on the basis of a tumor mass; for example, by procedures such as CAT scans, MR imaging, X-rays, ultrasound, or palpation, and / or due to the expression of one or more cancer-specific antigens in a sample obtainable from the patient. In other words, the term of the present invention includes cells, neoplasms, cancers, and tumors at any stage, including what clinicians call primary cancers, tumors, in situ growths, and late metastatic growths. The tumor can be a hematopoietic tumor, such as a blood cell tumor, i.e., a liquid tumor. Specific examples of clinical conditions based on such tumors include leukemias such as chronic myeloid leukemia or acute myeloid leukemia; myelomas such as multiple myeloma; lymphomas, etc.

[0146] As described above, compared to the Belantamab mafodotin antibody (abbreviated as GSK2857916), the antibody of the present invention can bind to both human BCMA and monkey BCMA, and its affinity level is close to or even better than that of GSK2857916. The antibody of the present invention is close to or even better than GSK2857916 in blocking the binding of BCMA to its ligands BAFF or APRIL. It blocks the binding of BCMA to BAFF and APRIL, and can inhibit the activation of intracellular signaling pathways (such as the NFκB signaling pathway) caused by ligand binding, thereby inhibiting the survival of plasma cells. The antibody of the present invention has a better endocytosis effect than GSK2857916 in terms of endocytosis effect. Endocytosis effect is a key function of antibody-drug conjugates (ADCs) in mediating cell killing. Antibodies with better endocytosis effect have a better corresponding ADC drug killing effect. The antibodies involved in the present invention have better thermal stability than GSK2857916 in terms of thermal stability. Thermal stability is a key indicator of antibody drugability. Antibodies with better thermal stability may have more advantages in antibody expression and reduce the possibility of producing aggregates. The antibodies involved in the present invention have a lower immunogenicity risk after humanization in terms of immunogenicity. Immunogenicity can provoke an immune response in the human body, on the one hand, producing anti-antibodies and reducing the effect of antibody drugs. On the other hand, strong immunogenicity may bring a greater risk of side effects of drugs. Humanized antibodies, on the other hand, mutate sequences outside the antibody CDR region to make them closer to human antibody sequences, thereby reducing the risk of antibodies producing strong immunogenicity.

[0147] Example

[0148] Example 1

[0149] Preparation of raw materials

[0150] In this example, BCMA antigen and its ligand were purchased from ACRO Biosystems. At the same time, GlaxoSmithKline (GSK)'s positive antibody GSK2857916 was prepared according to the U.S. patent application (US20140105915A). It is worth noting that the prepared positive antibody is not conjugated to a toxin molecule but does not affect other possible functions.

[0151] 1.1 Antigen preparation

[0152] The following four antigens were used in this application: human-BCMA-Fc, human-BCMA-His, cyno-BCMA-Fc, and cyno-BCMA-His, all purchased from ACRO Biosystems with catalog numbers BC7-H5254, BCA-H522y, BCA-C5253, and BCA-C52H7, respectively. Their activity was cross-validated with that of GSK2857916 and was consistent with that disclosed in US patent application US20140105915A. The results are shown in Figure 2(A).

[0153] 1.2 Preparation of ligand

[0154] The ligands used in this application are the following two: human-BAFF-Fc and human-APRIL-Fc, both purchased from ACRO Biosystems, with catalog numbers BAF-H4268 and APRIL-H5267, respectively. In addition, their activity has been verified by ligand binding to human-BCMA, and is consistent with that disclosed in US patent application US20140105915A. The results show that Figure 2(B) and 2(C) middle.

[0155] 1.3 Preparation of positive control antibody

[0156] In this application, the positive control antibody GSK2857916 was expressed using a transient transfection system (ExpiCHO), wherein the main materials used include: Gibco culture medium (Cat. No.: A29100-01), Gibco transfection kit (Cat. No.: A29129). First, the light chain sequence (as shown in the sequence of SEQ ID NO: 17) and the heavy chain sequence (as shown in the sequence of SEQ ID NO.: 18) of GSK2857916 were synthesized according to the sequence disclosed in U.S. patent application US20140105915A, and a plasmid containing the complete GSK2857916 antibody light chain and heavy chain genes was constructed by molecular cloning. The plasmid containing the light chain and heavy chain of the GSK2857916 antibody were mixed in a mass ratio of 2: 1. In a 25 mL expression system, the above plasmid mixture (25 μg) was mixed with the transfection reagent according to the standard process and added dropwise to 25 mL of the ExpiCHO cell expression system. After thorough mixing, express in a 37°C cell culture incubator for 18-22 hours. Subsequently, feed medium was added to the above transfection mixture and continued to be cultured in a 32°C cell culture incubator. On the 5th day after transfection, the second feed was added and the cells were placed in a 32°C cell culture incubator for further culture for 10-12 days. Next, the expressed cell suspension was centrifuged at high speed and the supernatant was taken. The obtained supernatant was filtered through a 0.22μm filter membrane and purified by affinity chromatography using a Protein A / G affinity column. After purification, the target protein was eluted with 100mM glycine salt (pH3.0), concentrated, replaced, and packaged. After passing SDS-PAGE identification and activity identification, it was stored and frozen.

