BCMA-targeted humanized monoclonal antibodies with human monkey crossover

文档序号：795811 发布日期：2021-04-13 浏览：4次中文

阅读说明：本技术 靶向bcma的具有人猴交叉的人源化单克隆抗体 (BCMA-targeted humanized monoclonal antibodies with human monkey crossover ) 是由李宁曹国庆郎国竣刘婵娟胡宇豪于 2020-09-25 设计创作，主要内容包括：本发明涉及一种分离的靶向B细胞成熟抗原(BCMA)的单克隆抗体,其中,所述抗体既能结合人BCMA也能结合猴BCMA。本发明还涉及编码该抗体的核酸,以及一种生产单克隆抗体的方法,以及包含本发明抗体的药物组合物。(The present invention relates to an isolated monoclonal antibody targeting B Cell Maturation Antigen (BCMA), wherein the antibody binds both human BCMA and monkey BCMA. The invention also relates to nucleic acids encoding the antibodies, as well as a method for producing monoclonal antibodies, and pharmaceutical compositions comprising the antibodies of the invention.)

1. An isolated monoclonal antibody that targets B Cell Maturation Antigen (BCMA), wherein the antibody binds both human BCMA and monkey BCMA.

2. The monoclonal antibody of claim 1, selected from any one of the following:

(1) an antibody comprising a heavy chain variable region comprising heavy chain complementarity determining region 1(CDR-H1) shown in SEQ ID NO:1 or 2, and/or comprising heavy chain complementarity determining region 2(CDR-H2) shown in SEQ ID NO:3 or 4, and/or comprising heavy chain complementarity determining region 3(CDR-H3) shown in SEQ ID NO:5 or 6;

(2) an antibody comprising a light chain variable region comprising light chain complementarity determining region 1(CDR-L1) shown in SEQ ID NO:7 or 8, and/or comprising light chain complementarity determining region 2(CDR-L2) shown in SEQ ID NO:9 or 10, and/or comprising light chain complementarity determining region 3(CDR-L3) shown in SEQ ID NO:11 or 12;

(3) an antibody comprising (1) the heavy chain variable region of said antibody and (2) the light chain variable region of said antibody.

3. The antibody of claim 1 or 2, wherein the antibody is selected from any one of:

(1) an antibody comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO 13 or 14, or a variant of the foregoing;

(2) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO 15 or 16, or a variant thereof;

(3) an antibody comprising (1) the heavy chain variable region of said antibody and (2) the light chain variable region of said antibody.

4. The antibody of any one of claims 1 to 3,

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, or

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.

5. The antibody of any one of claims 1 to 3,

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, or

The variable region of the light chain 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.

6. The antibody of any one of claims 1 to 3, wherein the light chain variable region of said antibody has the sequence shown in SEQ ID NO 13 or 14.

7. The antibody of any one of claims 1 to 3, wherein the heavy chain variable region of said antibody has the sequence shown in SEQ ID NO. 15 or 16.

8. A nucleic acid encoding the antibody of any one of claims 1-6.

9. An expression vector comprising the nucleic acid of claim 8.

10. A host cell comprising the expression vector or genome of claim 9 having the nucleic acid of claim 9 integrated therein.

11. A method of producing a monoclonal antibody, the method comprising culturing the host cell of claim 10 so that the monoclonal antibody of any one of claims 1-7 is produced.

12. A pharmaceutical composition comprising a monoclonal antibody according to any one of claims 1-7 and a pharmaceutically acceptable carrier.

13. A kit or article of manufacture comprising a monoclonal antibody according to any one of claims 1-7 or a pharmaceutical composition according to claim 12.

14. Use of a monoclonal antibody according to any one of claims 1-7 in the preparation of a medicament for the treatment of a disease associated with expression of BCMA,

preferably the disease is selected from the group consisting of B cell acute lymphocytic leukemia, T cell acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis and POEMS syndrome, further preferably, the disease is multiple myeloma.

Technical Field

The present invention relates to monoclonal antibodies and fragments thereof that specifically bind B Cell Maturation Antigen (BCMA), including both human BCMA and monkey BCMA antigens.

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.

The present invention also relates to monoclonal antibodies and fragments thereof that specifically bind BCMA and have excellent endocytic effects.

Background

B cells mature in the bone marrow and become plasma cells, capable of secreting antibodies against foreign viruses or bacteria. When plasma cells become myeloma cells, more malignant myeloma cells proliferate and secrete a large amount of useless antibodies. Myeloma usually grows in the spine, skull, pelvis, thorax, etc., and is characterized by a tumor or osteolytic lesion. Myeloma Disease is usually progressive, ranging from Monoclonal Gammoproteinemia (MGUS) of unknown significance, to low-risk smoldering multiple myeloma, to high-risk Smoldering Multiple Myeloma (SMM), and finally to multiple myeloma (Nature Reviews Disease cameras, 2017,3,17046.). The main characteristics of Multiple Myeloma (MM) include hypercalcemia, reduced renal function, anemia, skeletal dysfunction, etc., with severe bone pain and susceptibility to repetitive bone fracture (Nature Reviews Clinical Oncology,2012,9(3), 135-. According to the statistics of the international myeloma fund, only 8 months in 2017 prove that the worldwide number of the sick people reaches about 75 ten thousand, the number of new cases is about 11.40 ten thousand every year, and nearly 9 ten thousand of people die of the disease.

B Cell Maturation Antigen (BCMA), a member of the tumor necrosis factor superfamily, is mainly expressed on the surface of memory cells, plasmablasts and plasma cells, and hardly expressed on the surface of other cells, on the other hand, BCMA belongs to a transmembrane receptor on the cell surface, and its gene is located at TNFRSF17 site of chromosome 16. It has been reported in the literature (Blood Cancer Journal,2015,5(2), e 282-e 282.) that BCMA deficient mice are more normal in appearance and B cell number, but have very poor plasma cell viability.

Ligands for BCMA include B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). Wherein the receptor for BAFF further comprises BAFF-R and TACI, and the receptor for APRIL further comprises TACI. The main roles of the BCMA signaling pathway are to promote survival, differentiation of B cells, activation of regulatory T cells, and the like, and conversely, the TACI signaling pathway inhibits maturation of B cells (Nature reviews immunology,2009,9(7): 491.). For these three receptors (BAFF-R, TACI and BCMA), only BAFF-R is expressed on the immature B-cells, migrating B-cells and primary B-cell surfaces during B-cell development; the surfaces of GC B cells both express BAFF-R and BCMA; the surfaces of the memory cells all express BAFF-R, TACI and BCMA; TACI and BCMA are expressed on the surface of both plasmablasts and plasma cells; when the plasma cells become multiple myeloma cells by canceration, BCMA is highly expressed on the surface, and it is possible that TACI is expressed without BAFF-R (Nature reviews immunology,2009,9(7): 491.). From this, it was found that most of B cells do not express BCMA, and further, it was found that other organ cells hardly express BCMA. Clinically, patients with multiple myeloma have higher serum levels of BCMA, BAFF and APRIL and poorer overall survival and prognosis. Therefore, BCMA is a new target over CD19 and the like in treating multiple myeloma with high specificity and less side effects on the target. Therefore, the development of antibody drugs with blocking effect or endocytosis effect aiming at BCMA targets can not only improve the treatment effect of multiple myeloma, but also greatly reduce the side effect of treatment, and can generate great economic and social values.