[0157] Example 2

[0158] Preparation of cell lines

[0159] In this example, human myeloma cell line H929 expressing BCMA was purchased, and a cell line BCMA-HEK293 cells overexpressing human BCMA and a cell line BCMA-CHO cells overexpressing monkey BCMA were constructed.

[0160] 2.1 Preparation of H929 cell line

[0161] In this application, H929 was purchased from the Beijing Union Medical College Cell Bank with the catalog number 3111C0001CCC000360, which is a myeloma cell line that naturally overexpresses human BCMA.

[0162] 2.2 Preparation of BCMA-CHO cell lines

[0163] 2.2.1 Construction of plasmid expressing full-length monkey BCMA

[0164] A DNA fragment containing the cynomolgus macaque BCMA protein was synthesized using gene synthesis and cloned into an expression vector. This fragment was then transfected into E. coli, and a single E. coli clone was isolated and sequenced to obtain the correct plasmid clone. The plasmid was then extracted and resequenced for confirmation.

[0165] 2.2.2 Electroporation

[0166] CHO-S cells were cultured using Gibco's CD-CHO serum-free medium (Cat. No. 10743029). The day before electroporation, cells were passaged to 5 × 10 6 / mL, and the next day, the constructed plasmid was introduced into CHO-S cells using an Invitrogen electroporation kit (Cat. No. MPK10096) and electroporator (Cat. No. MP922947). The electroporated cells were transferred to CD-CHO medium and cultured in a 37°C cell culture incubator for 48 hours.

[0167] 2.2.3 Cell plating after electroporation

[0168] The electrotransfected CHO-s cells were plated into a 96-well plate at 2000 cells / well, and MSX (Millipore, GSS-1015-F) and GS supplement (Sigma, 58672C-100 ml) were added at a final concentration of 30 μM. The plates were then cultured in a 37°C carbon dioxide incubator. After 10 days, culture medium containing 30 μM MSX and 1× GS supplement was added.

[0169] 2.2.4 Clone selection, cell expansion, and FACS identification

[0170] Single cell clones grown in 96-well plates were picked and transferred to 24-well culture plates for further expansion and culture. FACS was then used to identify cell lines that had successfully transfected with monkey BCMA.

[0171] 2.3 Preparation of BCMA-HEK293 cell line

[0172] 2.3.1 Construction of plasmid expressing full-length human BCMA

[0173] A DNA fragment containing the human BCMA protein was synthesized using gene synthesis technology and cloned into an expression vector. This fragment was then introduced into E. coli via chemical transformation. A single E. coli clone was selected and sequenced to obtain the correct plasmid clone. The plasmid was then extracted and sequenced again for confirmation.

[0174] 2.3.2 Electroporation

[0175] HEK293 cells were cultured using Gibco's DMEM serum-free medium (Cat. No. 12634010). The day before electroporation, cells were passaged to 2 × 10 5 / mL, and the next day, the constructed plasmid was introduced into HEK293 cells using an Invitrogen electroporation kit (Cat. No.: MPK10096) and electroporator (Cat. No.: MP922947). The electroporated cells were transferred to DMEM medium and cultured in a 37°C cell culture incubator for 48 hours.

[0176] 2.3.3 Cell plating after electroporation

[0177] The electrotransfected HEK293 cells were plated into a 96-well plate at 1000 cells / well, puromycin was added at a final concentration of 2 μg / mL, and the cells were cultured in a 37°C carbon dioxide incubator. After 14 days, the culture medium was supplemented with 2 μg / mL puromycin.

[0178] 2.3.4 Clone selection, cell expansion, and FACS identification

[0179] Single cell clones grown in 96-well plates were picked and transferred to 24-well culture plates for further expansion and culture. Cell lines that had successfully transfected with human BCMA were then identified by FACS.

[0180] Example 3

[0181] Animal Immunization

[0182] In this embodiment, animal immunization includes several schemes and is carried out simultaneously. During the immunization process, mouse serum is collected for titer detection and evaluation of the immunization effect. Finally, the mouse number for library construction is determined based on the serum titer.

[0183] 3.1 Animal immunization

[0184] 3.1.1. Immunization plan 1

[0185] Babl / c mice (Shanghai Lingchang Biotechnology Co., Ltd., female, 6-8 weeks old, n=2) were immunized subcutaneously and intraperitoneally with the aforementioned human BCMA-Fc and monkey BCMA-Fc antigens (ACROBiosystems). Cross-immunization was performed. The initial dose was 100 μg / mouse in complete Freund's adjuvant (CFA). Subsequently, the dose was reduced to 50 μg / mouse in incomplete Freund's adjuvant (IFA), with cross-immunizations repeated every two weeks. The corresponding mice are numbered Mouse 1 and Mouse 2.

[0186] 3.1.2 Immunization Plan II

[0187] Babl / c mice (from the same batch as in Protocol 1, n=2) were immunized subcutaneously and intraperitoneally with the aforementioned human BCMA-Fc and monkey BCMA-Fc antigen proteins (ACRO Biosystems), HEK293 cells that overexpress human BCMA, and CHO cells that overexpress monkey BCMA, using a cross-immunization approach. The protein immunization dose was 20 μg / mouse, and the cell immunization dose was 1×10 7 Each mouse was cross-immunized intraperitoneally for weekly immunization. The corresponding mice were numbered Mouse 3 and Mouse 4.

[0188] 3.2 Serum titer ELISA test

[0189] 3.2.1 Antigen coating and blocking

[0190] ELISA plates were coated with human BCMA and monkey BCMA one day in advance at a concentration of 2 μg / mL, 30 μL / well, overnight at 4°C. On the day of the immunoassay, the plates were washed three times with PBST, blocked with 5% skim milk at room temperature for two hours, and then washed three more times with PBST.

[0191] 3.2.2 Primary and secondary antibodies

[0192] Each serum sample was diluted 500-fold based on the original solution, and then diluted in a 3-fold gradient. It was added to the ELISA plate as the primary antibody and incubated at room temperature for 1 hour. After washing the plate three times with PBST, the secondary antibody (Goat-anti-mouse-IgG-Fab-HRP, Sigma, M4115) was added and incubated at room temperature for 1 hour.

[0193] 3.2.3 Color development, termination, and plate reading

[0194] After incubation, the plates were washed six times with PBST and developed with TMB (SurModics, TMBS-1000-01). Based on the color development results, the reaction was terminated by adding 2M HCl, and the plate was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190). Serum titer results showed that the immunized mice had high titers against both human and monkey BCMA, making them suitable for the next step of antibody library construction. The results are shown in Tables 1-1 and 1-2 below.

[0195] Table 1-1 Detection of mouse serum titer using human BCMA

[0196]

[0197] Table 1-2 Detection of mouse serum titer using monkey BCMA

[0198]

[0199] Example 4

[0200] In this example, the mice with the highest titers from Example 3 were selected, and phage display technology was used to clone antibody genes from B cells in the mouse spleen into a phage display vector to construct an antibody library. Using human BCMA and monkey BCMA as screening antigens, a process of library screening, monoclonal primary screening, and sequencing ultimately yielded 17 antibody molecules with binding activity against both human and monkey BCMA antigens.

[0201] 4.1 Construction of phage display antibody gene library

[0202] After immunization, mice were euthanized according to standard procedures. The spleen of the mice was collected, ground and filtered, and spleen cells were collected and 1 mL of TRIzol was added. TM Reagent (Thermo Fisher, 15596026) was used to lyse splenocytes, and total RNA was extracted by the phenol-chloroform method. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 6210A). Subsequently, the cDNA was used as a PCR template, and specific primers for mouse antibody sequence amplification were used to amplify the light chain and heavy chain genes of the antibody, respectively. Finally, the antibody gene fragment was inserted into the phage display vector by double digestion with NcoI+NotI and ligation with T4 ligase. The ligation product was recovered using a DNA recovery kit (Omega, D6492-02) and finally transformed into competent Escherichia coli SS320 (Lucigen, MC1061 F) by electroporation (Bio-Rad, MicroPulser) and coated on a 2-YT (C+ / K+2-YT) solid plate containing ampicillin and tetracycline to amplify the SS320 bacteria of the correctly transformed antibody plasmid, and finally a library containing Fab segment antibody sequences was constructed.

[0203] 4.2 Phage display antibody gene library screening

[0204] 4.2.1 Screening of phage-displayed antibody gene libraries using magnetic beads

[0205] Magnetic bead screening is based on combining biotin-labeled antigen protein with avidin-coupled magnetic beads, incubating the antigen-bound magnetic beads with the library, washing, and eluting the magnetic beads, and undergoing 2-4 rounds of screening to ultimately enrich specific monoclonal antibodies against the antigen. In this example, the principle of cross-screening of biotin-labeled human BCMA antigen and monkey BCMA antigen was used, with human BCMA screening used in the first and third rounds and monkey BCMA screening used in the second round. A total of 3 rounds of screening were performed, and the enriched antibody sequence mixture was then subjected to primary screening of human and monkey BCMA monoclonal antibodies.

[0206] The specific implementation methods are as follows:

[0207] First, human BCMA protein labeled with Biotin was incubated with Avidin-coupled magnetic beads to allow binding of the human BCMA protein to the beads. The BCMA antigen-bound magnetic beads and the constructed phage library were incubated at room temperature for 2 hours. After washing 6-8 times with PBST, nonspecifically adsorbed phage were removed. Trypsin (Gibco, 25200072) was added, gently mixed, and reacted for 20 minutes to elute the specifically bound antibody-displaying phage. Subsequently, the eluted phage was infected with logarithmic-phase SS320 bacteria (Lucigen, MC1061 F) and allowed to stand for 30 minutes. The cells were then incubated at 220 rpm for 1 hour. VSCM13 helper phage was then added and allowed to stand for 30 minutes. The cells were further incubated at 220 rpm for 1 hour, centrifuged, and exchanged into C+ / K+2-YT medium. The resulting phage was then used for the next round of screening.