Currently, in terms of ADC (antibody-drug conjugate, ADC) drugs, betamamab malontin (abbreviated as GSK2857916) co-developed by Glaxo Group and Seattle Genetics has significant effect, and among 35 cases of over-pretreatment (most of patients received at least 5 treatments and failed treatment) R/R MM patients, ORR reaches 60%, and median-Free-Survival (Progression-Free-Survival) is 12 months (NCT 038445). In the CAR-T cell context, idecabagene viclear (abbreviated as bb2121), a new-based and blue-bird organism CAR-T cell therapy, achieved 85% overall remission rates in 33 patients with R/R MM who had previously failed at least 3 therapies, with a median PFS of 11.8 months (NCT 02658929). AMG 420, advanced in bispecific antibodies, which are smaller than traditional antibodies, are composed of two antibody domain fragments linked together and have superior activity but a shorter half-life than full-length antibodies (NCT02514239), is the most advanced therapy. In terms of clinical outcome, drugs directed against BMCA targets, whether monoclonal, double antibody, ADC or CAR-T cell therapy, achieved compelling results, and clinical results indicate that BCMA targets also have far fewer side effects than other targets.

Disclosure of Invention

Based on the above background, the present invention is intended to develop a novel antibody targeting BCMA.

The invention takes BCMA as immunogen to immunize mice, constructs and screens an antibody library through a phage display technology, and obtains a monoclonal antibody which is simultaneously combined with human and monkey BCMA antigens. And subsequently, a murine monoclonal antibody is modified into a humanized antibody through antibody humanized modification, functional experiments such as affinity, blocking and endocytosis prove that the humanized candidate antibody has excellent functions, and the endocytosis function at the cell level is superior to that of competitive GSK 2857916.

All patents and references disclosed within this specification are expressly and fully incorporated herein by reference.

The present invention relates to an isolated monoclonal antibody targeting B Cell Maturation Antigen (BCMA), wherein said antibody binds both human and monkey BCMA.

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

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

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

In a specific aspect, the antibodies of the invention comprise variants of the above-described antibodies and have the same or similar activity as the above-described antibodies of the invention.

In a particular aspect, the antibody of the invention comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO 13 or 14, or a variant of the foregoing.

In a particular aspect, the antibody of the invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO. 15 or 16, or a variant of the foregoing.

In a particular aspect, the antibody of the invention comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 13 or 14, or a variant thereof, and a heavy chain variable region having the amino acid sequence set forth in SEQ ID No. 15 or 16, or a variant thereof.

In a particular aspect, the heavy chain variable region of the antibody of the invention comprises CDR-H1 of SEQ ID NO. 1, CDR-H2 of SEQ ID NO. 3 and CDR-H3 of SEQ ID NO. 5.

In a particular aspect, the heavy chain variable region of the antibody of the invention comprises CDR-H1 of SEQ ID NO. 2, CDR-H2 of SEQ ID NO. 4 and CDR-H3 of SEQ ID NO. 6.

In a particular aspect, the light chain variable region of the antibody of the invention comprises CDR-L1 of SEQ ID NO. 7, CDR-L2 of SEQ ID NO. 9 and CDR-L3 of SEQ ID NO. 11.

In a particular aspect, the light chain variable region of the antibody of the invention comprises CDR-L1 of SEQ ID NO. 8, CDR-L2 of SEQ ID NO. 10 and CDR-L3 of SEQ ID NO. 12.

In a particular aspect, the light chain variable region of an antibody of the invention has the sequence shown in SEQ ID No. 13 or 14, or a sequence that is at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similar to any of the above sequences.

In a particular aspect, the heavy chain variable region of an antibody of the invention has the sequence shown in SEQ ID No. 15 or 16, or a sequence that is at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similar to any of the above sequences.

In a particular aspect, the antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR-H1 of SEQ ID NO. 1, the CDR-H2 of SEQ ID NO. 3 and the CDR-H3 of 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.

Further, the variable region of the light chain of the antibody has the sequence shown in SEQ ID NO 13, or a sequence having at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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 that is at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similar to any of the above sequences.

In a particular aspect, the antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR-H1 of SEQ ID NO. 2, the CDR-H2 of SEQ ID NO. 4 and the CDR-H3 of 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.

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%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity to any of the above sequences.

The invention also relates to a monoclonal antibody targeting B Cell Maturation Antigen (BCMA), which antibody recognizes the same epitope as the above described antibody of the invention.

The invention also relates to a monoclonal antibody targeting B Cell Maturation Antigen (BCMA), which antibody competes with the above-described antibody of the invention for binding to B Cell Maturation Antigen (BCMA).

The invention also relates to nucleic acids encoding the above-described antibodies of the invention.

The invention also relates to an expression vector comprising the nucleic acid of the invention as described above.

The invention also relates to a host cell comprising an expression vector or genome according to the invention as described above, into which a nucleic acid according to the invention has been integrated.

The present invention also relates to a method for producing a monoclonal antibody, which comprises culturing the host cell of the present invention to produce the monoclonal antibody of the present invention described above.

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

The invention also relates to a kit or article of manufacture comprising a monoclonal antibody according to the invention or a pharmaceutical composition according to the invention.

In a particular aspect, the disease is selected from the group consisting of B cell acute lymphocytic leukemia, T cell acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-hodgkin lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor, waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis, and POEMS syndrome.

In a particular aspect, the disease is multiple myeloma.

The invention also relates to the use of the monoclonal antibodies of the invention in the manufacture of a medicament for the treatment of a disease associated with the expression of BCMA.

In a particular aspect, the disease is multiple myeloma.

Specifically, the present invention relates to the following aspects:

1. an isolated monoclonal antibody that targets B Cell Maturation Antigen (BCMA), wherein the antibody binds both human BCMA and monkey BCMA.

2. The monoclonal antibody of item 1, said antibody being selected from any one of the following:

(3) an antibody comprising (1) the heavy chain variable region of said antibody and (2) the light chain variable region of said antibody;

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

3. The antibody of claim 1 or 2, wherein the antibody is selected from any one of the following:

(1) an antibody comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO 13 or 14, or a variant of the foregoing;

(2) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO 15 or 16, or a variant thereof;

(3) an antibody comprising (1) the heavy chain variable region of said antibody and (2) the light chain variable region of said antibody.

4. The antibody of any one of claims 1 to 3, wherein the heavy chain variable region of the antibody comprises CDR-H1 of SEQ ID NO. 1, CDR-H2 of SEQ ID NO. 3 and CDR-H3 of SEQ ID NO. 5.

5. The antibody of any one of claims 1 to 3, wherein the heavy chain variable region of the antibody comprises CDR-H1 of SEQ ID NO. 2, CDR-H2 of SEQ ID NO. 4 and CDR-H3 of SEQ ID NO. 6.

6. The antibody of any one of claims 1 to 3, wherein the light chain variable region of the antibody comprises CDR-L1 of SEQ ID NO. 7, CDR-L2 of SEQ ID NO. 9 and CDR-L3 of SEQ ID NO. 11.

7. The antibody of any one of claims 1 to 3, wherein the light chain variable region of the antibody comprises CDR-L1 of SEQ ID NO. 8, CDR-L2 of SEQ ID NO. 10 and CDR-L3 of SEQ ID NO. 12.

8. The antibody of any one of claims 1 to 3, wherein the light chain variable region of the antibody has a sequence of SEQ ID NO 13 or 14, or a sequence that is at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similar to any one of the above sequences.

9. The antibody according to any one of claims 1 to 3, wherein the heavy chain variable region of the antibody has a sequence represented by SEQ ID NO. 15 or 16, or a sequence having at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity to any one of the above sequences.

10. A monoclonal antibody that targets B Cell Maturation Antigen (BCMA), wherein the antibody recognizes the same epitope as the antibody of any one of claims 1 to 9.

11. A monoclonal antibody that targets B Cell Maturation Antigen (BCMA), wherein the antibody competes for binding to B Cell Maturation Antigen (BCMA) with the antibody of any one of claims 1 to 9.