[0208] 4.4.2 Screening of phage-displayed antibody gene libraries using the immunotube method

[0209] The principle of immune tube screening is to coat the BCMA protein on the surface of an immune tube with high adsorption capacity, and then add the phage display antibody library to the immune tube and incubate with the antigen protein adsorbed on the surface of the immune tube, wash and elute the sea selection process, go through 2-4 rounds of sea selection, and finally enrich the specific monoclonal antibodies for the antigen. The purpose of the immune tube method and the magnetic bead method is to enrich specific antibodies for the antigen, which are two complementary experimental methods. In this embodiment, the principle of cross-screening of human BCMA antigen and monkey BCMA antigen is adopted, wherein the first and third rounds adopt human BCMA sea selection, and the second round adopts monkey BCMA sea selection, for a total of 3 rounds of sea selection, and then the enriched antibody sequence mixture is subjected to monoclonal primary screening of human and monkey BCMA. The specific implementation method is similar to the magnetic bead method screening.

[0210] 4.5 Selection of Monoclonal Cells

[0211] After three rounds of screening, a subset of single clones from the third round pool were selected for ELISA testing, including binding to both human and monkey BCMA. Ultimately, 93 positive clones were identified from 1,344 clones that bound to both human and monkey BCMA. After sequencing analysis and affinity ranking for both human and monkey BCMA, 17 clones were selected for full-length sequence reconstruction for further experiments.

[0212] Example 5

[0213] Full-length antibody construction, expression and purification

[0214] In this example, the 17 human-monkey cross-Fab antibodies obtained in Example 4 were constructed into human IgG1 type, wherein the light chains were all Kappa, and the antibody type was human-mouse chimeric antibody.

[0215] 5.1 Plasmid construction

[0216] The heavy chain sequence of the screened sequence was fused with the human IgG1 Fc segment, and the light chain was fused with the human Kappa constant region. The plasmids of the heavy and light chains were transformed into ExpiCHO cells, induced to express, and the full-length antibody was obtained.

[0217] 5.2 Antibody Expression and Purification

[0218] In this application, the antibody uses the ExpiCHO transient expression system, the culture medium is (Gibco, A29100-01), and the transfection kit is (Gibco, A29129). The specific method is as follows: the ExpiCHO cells are passaged the day before transfection, and in a 25mL system, 25μg of the constructed plasmid is mixed with the transfection reagent and then added dropwise to 25mL ExpiCHO cells. After thorough mixing, it is expressed in a 37°C cell culture incubator for 18-22h. Subsequently, feed medium is added to the above transfection mixture and placed in a 32°C cell culture incubator for continued culture. On the 5th day after transfection, the second feed is added, and the cells are placed in a 32°C cell culture incubator for continued culture for 10-12 days. Then, the expressed cell suspension is centrifuged at high speed and the supernatant is taken. The obtained supernatant is filtered through a 0.22μm filter membrane and purified by affinity chromatography using a Protein A / G affinity column. After purification, the target protein was eluted with 100 mM glycine salt (pH 3.0), concentrated, replaced, and packaged. After SDS-PAGE identification, SEC purity detection, and activity identification, it was stored and frozen.

[0219] Example 6

[0220] ELISA level affinity blocking effect detection of candidate antibodies

[0221] In this example, the affinity of the candidate antibody to human and monkey BCMA was verified by an ELISA-based method, and the effect of the candidate antibody in blocking the binding of BCMA and BAFF or APRIL was verified by an ELISA-based method.

[0222] 6.1 ELISA-based detection of the affinity of candidate antibodies for human and monkey BCMA

[0223] On a 96-well ELISA plate, human BCMA and monkey BCMA were coated respectively, 2μg / mL, 30μL / well, at 4 degrees overnight. The next day, the well plate was washed 3 times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate 3 times with PBST, gradient dilutions of the candidate antibody and the positive control antibody (GSK2857916) were added and incubated for 1 hour. After that, the secondary antibody (anti-human-IgG-Kappa-HRP, abcam, ab79115) was added and incubated for 1 hour after washing 3 times with PBST. After incubation, the plate was washed six times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the plate was read at OD450 using an enzyme reader (Molecular Devices, SpecterMax 190), and the results were displayed. Figure 4(A) to Figure 4(B) The results showed that the candidate antibody has an affinity for human BCMA and monkey BCMA that is close to or even better than that of the GSK2857916 antibody.

[0224] 6.2 ELISA-based detection of candidate antibodies blocking the binding of BCMA and BAFF

[0225] In this embodiment, the blocking system includes a preliminary development process, and based on the development results, the parameters of the blocking system that are actually used and have both sensitivity and stability are determined.

[0226] For BAFF blocking, human BAFF protein (1 μg / mL, 30 μL / well) was coated overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with 5% skim milk for 2 hours. The candidate antibody or positive control antibody (GSK2857916) was then serially diluted and premixed with biotin-labeled human BCMA (0.6 μg / mL) for 0.5 hours. After blocking and washing, the plates were added to a 96-well ELISA plate and incubated for 1 hour. After washing three times with PBST, the secondary antibody (NeutrAvidin-HRP, Therofisher, 31001) was added and incubated for 1 hour. After incubation, the plate was washed six times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. Based on the color development results, 2M HCl was added to terminate the reaction. The plate was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190). The results are shown in Figure 5(A). The results show that the candidate antibody has an effect of blocking the binding of human BCMA and BAFF that is close to or even better than that of the GSK2857916 antibody.

[0227] 6.3 ELISA-based detection of candidate antibodies blocking the binding of BCMA and APRIL

[0228] In this embodiment, the blocking system includes a preliminary development process, and based on the development results, the parameters of the blocking system that are actually used and have both sensitivity and stability are determined.

[0229] For APRIL blocking, human APRIL protein was coated at 2 μg / mL, 30 μL / well, at 4°C overnight. The next day, the wells were washed three times with PBST and blocked with 5% skim milk for 1 hour. The candidate antibody or positive control antibody (GSK2857916) was then serially diluted and premixed with Biotin-labeled human BCMA (0.6 μg / mL) for half an hour. After blocking and washing, the wells were added to a 96-well flat-bottom plate and incubated for 1 hour. After washing three times with PBST, the secondary antibody NeutrAvidin-HRP (Therofisher, 31001) was added and incubated for 1 hour. After incubation, the plate was washed six times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. Based on the color development results, 2M HCl was added to terminate the reaction. The plate was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190). The results are shown in Figure 5(B). The results show that the candidate antibody has an effect of blocking the binding of human BCMA and APRIL that is close to or even better than that of the GSK2857916 antibody.

[0230] Example 7

[0231] FACS analysis of candidate antibodies and affinity testing of human and monkey BCMA-expressing cell lines

[0232] In this example, the affinity of the candidate antibodies to cells expressing human and monkey BCMA was verified based on the FACS method.

[0233] 7.1 FACS-based detection of the affinity of candidate antibodies for human BCMA-HEK293 cells

[0234] Human BCMA-HEK293 cells in the exponential growth phase were collected and centrifuged at 300 g to remove the supernatant. The cells were resuspended in the prepared FACS buffer, counted, and the cell suspension density was adjusted to 2 × 10 6 / mL. BCMA-HEK293 cells were then added to a 96-well round-bottom plate at 100 μL per well and centrifuged at 300 g to remove the supernatant. Serial dilutions of candidate antibodies and positive control antibody were added to the corresponding wells, the cells were evenly dispersed with a pipette, and the cells were incubated at 4°C for 30 min. The incubated cell mixture was centrifuged at 300 g to remove the supernatant. 200 μL of FACS buffer was added to the corresponding wells and the cells were resuspended with a pipette. This was repeated twice, and the supernatant was removed by centrifugation at 300 g. A PE-labeled anti-human IgG-Fc flow cytometry antibody (Abcam, 98596) was added, the cells were evenly dispersed with a pipette, and the cells were incubated at 4°C for 30 min. The supernatant was removed by centrifugation at 300 g to remove the supernatant. FACS buffer was then added and the cells were resuspended. This was repeated twice, and 200 μL of FACS buffer was added to each well and the cells were resuspended. Finally, the cells were analyzed by flow cytometry (Beckman, CytoFLEX AOO-1-1102). In this example, the results are shown in FIG3(A), which show that the affinity of the antibody SY14-3rd-5-6-7 for human BCMA-HEK293 cells is superior to that of the positive control antibody (GSK2857916).

[0235] 7.2 FACS-based detection of the affinity of candidate antibodies for monkey BCMA-CHO cells

[0236] In this example, the procedure was identical to that in 7.1, except that monkey BCMA-CHO cells were used. The results are shown in Figure 3(B), demonstrating that the antibody SY14-3rd-5-6-7 has superior affinity for monkey BCMA-CHO cells to the positive control antibody (GSK2857916). The signal difference between humans and monkeys is hypothesized to be due to differences in BCMA expression levels in BCMA-HEK293 cells and BCMA-CHO cells.

[0237] Example 8

[0238] Internalization effect detection of candidate antibodies

[0239] In this example, a method system for detecting the endocytosis of antibodies in the human myeloma cell line H929 was developed and the endocytosis effect of candidate antibodies was detected accordingly. The basic principle is to detect the endocytosis effect of antibodies through cytotoxicity. Fab-ZAP (Atsbio, IT-51-100) is a Fab fragment connected to saporin. Saporin is a ribosome inhibitor that can inhibit protein synthesis and cause cell death. In this example, Fab-ZAP is a Fab fragment that can bind to the Fc of human antibodies. After co-incubation of Fab-ZAP and anti-BCMA antibodies, the antibodies are charged with toxins. When the anti-BCMA antibodies are internalized by H929 cells, the toxins enter the cells along with the antibodies and cause cell death. Therefore, the endocytosis effect of the antibodies can be detected by detecting cell activity using the MTS kit (Promega, G3580).