12. A nucleic acid encoding the antibody of any one of claims 1-11.

13. An expression vector comprising the nucleic acid of item 12.

14. A host cell comprising the expression vector of item 13 or a nucleic acid of item 12 integrated into the genome.

15. A method of producing a monoclonal antibody, the method comprising culturing the host cell of item 14 such that the monoclonal antibody of any one of items 1-11 is produced.

16. A pharmaceutical composition comprising the monoclonal antibody according to any one of claims 1-11 and a pharmaceutically acceptable carrier.

17. A kit or article of manufacture comprising a monoclonal antibody according to any one of claims 1-11 or a pharmaceutical composition according to claim 16.

18. A method of treating a disease associated with expression of BCMA comprising:

administering to a subject in need thereof a monoclonal antibody according to any one of claims 1-11 or a pharmaceutical composition according to claim 16 or a kit or article of manufacture according to claim 17.

19. The method of claim 18, wherein the disease is selected from B-cell acute lymphocytic leukemia, T-cell acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-hodgkin lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor, waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis, and POEMS syndrome.

20. The method of claim 18 or 19, wherein the disease is multiple myeloma.

21. Use of a monoclonal antibody according to any one of claims 1-11 in the preparation of a medicament for treating a disease associated with expression of BCMA.

22. The use according to item 21, wherein the disease is selected from B-cell acute lymphocytic leukemia, T-cell acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-hodgkin lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor, waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis, and POEMS syndrome.

23. The use of clause 21 or 22, wherein the disease is multiple myeloma.

Effects of the invention

Compared with the Belatamab mfedotin antibody (GSK2857916), the antibody provided by the invention can be combined with human BCMA and monkey BCMA, and is close to or even better than the antibody in the aspect of affinity level. The antibody related to the invention is close to or even better than GSK2857916 in the aspect of blocking the effect of BCMA binding to ligand BAFF or APRIL. The antibody of the present invention has a better endocytosis effect than GSK2857916 in terms of endocytosis effect. The antibody of the invention has better thermal stability than GSK2857916 in terms of thermal stability. The antibody of the invention is humanized in immunogenicity, and has lower immunogenicity.

Drawings

Figure 1 shows the process of antibody production, showing the process of antibody production with human monkey crossover targeting BCMA.

FIG. 2 shows the activity assay of the recombinant proteins BCMA, BAFF and APRIL, and the results show that the activity of each protein is normal. Figure 2(a) shows the binding results of human and monkey BCMA antigens of different tags (Fc and His) and the positive control antibody GSK 2857916. 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 at different concentrations of plates.

Figure 3 shows candidate antibody and overexpression of human or monkey BCMA cell binding assay, the results show that candidate antibody with human monkey cross characteristics. Fig. 3(a) shows the binding of partial candidate antibodies to HEK293 cells overexpressing human BCMA as determined by FACS. Fig. 3(B) shows the binding of part of the candidate antibody to CHO cells overexpressing monkey BCMA as determined by FACS.

FIG. 4 shows the results of the detection of the cross-affinity activity of a portion of the candidate antibody with human and monkey BCMA. Fig. 4(a) shows the affinity effect of partial candidate antibody to human BCMA at Elisa level, and the results show that partial candidate antibody has near or even better affinity effect compared to GSK2857916 antibody. Fig. 4(B) shows the affinity effect of partial candidate antibody to monkey BCMA at Elisa level, and the results show that partial candidate antibody has near or even better affinity effect compared to GSK2857916 antibody.

FIG. 5 shows the results of the detection of the blocking effect of a part of the candidate antibodies. Fig. 5(a) shows that part of the candidate antibody blocked BCMA binding to BAFF at the Elisa level, and the results indicate that part of the candidate antibody had a near or even better blocking effect compared to the GSK2857916 antibody. Fig. 5(B) and shows that part of the candidate antibody blocked BCMA binding to APRIL at the Elisa level, and the results indicate that part of the candidate antibody had a near or even better blocking effect compared to GSK2857916 antibody.

FIG. 6 shows the results of the measurement of the endocytic effect. FIGS. 6(A) and (B) show the effect of partial candidate antibody on the endocytosis of human myeloma cell line H929 cells, and the results show that partial candidate antibody has better endocytosis effect than GSK2857916 antibody.

Fig. 7 shows a summary of the individual functions of the candidate antibodies, wherein fig. 7 shows the affinity effect of a portion of the candidate antibodies on HEK293 cells expressing human BCMA, on CHO cells expressing monkey BCMA, on Elisa level and on human and monkey BCMA, on Elisa level blocking BAFF binding to BCMA, on Elisa level blocking APRIL binding to BCMA, and on the endocytosis of the antibodies on human myeloma cells H929; the number of '+' is from more than small, representing that the affinity, blocking and endocytosis effects of the antibody are from strong to weak, and the results show that the antibodies SY14-3rd-5-6-7 and SY14-3rd-5-6-32 show more excellent comprehensive effects.

FIG. 8 shows the results of the detection of the cross-affinity activity of the humanized antibody (5-6-7-hu-2) against human and monkey BCMA. FIG. 8(A) shows the effect of detecting the affinity of SY14-3rd-5-6-7 (also referred to as 5-6-7 or 5-6-7-WT) for human BCMA at Elisa levels before and after antibody humanization, indicating that the affinity of antibody humanized is consistent with that of human BCMA before and after humanization. FIG. 8(B) shows the affinity effect of SY14-3rd-5-6-7 antibody before and after humanization on monkey BCMA at Elisa level, and the results show that the affinity of antibody after humanization was consistent with monkey BCMA and before humanization.

FIG. 9 shows the results of the detection of the cross-affinity activity of the humanized antibody (5-6-32-hu-2) with human and monkey BCMA, and FIG. 9(A) shows the effect of detecting the affinity of SY14-3rd-5-6-32 (also referred to as 5-6-32 or 5-6-32-WT) with human BCMA at Elisa level before and after the antibody is humanized, indicating that the affinity with human BCMA is still better than that of the positive antibody (GSK 2857916). FIG. 9(B) shows that before and after the antibody humanization, SY14-3rd-5-6-32 was tested, the affinity effect with monkey BCMA was observed at Elisa level, and the affinity with monkey BCMA was still better than that of the positive antibody GSK 2857916.

FIG. 10 shows the result of detection of blocking effect of humanized antibody (5-6-7-hu-2), and FIG. 10(A) shows the ability of SY14-3rd-5-6-7 (also referred to as 5-6-7 or 5-6-7-WT) to block BCMA from binding to BAFF at the Elisa level after antibody humanization, indicating that the effect of blocking BCMA from binding to BAFF after antibody humanization is slightly worse than that of positive antibody (GSK 2857916). FIG. 10(B) shows that the ability of SY14-3rd-5-6-7 antibody to block BCMA binding to APRIL at the Elisa level after antibody humanization was examined, and the results indicate that antibody humanization was slightly less effective in blocking BCMA binding to APRIL than positive antibody (GSK 2857916).

FIG. 11 shows the result of detection of blocking effect of humanized antibody (5-6-32-hu-2), and FIG. 11(A) shows the ability of SY14-3rd-5-6-32 (also referred to as 5-6-32 or 5-6-32-WT) to block BCMA from binding to BAFF at the Elisa level after antibody humanization, indicating that the effect of blocking BCMA from binding to BAFF after antibody humanization is slightly worse than that of positive antibody (GSK 2857916). FIG. 11(B) shows that the ability of SY14-3rd-5-6-32 antibody to block BCMA binding to APRIL at the Elisa level after antibody humanization was examined, and the results indicate that antibody humanization was slightly less effective in blocking BCMA binding to APRIL than positive antibody (GSK 2857916).