[0240] The specific detection method is as follows: H929 cells were revived one week in advance and passaged every 3 days after resuscitation. The cell seeding density was 2×10 5 / mL, and the cell passage should not exceed 3 weeks. Pipette cells in the logarithmic growth phase, mix the cells thoroughly, count and determine their viability. Take a 96-well flat-bottom plate and adjust the cell density to 4×10 5 / mL, add 50μL of cells to each well, gently tap to mix, and incubate the cell culture plate in a 37°C cell culture incubator for 16 hours. Then, prepare the Fab-ZAP solution to a 27nM (2.16μg / ml) dilution in 1640 complete medium with 10% FBS, 1% PS, and 50μM β-mercaptoethanol. Add this to the serially diluted antibody solution to achieve a final Fab-ZAP concentration of 13.5nM (1.08μg / ml). Using a 300μL dispenser, dispense 50μL of the dilution solution from the dilution plate into the cell plate according to the designed layout. Gently tap to mix, and incubate in a cell culture incubator for 3 days. Afterwards, melt MTS at room temperature in advance, remove the cell plate from the incubator, use a 100μL 12-channel pipette to take 20μL of MTS and add it to each well. After gently tapping, place it in the incubator and incubate for 2 hours. Finally, place the cell plate in a microplate reader, read the reading at a wavelength of 492nm, and save it.

[0241] In this embodiment, the results are shown in Figure 6(A) and 6(B) The results showed that the candidate antibodies SY14-3rd-5-6-7 and SY14-3rd-5-6-32 had better endocytosis effects than the positive control antibody (GSK2857916).

[0242] At this point, all the test results of candidate antibodies are summarized in Figure 7 middle.

[0243] Example 9

[0244] Antibody humanization

[0245] In this example, amino acid point mutations were performed on the framework of the heavy and light chain V regions of the murine antibody to make it closer to the human germline. The engineered 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). The preferred candidate antibodies after the engineering are designated 5-6-7-hu-2 and 5-6-32-hu-2, respectively.

[0246] Example 10

[0247] Functional validation of humanized antibodies

[0248] 10.1 ELISA-based testing of the affinity of two candidate antibodies for human and monkey BCMA before and after humanization

[0249] The specific operation method is shown in Example 6. The results are shown in Figures 8(A), 8(B), 9(A) and 9(B). The results show that the binding activity of the antibody before and after humanization to human and monkey BCMA is basically the same.

[0250] 10.2 ELISA-based testing of the blocking effects of two candidate antibodies on BCMA and BAFF before and after humanization

[0251] The specific operation is shown in Example 6. Figure 10(A) and 11(A) The results showed that the humanized antibody was slightly less effective than the positive antibody in blocking the binding of BCMA and BAFF.

[0252] 10.3 ELISA-based testing of the BCMA and APRIL blocking effects of two candidate antibodies before and after humanization

[0253] For specific operations, see Example 6. The results are shown in 10(B) and 11(B). The results show that the effect of the humanized antibody in blocking the binding of BCMA and APRIL is slightly worse than that of the positive antibody.

[0254] 10.4 FACS-based detection of the affinity of candidate antibodies for human BCMA-HEK293 cells

[0255] For specific operations, see Example 7. The results are shown in Figure 12 (A). The results show that the binding effects of the humanized antibody and human BCMA-HEK293 cells are basically the same, and both are better than the positive control antibody (GSK2857916).

[0256] 10.5 FACS-based detection of the affinity of candidate antibodies for monkey BCMA-CHO cells

[0257] For specific operations, see Example 7. The results are shown in Figure 12(B). The results show that the binding effects of the humanized antibody and monkey BCMA-CHO cells are basically the same, and both are better than the positive control antibody (GSK2857916).

[0258] 10.6 FACS-based Detection of the Affinity of Candidate Antibodies to the Human Myeloma Cell Line H929

[0259] For specific operations, see Example 7. The results are shown in Figure 12 (C). The results show that the binding effects of the humanized antibody and H929 cells are basically the same before and after humanization, and both are better than the positive control antibody (GSK2857916).

[0260] 10.7 Detection of endocytic effects of antibodies before and after humanization

[0261] In this example, the endocytosis of two candidate antibodies before and after humanization was also tested in the human myeloma cell line H929. For details, see Example 8. The results are shown in Figures 13-A and 13-B. The results show that the endocytosis of the humanized antibody 5-6-7-huV2 was slightly improved compared to the pre-humanized antibody (5-6-7-WT). The endocytosis of the other humanized antibodies before and after humanization was essentially the same in the human myeloma cell line H929, and all were superior to the positive control antibody (GSK2857916).

[0262] At this point, all the test results of the two candidate antibodies before and after humanization transformation are summarized in Figure 14 middle.

[0263] Example 11

[0264] DSF testing of candidate antibodies before and after humanization

[0265] In this example, the thermal stability data of two candidate antibodies before and after humanization and a positive control antibody (GSK2857916) were tested. The specific process is as follows: prepare an antibody solution of 0.25 mg / mL, 19 μL / well, set up three parallel wells for each test sample, and use PBS and IPI as references. Then add 1 μL of 100× SYPRO orange dye to each well and prepare for the machine. The test was performed using an ABI 7500FAST RT-PCR instrument. The test type selected was melting curve, and the continuous mode was used. The scanning temperature range was 25-95°C, the heating rate was 1%, and the temperature was balanced at 25°C for 5 minutes. Data was collected during the heating process. The reporter group selected ROX, the quencher group selected None, the reaction volume was 20 μL, and the temperature corresponding to the first peak and valley of the first derivative of the melting curve was determined as the denaturation temperature of the candidate antibody. The results are detailed in Tables 1-3.

[0266] Table 1-3 Humanized antibody thermal stability test results

[0267]

[0268] Example 12

[0269] Affinity testing of candidate antibodies before and after humanization

[0270] In this example, the affinity of the candidate antibodies 5-6-7 and 5-6-32 to human BCMA and monkey BCMA was detected using the Fortebio Octet RED96 instrument before and after humanization.

[0271] 12.1 Material Preparation

[0272] Weigh 1g of BSA, add 500μL of Tween 20, and mix thoroughly in 1000mL of 1x PBS. Filter and aliquot for storage. Pipette 0.1mL of 0.1M glycine solution (pH 2.0) and add 0.9mL of ultrapure water. Mix thoroughly. Dilute the antibody to 10μg / mL in KB buffer. Dilute the antigen to a concentration series of 200, 50, 12.5, and 0nM in KB buffer.

[0273] 12.2 Experimental Procedure

[0274] Pre-wet the sensor (Anti-Human Fba-CH1 2nd Generation, FAB2G) in the dark for at least 10 minutes before testing the sample plate (GreinierBio, PN 655209). Once the test is complete, proceed according to the pre-set protocol. For sample plate 1, add 200 μL / well of KB buffer to columns 1, 10, and 12, 0.01 M glycine (pH 2.0) to column 11, and the prepared sample solution to columns 2-8 (one sample per four wells, i.e., two samples per column). Add human BCMA-Fc to column 9 in descending order of concentration: 200 nM antigen solution to wells 1 and 5, 50 nM antigen solution to wells 2 and 6, 12.5 nM antigen solution to wells 3 and 7, and 0 nM antigen solution to wells 4 and 8. Sample plate 2 was prepared as usual, except that the antigen in column 9 was replaced with monkey BCMA protein. The data results are detailed in Tables 1-4.

[0275] Table 1-4 Affinity test results of humanized antibodies to human and monkey BCMA

[0276]

[0277] Example 13

[0278] Based on the above examples, 5-6-7-hu-2 and 5-6-32-hu-2 were selected, analyzed, and sequenced. The variable regions of human antibody sequences were defined using the IMGT database (http: / / www.imgt.org / ), and the sequences of the light and heavy chain variable regions of the antibodies of the present invention (SEQ ID NOs: 13-16) were determined. The variable region sequences were analyzed, and the complementarity determining region sequences of the heavy and light chains of the antibodies (SEQ ID NOs: 1-12) were determined using the AbM CDR definition method.

[0279] The sequences protected by the present invention are as follows:

[0280] SEQ ID NO: 1

[0281] GHIFTNFHFH

[0282] SEQ ID NO:2

[0283] GYIFTNYHMH

[0284] SEQ ID NO:3

[0285] GIYPGNGDTF

[0286] SEQ ID NO:4

[0287] <h2 style=";text-align:left;direction:ltr">GIYPGNGDIF<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0288] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0289] <h2 style=";text-align:left;direction:ltr"> GSYYGYIDAMDY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0290] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0291] <h2 style=";text-align:left;direction:ltr"> GSYYGYIDAMDY<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0292] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0293] <h2 style=";text-align:left;direction:ltr"> RASQDISNYLN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0294] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0295] <h2 style=";text-align:left;direction:ltr"> RASQDISNDLN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0296] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0297] <h2 style=";text-align:left;direction:ltr"> YTSRLHS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0298] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0299] <h2 style=";text-align:left;direction:ltr"> YTSRLPS<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0300] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0301] <h2 style=";text-align:left;direction:ltr"> QQGNTLPWT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0302] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:12<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0303] <h2 style=";text-align:left;direction:ltr"> QQGHTLPWT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0304] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:13<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0305] <h2 style=";text-align:left;direction:ltr"> DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGQGTKLEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0306] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:14<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0307] <h2 style=";text-align:left;direction:ltr"> DIQMTQSPSSLSSASVGDRVTITCRASQDISNDLNWYQQKPGKAPKLLIYYTSRLPSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGHTLPWTFGQGTKLEIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0308] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0309] QVQLVQSGAEVKKPGSSVKISCKASGHIFTNFHFHWVRQAPGQGLEWIGGIYPGNGDTFYNQKFQGRATITADKSTSTAYMELSSLRSEDTAVYYCVRGSYYGYIDAMDYWGQGTSVTVSS