FIG. 12 shows the results of the measurement of cell binding activity of the antibody before and after humanization in highly human and monkey BCMA, and FIG. 12(A) shows the results of the measurement of binding ability of the 5-6-7 and 5-6-32 antibodies before and after humanization to HEK293 cells overexpressing human BCMA, indicating that the binding ability of the antibody after humanization is consistent with that before humanization and is superior to GSK2857916 antibody. FIG. 12(B) shows that the binding ability to CHO cells overexpressing monkey BCMA before and after humanization of 5-6-7 and 5-6-32 antibodies was examined, and the results indicate that the binding ability to antibody after humanization was consistent with that before humanization and was superior to the GSK2857916 antibody. FIG. 12(C) shows that the binding ability to myeloma cell line H929 cells before and after humanization of 5-6-7 and 5-6-32 antibodies was examined, and the results indicate that the binding ability to the humanized antibody after humanization was consistent with that before humanization and was superior to that of the GSK2857916 antibody.

Fig. 13 shows the results of measurement of the endocytosis effect on H929 cells before and after antibody humanization, and fig. 13(a) shows the effect of endocytosis on BCMA-expressing human myeloma cell line H929 cells before and after antibody humanization, indicating that the endocytosis effect after antibody humanization is consistent with that before humanization and superior to that of GSK2857916 antibody. FIG. 13(B) shows that the effect of endocytosis on BCMA-expressing human myeloma cell line H929 cells was examined before and after humanization of the 5-6-32 antibody, and the results indicate that the endocytosis effect after humanization of the antibody was consistent with that before humanization and was superior to that of the GSK2857916 antibody.

Fig. 14 shows the results of summarizing the respective functions of the humanized antibodies, and fig. 14 shows the degree of humanization of the humanized antibodies, the affinity effect on HEK293 cells expressing human BCMA, the affinity effect on CHO cells expressing monkey BCMA, the affinity effect with human and monkey BCMA at the Elisa level, the effect of blocking the binding of BAFF to BCMA, the effect of blocking the binding of APRIL to BCMA, and the effect of endocytosis of the antibodies on human myeloma cells H929; the number of '+' is from high to low, which means that the affinity, blocking and endocytosis effects of the antibody are from strong to weak. The results show that the functions of the humanized antibody are consistent with those of the humanized antibody before humanization, and the functions of the humanized antibody part are close to or better than those of the GSK2857916 antibody.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

Technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, and in case of conflict, the definitions in this specification shall control.

In general, the terms used in the present specification have the following meanings.

In the present specification, an "isolated" antibody is an antibody that has been separated from 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 reverse phase HPLC).

The term "BCMA", also known as B cell maturation antigen, also referred to as CD269, is herein a member of the tumor necrosis factor receptor superfamily, TNFRSF17(Thompson et al, J.Exp.Medicine,192(1): 129-. Human BCMA is expressed almost exclusively in plasma cells and multiple myeloma cells (see, e.g., Novak et al, Blood,103(2): 689-. BCMA binds 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 can be a suitable tumor antigen target for immunotherapeutics for multiple myeloma.

"antigen (Ag)" refers to a compound, composition or substance that can stimulate antibody production or T cell response in an animal, including compositions that are injected or absorbed into an animal (e.g., compositions that include cancer-specific proteins). The antigen reacts with products of specific humoral or cellular immunity, including products induced by heterologous antigens (e.g., the disclosed antigens). In particular embodiments, the target antigen is an epitope of a BCMA polypeptide.

An "epitope" or "antigenic determinant" refers to a region of an antigen to which a binding agent binds. Epitopes can be formed from contiguous amino acids or noncontiguous amino acids joined in parallel via tertiary folding of a protein. Epitopes formed by consecutive amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally absent upon treatment with denaturing solvents. Epitopes typically comprise at least 3, and more typically at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

Antibodies include antigen-binding fragments thereof, such as camel Ig, Ig NAR, Fab fragments, Fab ' fragments, f (ab) '2 fragments, f (ab) '3 fragments, Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv proteins ("dsFv"), and single domain antibodies (sdAb, nanobody), as well as portions of full-length antibodies responsible for antigen binding. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof. See also the Pierce Catalog and Handbook (Pierce Catalog and Handbook),1994-1995 (Pierce chemical Co.), rocoverd (Rockford, IL)); kuby, journal of immunology, 3rd edition, w.h. freiman (w.h. freeman & Co.), new york, 1997.

As understood by the skilled person and as described elsewhere herein, a full antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second and third constant regions, 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 lambda or kappa. Immunoglobulins comprising alpha, delta, epsilon, gamma and mu heavy chains are classified as immunoglobulins (Ig) a, IgD, IgE, IgG and IgM. The complete antibody formed a "Y" shape. The stem of Y consists of the second and third constant regions of the two heavy chains (and for IgE and IgM, the fourth constant region) joined together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have a constant region consisting of three tandem (in-line) Ig domains, and a hinge region for increased flexibility; heavy chains mu and epsilon have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH 2 domain" and the "CH 3 domain", respectively. Each arm of Y comprises the variable region and the first constant region of a single heavy chain joined to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

The light and heavy chain variable regions contain "framework" regions interspersed with three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). CDRs may be defined or identified by conventional methods, for example by Sequences according to Kabat et al (Wu, TT and Kabat, E.A., J.Imedial Med.132 (2):211-50 (1970); Borden, P. and Kabat E.A., PNAS,84: 2440-.

The sequences of the framework regions of different light or heavy chains have relative preservability within a species (e.g., human). The framework regions of the antibody, which are the combined framework regions of the component light and heavy chains, are used to locate and align the CDRs in three-dimensional space. The 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 also typically identified by the chain in which the particular CDR is located. Thus, the CDRs located in the variable domain of the heavy chain of the 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 the antibody are referred to as CDR-L1, CDR-L2 and CDR-L3. Antibodies with different specificities (i.e., different combinatorial sites for different antigens) have different CDRs. Despite the CDR differences from antibody to antibody, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. These positions within the CDRs are called Specificity Determining Residues (SDRs). Illustrative examples of light chain CDRs suitable for use in constructing the humanized BCMA CARs encompassed herein include, but are not limited to, the CDR sequences set forth in SEQ ID NOs 1-3. Illustrative examples of heavy chain CDRs suitable for use in constructing the humanized BCMA CARs encompassed herein include, but are not limited to, the CDR sequences set forth in SEQ ID NOs 4-6.

Mention of "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 fragment as disclosed herein. Mention of "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 fragment as disclosed herein.

A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by cells into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by preparing hybrid antibody-forming cells from fusions of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

"Fv" is the smallest antibody fragment containing the complete antigen binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In the single-chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to that of a two-chain Fv species. In this configuration, the three hypervariable regions (HVRs) of each variable domain interact to define an 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 antigen, but with a lower affinity than the entire binding site.

The Fab fragment contains the heavy and light chain variable domains and also the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the name given herein for Fab', in which the cysteine residues of the constant domains carry free thiol groups. F (ab ') 2 antibody fragments were originally produced as Fab' fragment pairs with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

As described above, the present invention relates to an isolated monoclonal antibody targeting B Cell Maturation Antigen (BCMA), wherein the antibody binds both human and monkey BCMA. Specifically, in the present invention, the affinity effect of the antibody of the present invention on human and monkey BCMA-expressing cells was verified by a flow cytometry method (FACS method). The monoclonal antibody obtained by the invention has better affinity effect on human BCMA-HEK293 cells than that of a positive control antibody (GSK 2857916). The affinity effect of the monoclonal antibody obtained by the invention on the monkey BCMA-CHO cells is close to or better than that of a positive control antibody (GSK 2857916). The monoclonal antibody obtained by the invention can be simultaneously combined with human and monkey BCMA antigens with high affinity and monkey BCMA antigens, so that before the antibody enters clinical research, monkey (cynomolgus monkey) is taken as a model to well perform toxicological evaluation and pharmacokinetic evaluation. Meanwhile, the monoclonal antibody of the invention has very high affinity, the antibody with high affinity has more advantages in terms of drug effect, such as the antibody is dissociated more slowly after being combined to a target antigen molecule, so that the endocytosis effect of the antibody in cells is better, and the required antibody dosage is possibly lower under the condition of achieving the same cell or animal drug effect.

As used herein, the term "specific binding," "specific recognition," or "specific for" refers 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 a target (which may be an epitope) is one that binds to the target with greater affinity, avidity, more readily, and/or with a longer duration than the binding of other targets. In some embodiments, the extent of binding of the antigen binding protein to an unrelated target is less than about 10% of the binding of the antigen binding protein to the target as determined, for example, by Radioimmunoassay (RIA). In some embodiments, an antigen binding protein that specifically binds a target has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, or less than or equal to 0.1 nM.

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

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

Specifically, the antibody of the present invention may comprise a heavy chain variable region comprising heavy chain complementarity determining region 1(CDR-H1) represented by SEQ ID NO:1 or 2, and/or comprising heavy chain complementarity determining region 2(CDR-H2) represented by SEQ ID NO:3 or 4, and/or comprising heavy chain complementarity determining region 3(CDR-H3) represented by SEQ ID NO:5 or 6; the light chain variable region comprises light chain complementarity determining region 1(CDR-L1) shown in SEQ ID NO. 7 or 8, and/or comprises light chain complementarity determining region 2(CDR-L2) shown in SEQ ID NO. 9 or 10, and/or comprises light chain complementarity determining region 3(CDR-L3) shown in SEQ ID NO. 11 or 12.

In particular, the antibodies of the invention comprise a light chain variable region comprising

SEQ ID NO:13：

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGQGTKLEIK

SEQ ID NO:14：

DIQMTQSPSSLSASVGDRVTITCRASQDISNDLNWYQQKPGKAPKLLIYYTSRLPSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGHTLPWTFGQGTKLEIK

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

In particular, the antibodies of the invention comprise a heavy chain variable region having

SEQ ID NO:15：

QVQLVQSGAEVKKPGSSVKISCKASGHIFTNFHFHWVRQAPGQGLEWIGGIYPGNGDTFYNQKFQGRATITADKSTSTAYMELSSLRSEDTAVYYCVRGSYYGYIDAMDYWGQGTSVTVSS

SEQ ID NO:16：

QVQLVQSGAEVKKPGSSVKISCKASGHIFTNFHFHWVRQAPGQGLEWIGGIYPGNGDTFYNQKFQGRATITADKSTSTAYMELSSLRSEDTAVYYCVRGSYYGYIDAMDYWGQGTSVTVSS

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

Specifically, 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 thereof and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO 15 or 16 or a variant thereof.

Specifically, the heavy chain variable region of an 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. The heavy chain variable region of another antibody of the present invention comprises CDR-H1 shown in SEQ ID NO. 2, CDR-H2 shown in SEQ ID NO. 4 and CDR-H3 shown in SEQ ID NO. 6.

Specifically, 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. The light chain variable region of another antibody of the present invention comprises CDR-L1 shown in SEQ ID NO. 8, CDR-L2 shown in SEQ ID NO. 10 and CDR-L3 shown in SEQ ID NO. 12.

Specifically, the light chain variable region of the antibody of the present invention has a sequence represented by SEQ ID NO 13 or 14, or a sequence having at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity to any of the above sequences. Specifically, the heavy chain variable region of the antibody of the present invention has a sequence represented by SEQ ID NO. 15 or 16, or a sequence having at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity to any of the above sequences.

Specifically, an antibody of the present invention comprises a heavy chain variable region comprising 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 variable region of the light chain of the antibody has the sequence shown in SEQ ID NO 13, or a sequence having at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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 that is at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similar to any of the above sequences.

Specifically, another antibody of the present invention comprises a heavy chain variable region comprising 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 variable region of the light chain of the antibody has the sequence shown in SEQ ID NO. 14, or a sequence having at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity to any of the above sequences.

As used herein, the term "variant" refers to a heavy chain variable region or a 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 prior to modification. In one embodiment, the antigen binding protein comprising a variable heavy chain variable region or light chain variable region sequence retains 60%, 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein prior to modification. It will be appreciated that each heavy chain variable region or light chain variable region may 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 a heavy chain variable region amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the heavy chain variable region amino acid sequence 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 percent homology may be across the entire heavy chain variable region and/or the entire light chain variable region, or the percent homology may be limited to the framework regions, while the sequences corresponding to the CDRs are 100% identical to the CDRs 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, e.g., 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 prior to modification. In one embodiment, the antigen binding protein containing the variant CDRs retains 60%, 70%, 80%, 90%, 100% of the biological characteristics of the antigen binding protein prior to modification. It is understood that each CDR that can be modified alone or in combination with another CDR. In one embodiment, the modification is a substitution, in particular a conservative substitution.

As described above, the antibodies of the present invention have excellent endocytic effects, all superior to the positive control antibody (GSK 2857916). Endocytosis (also known as endocytosis or endocytosis) is the process of transport of extracellular substances into cells by the deformation of the plasma membrane. Endocytosis can be classified into three types according to the different sizes of the cellular materials and the cellular mechanisms: phagocytosis, engulfment, receptor-mediated endocytosis. The endocytosis of the antibody refers to receptor-mediated endocytosis, after a monoclonal antibody targeting BCMA is combined with BCMA, a BCMA-antibody complex is mediated to form an endocytosome and then fused with a lysosome, the BCMA-antibody complex is mediated and degraded by the lysosome in the lysosome, and part of the BCMA or BCMA-antibody complex can be transported back to a cell membrane. After the monoclonal antibody is coupled with the small toxin molecule to form an ADC drug, the ADC drug is transported into cells through endocytosis, and the released toxin molecule can kill target cells (such as multiple myeloma cells). Therefore, when the monoclonal antibody is developed into an ADC drug, the better endocytosis effect is very critical to help the mediated drug to enter target cells.

In addition, the antibody is a humanized and modified antibody, the binding activity of the antibody before and after the humanized and modified antibody is basically consistent with that of human and monkey BCMA, and the effect of the antibody before and after the humanized and modified antibody on the combination of the BCMA and the BAFF is basically consistent. The binding effect of the antibody before and after humanized modification and the monkey BCMA-CHO cell is basically consistent and is superior to that of a positive control antibody (GSK2857916), and the binding effect of the antibody before and after humanized modification and the H929 cell is basically consistent and is superior to that of the positive control antibody (GSK 2857916). And the endocytosis effect of the antibody before and after humanization on the human myeloma cell line H929 cell is basically consistent and is superior to that of a positive control antibody (GSK 2857916).

"humanized antibody" refers to a class of engineered antibodies having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived portion of the molecule being derived from one (or more) human immunoglobulin(s). Furthermore, framework support residues can be altered to preserve binding affinity (see, e.g., 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 the database, Los Alamos database and Swiss protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. Human antibodies characterized by homology (based on amino acids) to the framework regions of the donor antibody may be suitable to provide heavy chain constant regions and/or heavy chain variable framework regions for insertion of the donor CDRs. Suitable acceptor antibodies that provide 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 need not be derived from the same acceptor antibody.

The heat stability of the antibody after humanized modification is slightly superior to that of a positive control antibody (GSK2857916), the heat stability condition of an antibody patent drug is achieved, and the poor heat stability can cause the antibody patent drug problems, such as low antibody expression and antibody aggregation.