[0310] SEQ ID NO:16

[0311] QVQLVQSGAEVKKPGASVKMSCKASGYIFTNYHMHWVRQAPGQGLEWIGGIYPGNGDIFYAQKFQGRATITADKSTSTAYIELSSMRSEDTAVYYCARGSYYGYIDAMDYWGQGTSVTVSS

[0312] SEQ ID NO: 17

[0313] DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIK

[0314] SEQ ID NO: 18

[0315] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS

[0316] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. Sequence Listing <110> Suzhou Qinwei Pharmaceutical Co., Ltd. <120> Humanized monoclonal antibody targeting BCMA with human-monkey cross-talk <130> TPD01188 <141> 2020-09-25 <150> 201910960313.4 <151> 2019-10-10 <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <400> 1 Gly His Ile Phe Thr Asn Phe His Phe His 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <400> 2 Gly Tyr Ile Phe Thr Asn Tyr His Met His 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <400> 3 Gly Ile Tyr Pro Gly Asn Gly Asp Thr Phe 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <400> 4 Gly Ile Tyr Pro Gly Asn Gly Asp Ile Phe 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <400> 5 Gly Ser Tyr Tyr Gly Tyr Ile Asp Ala Met Asp Tyr 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <400> 6 Gly Ser Tyr Tyr Gly Tyr Ile Asp Ala Met Asp Tyr 1 5 10 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <400> 7 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <400> 8 Arg Ala Ser Gln Asp Ile Ser Asn Asp Leu Asn 1 5 10 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <400> 9 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <400> 10 Tyr Thr Ser Arg Leu Pro Ser 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <400> 11 Gln Gln Gly Asn Thr Leu Pro Trp Thr 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12 Gln Gln Gly His Thr Leu Pro Trp Thr 1 5 <210> 13 <211> 107 <212> PRT <213> Artificial Sequence <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Asp 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 15 <211> 121 <212> PRT <213> Artificial Sequence <400> 15 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly His Ile Phe Thr Asn Phe 20 25 30 His Phe His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Tyr Pro Gly Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Gly Ser Tyr Tyr Gly Tyr Ile Asp Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 16 <211> 121 <212> PRT <213> Artificial Sequence <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr 20 25 30 His Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Tyr Pro Gly Asn Gly Asp Ile Phe Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Ile Glu Leu Ser Ser Met Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Gly Tyr Ile Asp Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 17 <211> 107 <212> PRT <213> Artificial Sequence <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Asn Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Arg Lys Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 18 <211> 121 <212> PRT <213> Artificial Sequence <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Thr Tyr Arg Gly His Ser Asp Thr Tyr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Ile Tyr Asp Gly Tyr Asp Val Leu Asp Asn Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120

Claims

1. An isolated monoclonal antibody targeting B cell maturation antigen (BCMA), wherein: The antibody can bind to both human BCMA and monkey BCMA, and The heavy chain variable region of the antibody comprises a heavy chain complementary determining region 1 (CDR-H1) shown in SEQ ID NO: 1, a heavy chain complementary determining region 2 (CDR-H2) shown in SEQ ID NO: 3, and a heavy chain complementary determining region 3 (CDR-H3) shown in SEQ ID NO: 5, and the light chain variable region of the antibody comprises a light chain complementary determining region 1 (CDR-L1) shown in SEQ ID NO: 7, a light chain complementary determining region 2 (CDR-L2) shown in SEQ ID NO: 9, and a light chain complementary determining region 3 (CDR-L3) shown in SEQ ID NO:

11.

2. The antibody according to claim 1, characterized in that The light chain variable region of the antibody has the sequence shown in SEQ ID NO: 13, and the heavy chain variable region of the antibody has the sequence shown in SEQ ID NO:

15.

3. A nucleic acid encoding the antibody according to claim 1 or 2. An expression vector comprising the nucleic acid according to claim 3 . A host cell comprising the expression vector according to claim 4 or the nucleic acid according to claim 3 integrated into its genome. 6 . A method for producing a monoclonal antibody, comprising culturing the host cell according to claim 5 to thereby produce the monoclonal antibody according to claim 1 or 2.

7. A pharmaceutical composition comprising the monoclonal antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier.

8. A pharmaceutical kit or article of manufacture comprising the monoclonal antibody according to claim 1 or 2 or the pharmaceutical composition according to claim 7.

9. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a medicament for treating a disease associated with BCMA expression, wherein: The disease is selected from MALT lymphoma, multiple myeloma and non-Hodgkin's lymphoma.

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