In addition, the present invention relates to a monoclonal antibody targeting B Cell Maturation Antigen (BCMA), which recognizes the same epitope as the antibody of the present invention described above. The invention also relates to a monoclonal antibody targeting B Cell Maturation Antigen (BCMA), which antibody competes with the above-described antibody of the invention for binding to B Cell Maturation Antigen (BCMA).

In particular, the invention also relates to nucleic acids encoding the above-described antibodies of the invention. The invention also relates to an expression vector comprising the nucleic acid of the invention as described above. The invention also relates to a host cell comprising an expression vector or genome according to the invention as described above, into which a nucleic acid according to the invention has been integrated.

As known in the art, "polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a chain of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into a strand by a DNA or RNA polymerase. As used herein, "vector" refers to a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the gene or sequence 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 condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. The terms "host cell," "host cell line," and "host cell culture" are used interchangeably herein and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as the cell screened or selected for in the originally transformed cell.

The invention also relates to a pharmaceutical composition comprising the monoclonal antibody of the invention and a pharmaceutically acceptable carrier. The invention also relates to a kit or article of manufacture comprising a monoclonal antibody according to the invention or a pharmaceutical composition according to the invention. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and not react with the immune system of a subject. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions (e.g., oil/water emulsions), and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are Phosphate Buffered Saline (PBS) or physiological saline (0.9%). Compositions comprising such carriers are formulated by well-known conventional methods (see, e.g., 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).

The present invention also relates to a method of treating a disease associated with expression of BCMA comprising: administering to a subject in need thereof a monoclonal antibody of the invention or a pharmaceutical composition of the invention or a kit or article of manufacture of the invention. Specifically, the disease is selected from the group consisting of B cell acute lymphocytic leukemia, T cell acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-hodgkin lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor, waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis, and POEMS syndrome. Specifically, the disease is multiple myeloma.

In particular, the term "Multiple Myeloma (MM)", also known as plasma cell myeloma or Kahler's disease (after Otto Kahler), is a refractory clonal B cell tumor characterized by the accumulation of malignant plasma B cells in the bone marrow in intimate contact with stromal cells. MM is a progressive disease, in particular caused, for example, by a variety of genetic lesions to precursor plasma B cells, i.e., chromosomal translocations resulting primarily from translocations, such as t (11; 14), t (4; 14), t (8; 14), or deletions, such as del (13) and del (17), that cause tumor cells to proliferate extensively and become apoptosis-resistant. B lymphocytes originate in the bone marrow and move to lymph nodes. Due to their development, B lymphocytes mature on their cell surface and display different proteins. When they are activated to secrete antibodies, they are called plasma cells. Multiple myeloma develops in B cells after they leave a partial lymph node called the germinal center. The immune system maintains proliferation of B cells and secretion of antibodies under tight control. This control is lost when chromosomes and bases are damaged (usually by rearrangement). Typically, the promoter gene is moved (or translocated) to the chromosome, which stimulates antibody gene overproduction.

As described above, chromosomal translocations between immunoglobulin heavy chain genes (on chromosome 14, locus 14q 32) and oncogenes (often 11q13, 4p16.3, 6p21, 16q23 and 20q11[10]) are often observed in multiple myeloma patients. This mutation results in dysregulation of the oncogene, which is considered to be an important initiating event in the pathogenesis of myeloma. The result is proliferation and genomic instability of plasma cell clones, leading to further mutations and translocations. Chromosome 14 abnormalities are observed in about 50% of all myeloma cases. Deletion of (a part of) chromosome 13 is also observed in about 50% of cases.

As used herein, a "subject", "individual" or "subject" 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, the administration of a monoclonal antibody according to the invention or a pharmaceutical composition according to the invention or a kit or preparation according to the invention to a subject in need thereof refers to the administration of an effective amount of a pharmaceutical composition or a medicament or preparation or the like, as used herein, the term "effective amount" means the amount of a drug or medicament that elicits the biological or pharmaceutical response of a tissue, system, animal or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount that causes improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a decrease in the rate of progression of a disease or condition, as compared to a corresponding subject not receiving that amount. The term also includes within its scope an amount effective to enhance normal physiological function.

The invention also relates to the use of the monoclonal antibodies of the invention in the manufacture of a medicament for the treatment of a disease associated with the expression of BCMA. Specifically, the disease is selected from the group consisting of B cell acute lymphocytic leukemia, T cell acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-hodgkin lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor, waldenstrom's macroglobulinemia, myeloma, MGUS, plasmacytoma, systemic amyloid light chain amyloidosis, and POEMS syndrome. Specifically, the disease is multiple myeloma. As used herein, the terms "cancer," "neoplasm," and "tumor" are used interchangeably and, in single or plural form, refer to a cell that has undergone malignant transformation or has undergone cellular changes that result in abnormal or unregulated growth or hyperproliferation. Such changes or malignant transformations will typically render such cells pathogenic to the host organism and are therefore also intended to include precancerous or precancerous cells that become or may become pathogenic and require or may benefit from intervention. Primary cancer cells (i.e., cells taken from the vicinity of a malignant transformation site) can be distinguished from non-cancerous cells by well-established techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells, but also any cells derived from a cancer cell ancestor (anecessor). This includes metastatic cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that typically manifest as solid tumors, "clinically detectable" tumors are those detectable on a tumor mass basis; for example, a tumor that is detectable by procedures such as CAT scanning, MR imaging, X-ray, ultrasound, or palpation, and/or due to expression of one or more cancer specific antigens in a sample obtainable from a patient. In other words, the term of the present invention includes cells, neoplasms, cancers and tumors at any stage, including what clinicians refer to as precancers, tumors, growth in situ, and late metastatic growth. The tumor may be a hematopoietic tumor, such as a blood cell tumor, or the like, i.e., a liquid tumor. Specific examples of clinical conditions based on such tumors include leukemias such as chronic myelogenous leukemia or acute myelogenous leukemia; myeloma such as multiple myeloma; lymphoma, and the like.

As described above, the antibody of the present invention binds to both human and monkey BCMA, and is close to or even better than the Belantamab mfedotin antibody (abbreviated GSK2857916) in terms of affinity level. The antibody provided by the invention is close to or even better than GSK2857916 in the aspect of blocking the effect of BCMA in binding with a ligand BAFF or APRIL, blocks the binding of BCMA, BAFF and APRIL, and can inhibit the activation of an intracellular signal pathway (such as an NF kappa B signal pathway) caused by the binding of the ligand, thereby inhibiting the survival of plasma cells. The antibody provided by the invention has a better endocytosis effect than GSK2857916 in the aspect of endocytosis effect, the endocytosis effect is a key function of mediated cell killing of an Antibody Drug Conjugate (ADC), and the antibody with the better endocytosis effect has the advantage of better killing effect on ADC drugs. The antibody has better thermal stability than GSK2857916 in the aspect of thermal stability, the thermal stability is a key index of antibody drug-forming property, the antibody has better thermal stability, and the antibody possibly has more advantages in antibody expression, and the possibility of generating polymer is reduced. The antibody related to the invention is a humanized antibody in immunogenicity, has lower immunogenicity risk, the immunogenicity can stimulate immune reaction in human body, on one hand, the antibody can generate anti-antibody and reduce the effect of antibody drug, on the other hand, the strong immunogenicity can bring more side effect risk of the drug. The humanized antibody is obtained by mutating and transforming the sequences except the CDR regions of the antibody to be closer to the sequences of the human antibody, thereby reducing the risk of generating strong immunogenicity of the antibody.

Examples

Example 1

Preparation of the starting Material

In this example, the BCMA antigen and its ligand were purchased from ACRO Biosystems, and the positive antibody GSK2857916 to Glactin Smith (GSK) was prepared according to US patent application (US20140105915A), notably without coupling to a toxin molecule but without affecting other possible functions.

1.1 preparation of antigens

The antigens in the application are used in total in the following 4 types: human-BCMA-Fc, human-BCMA-His, cyno-BCMA-Fc and cyno-BCMA-His, all purchased from ACRO Biosystems under the respective code numbers BC7-H5254, BCA-H522y, BCA-C5253, BCA-C52H 7. In addition, its activity has been mutually verified with GSK2857916 and is consistent with that disclosed in US patent application US20140105915A, and the results are shown in fig. 2 (a).

1.2 preparation of the ligand

The ligands used in this application were commonly used to 2 types, human-BAFF-Fc and human-APRIL-Fc, both purchased from ACRO Biosystems under the respective BAF-H4268 and APL-H5267. In addition, its activity has been demonstrated by ligand binding to human-BCMA and is consistent with that disclosed on U.S. patent application US20140105915A, the results are shown in fig. 2(B) and 2 (C).

1.3 preparation of Positive control antibodies

In the present application, the positive control antibody GSK2857916 is expressed using the transient transfer system (expichho), wherein the main materials used include: gibco medium (cat # A29100-01), Gibco transfection kit (cat # A29129). First, the light chain sequence (shown as the sequence of SEQ ID NO: 17) and the heavy chain sequence (shown as the sequence of SEQ ID NO: 18) of GSK2857916 were synthesized based on the sequences disclosed in U.S. patent application US20140105915A, and a plasmid containing the entire GSK2857916 antibody light chain and heavy chain genes was constructed by a molecular cloning method. The plasmid containing the light chain and heavy chain of the GSK2857916 antibody was mixed at a mass ratio of 2:1, and the above plasmid mixture (25 μ g) was mixed with transfection reagent in 25mL expression system according to standard procedures and added dropwise to 25mL expichho cell expression system. After mixing well, the mixture was expressed in a cell culture chamber at 37 ℃ for 18 to 22 hours. Subsequently, the transfection mixture was supplemented with a feed medium and placed in a 32 ℃ cell incubator for further culture. On day 5 post-transfection, a second feed was added and the cells placed in a 32 ℃ cell incubator for further 10-12 days. Then, the expressed cell suspension was centrifuged at high speed, and the supernatant was collected, filtered through a 0.22 μm filter and purified by Protein A/G affinity column chromatography. After purification, eluting the target protein by using 100mM glycinate (pH3.0), concentrating, replacing, subpackaging, and warehousing and freezing for storage after SDS-PAGE identification and activity identification are qualified.

Example 2

Preparation of cell lines

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

2.1 preparation of H929 cell line

In the application, H929 is purchased from Beijing collaborating cell bank, has the product number of 3111C0001CCC000360, and is a myeloma cell strain of natural high-expression human BCMA.

2.2 preparation of BCMA-CHO cell line

2.2.1 construction of a plasmid expressing monkey BCMA full Length

A DNA fragment containing the cynomolgus monkey BCMA protein is synthesized by a gene synthesis technology and cloned to an expression vector. Introducing Escherichia coli by transformation, selecting Escherichia coli monoclonal, sequencing to obtain correct plasmid clone, extracting plasmid, and sequencing again.

2.2.2 electrotransfer

CHO-s cells were cultured using CD-CHO serum-free medium from Gibco (cat # 10743029). One day before electroporation, cells were passaged to 5X 10⁶The next day, the constructed plasmid was introduced into CHO-s cells using an electrotransfer kit (cat # MPK10096) and an electrotransfer instrument (cat # MP922947) from Invitrogen. The cells after electrotransformation are transferred to a CD-CHO culture medium and placed in a cell culture box at 37 ℃ for 48 hours.

2.2.3 post-electroporation cell plating

After electroporation, the CHO-s cells were plated at 2000 cells/well in a 96-well plate, and 30. mu.M MSX (Millipore, GSS-1015-F) and GS supplement (Sigma, 58672C-100ml) were added to the final concentration, and the plate was incubated at 37 ℃ in a carbon dioxide incubator, and after 10 days, a medium containing 30. mu.M MSX and 1 XGS supplement was additionally added.

2.2.4 clone selection, cell expansion and FACS identification

Picking single cell clones growing in a 96-well plate, transferring the single cell clones to a 24-well culture plate for continuous amplification culture, and then identifying successful monkey BCMA stable cell strains through FACS.

2.3 preparation of BCMA-HEK293 cell line

2.3.1 construction of plasmid expressing full Length human BCMA

A DNA fragment containing human BCMA protein was synthesized by a gene synthesis technique and cloned into an expression vector. Introducing Escherichia coli by transformation, selecting Escherichia coli monoclonal, sequencing to obtain correct plasmid clone, extracting plasmid, and sequencing again.

2.3.2 electrotransfer

HEK293 cells were cultured using DMEM serum-free medium from Gibco (cat # 12634010). One day before electroporation, cells were passaged to 2X 10⁵The next day, the constructed plasmid was introduced into HEK293 cells using an electrotransfer kit (cat # MPK10096) and an electrotransfer instrument (cat # MP922947) from Invitrogen. The cells after the electrotransformation are transferred to a DMEM medium and placed in a cell culture box at 37 ℃ for 48 hours.

2.3.3 post-electroporation cell plating

The electroporated HEK293 cells were plated at 1000 cells/well in 96-well plates, puromycin at a final concentration of 2. mu.g/mL was added, the plates were incubated in a 37 ℃ carbon dioxide incubator, and after 14 days, 2. mu.g/mL of puromycin-supplemented medium was added.

2.3.4 clone selection, cell expansion and FACS identification

Picking single cell clones growing in a 96-well plate, transferring the single cell clones to a 24-well culture plate for continuous amplification culture, and then identifying cell strains successfully transformed by the human BCMA through FACS.

Example 3

Animal immunization

In this example, the animal immunization includes several protocols and is performed simultaneously, and in the course of immunization, the mouse serum is taken for titer detection and the effect of immunization is evaluated, and finally the mouse number of the library is determined according to the serum titer.

3.1 animal immunization

3.1.1. Immunization protocol one

Babl/c mice (Shanghai Ling Biotech Co., Ltd., female, 6-8 weeks, n ═ 2) were immunized by subcutaneous and intraperitoneal injections, and the materials included human BCMA-Fc and monkey BCMA-Fc antigen proteins (ACRO Biosystems) mentioned above, using a cross-immunization method. The first immunization dose was 100. mu.g/mouse supplemented with CFA (complete Freund's adjuvant), after which the immunization dose was reduced to 50. mu.g/mouse supplemented with IFA (Freund's incomplete adjuvant), all were two-week-one cross-immunization. The corresponding mice are numbered Mouse 1 and Mouse 2.

3.1.2 immunization protocol two

Babl/c mice (mice in the same batch as protocol one, n ═ 2) were immunized by subcutaneous and intraperitoneal injections, and the materials included human BCMA-Fc, monkey BCMA-Fc antigen protein (ACRO Biosystems), HEK293 cells highly expressing human BCMA, and CHO cells highly expressing monkey BCMA, as mentioned above, in a cross-immunization manner. The protein immunization dose is 20 mug/mouse, and the cell immunization dose is 1 multiplied by 10⁷I/v, i.p., one week cross-immunization. The corresponding mice are numbered Mouse 3 and Mouse 4.

3.2 serum titre ELISA detection

3.2.1 antigen coating and blocking

Human and monkey BCMA were coated one day in advance at 2 μ g/mL, 30 μ L/well overnight at 4 ℃ on ELISA plates. On the day of immunotiter determination, plates were washed three times with PBST, blocked with 5% skim milk for two hours at room temperature, and washed three times with PBST.

3.2.2 Primary and Secondary antibodies

Each serum sample was diluted 500-fold based on stock solution, then diluted with a 3-fold gradient, added to an ELISA plate as a primary antibody, incubated at room temperature for 1h, washed with PBST three times, added with a secondary antibody (Goat-anti-mouse-IgG-Fab-HRP, Sigma, M4115), and incubated at room temperature for 1 h.

3.2.3 developing, terminating and reading plates

After incubation, the plates were washed six times with PBST, developed with TMB (SurModics, TMBS-1000-01), stopped by adding 2M HCl according to the development, and read by a microplate reader (Molecular Devices, SpectreMax 190) at OD 450. Serum titer results show that the immunized mice have high titers to human BCMA and monkey BCMA, and can be used for the next antibody library construction, and the results are shown in tables 1-1 and 1-2 below.

TABLE 1-1 measurement of potency of mouse serum using human BCMA

TABLE 1-2 measurement of potency of mouse serum by monkey BCMA

Example 4

In this example, the most effective mouse in example 3 is preferable, and the antibody gene of the B cell in the mouse spleen is cloned to a phage display vector by using phage display technology to construct an antibody library. Human BCMA and monkey BCMA are used as screening antigens, and 17 antibody molecules with binding activity for the human BCMA antigen and the monkey BCMA antigen are finally obtained through the processes of library sea-choosing, monoclonal primary screening, sequencing and the like.

4.1 phage display antibody Gene library construction

After immunization, mice were treated according to the standard protocol of euthanasia. Collecting spleen cells of mice after grinding and filtering, adding 1mL of TRIzol^TMReagent (Thermo Fisher, 15596026) lysed spleen cells, total RNA was extracted by phenol chloroform method, and the extracted RNA was reverse transcribed into cDNA by reverse transcription kit (TaKaRa, 6210A). And then, respectively amplifying light chain genes and heavy chain genes of the antibody by using cDNA as a PCR template and using specific primers amplified by a murine antibody sequence. Finally, the antibody gene fragment was inserted into a phage display vector by double restriction with NcoI + NotI and ligation with T4 ligase, the ligation product was recovered with a DNA recovery kit (Omega, D6492-02), and finally transformed into competent E.coli SS320 (Lucigen, MC 1061F) by an electrotransfer instrument (Bio-Rad, MicroPulser), and spread on a 2-YT (C +/K +2-YT) solid plate containing ampicillin and tetracycline, and SS320 bacteria of the correctly transformed antibody plasmid were amplified, and finally a library containing Fab fragment antibody sequences was constructed.

4.2 phage display antibody Gene library screening

4.2.1 screening of phage display antibody Gene library by magnetic bead method

The magnetic bead method screening is based on the process that Biotin labeled antigen protein is combined with magnetic beads coupled with Avidin, the magnetic beads combined with the antigen and a library are subjected to a sea-sorting process of incubation, washing and elution, and are subjected to 2-4 rounds of sea-sorting, and finally, specific monoclonal antibodies aiming at the antigen are enriched. In this example, the principle of cross-selection of human BCMA antigen and monkey BCMA antigen labeled by Biotin is utilized, wherein human BCMA is selected in the first and third rounds, monkey BCMA is selected in the second round, and 3 rounds of total selection are performed, and then the enriched antibody sequence mixture is subjected to monoclonal prescreening of human and monkey BCMA.

The specific implementation method comprises the following steps:

human BCMA protein labeled with Biotin was first incubated with Avidin-coupled magnetic beads, so that the human BCMA protein was bound to the magnetic beads. The magnetic beads with the BCMA antigen bound and the constructed phage library were incubated at room temperature for 2 h. After washing 6-8 times with PBST, the non-specifically adsorbed phage was removed, and Trypsin (Gibco, 25200072) was added to mix gently and react for 20min to elute the specifically bound antibody-displaying phage. Subsequently, SS320 thalli (Lucigen, MC 1061F) in the logarithmic phase are infected by the eluted phage and are kept still for 30min, then are cultured for 1h under the condition of 220rpm, then VSCM13 is added to assist the phage and are kept still for 30min, the phage are continuously cultured for 1h under the condition of 220rpm, and are centrifuged and replaced into a C +/K +2-YT culture medium, and finally the obtained phage are continuously used for the next round of sea election.

4.4.2 screening of phage display antibody Gene library by the Immunity tube method

The principle of immune tube screening is that BCMA protein is coated on the surface of an immune tube with high adsorption force, and the phage display antibody library is added into the immune tube and is incubated, washed and eluted with antigen protein adsorbed on the surface of the immune tube, and subjected to 2-4 rounds of sea separation, and finally, specific monoclonal antibodies aiming at antigens are enriched. The purpose of the immune tube method and the magnetic bead method is to enrich specific antibodies aiming at antigens, and the method is two complementary experimental methods. In this example, the principle of cross-selection of human BCMA antigen and monkey BCMA antigen is used, wherein the first and third rounds of human BCMA selection are performed by human BCMA selection, the second round of monkey BCMA selection is performed by monkey BCMA selection, and the total selection is performed for 3 rounds, and then the monoclonal primary screening of human and monkey BCMA is performed on the enriched antibody sequence mixture. The specific implementation method is similar to the screening by the magnetic bead method.

4.5 selection of the monoclonal

After three rounds of screening, a portion of the single clones were selected from Pool in the third round for ELISA detection, including binding to human BCMA and monkey BCMA. Finally, 93 positive clones capable of combining with human and monkey BCMA were selected from 1344 clones, and after sequencing analysis and analysis of human and monkey BCMA affinity sequencing results, sequences of 17 clones were finally selected to construct full length for further experiments.

Example 5

Full-length antibody construction, expression and purification

In this example, the 17 human monkey crossover Fab antibody obtained in example 4 was constructed as human IgG1 type, where the light chains were all Kappa and the antibody type was human murine chimeric antibody.

5.1 construction of plasmids

And (3) fusing a heavy chain sequence of the sequence obtained by screening with a human IgG1 Fc segment to construct, fusing a light chain with a human Kappa constant region to construct, transforming plasmids of the heavy chain and the light chain into ExpCHO cells, inducing expression and obtaining the full-length antibody.

5.2 expression purification of antibodies

In this application, the antibody was expressed using the ExpicHO transient expression system in the medium (Gibco, A29100-01) and the transfection kit (Gibco, A29129). The specific method comprises the following steps: the ExpCHO cells are passaged one day before transfection, 25 mu g of constructed plasmid is mixed with a transfection reagent in a 25mL system and then dripped into the 25mL ExpCHO cells, and after the mixture is fully mixed, the mixture is expressed for 18-22h in a cell culture box at 37 ℃. Subsequently, the transfection mixture was supplemented with a feed medium and placed in a 32 ℃ cell incubator for further culture. On day 5 post-transfection, a second feed was added and the cells placed in a 32 ℃ cell incubator for further 10-12 days. Then, the expressed cell suspension was centrifuged at high speed, and the supernatant was collected, filtered through a 0.22 μm filter and purified by Protein A/G affinity column chromatography. After purification, eluting the target protein by using 100mM glycinate (pH3.0), concentrating, replacing, subpackaging, and warehousing and freezing for storage after SDS-PAGE identification, SEC purity detection and activity identification.

Example 6

Detection of candidate antibody ELISA level affinity blocking effect

In this example, the affinity effect of the candidate antibody on human and monkey BCMA was verified by ELISA-based methods, and the effect of the candidate antibody on blocking the binding of BCMA to BAFF or APRIL was verified by ELISA-based methods.

6.1 detection of affinity Effect of candidate antibodies on human and monkey BCMA based on ELISA

Human and monkey BCMA were coated on 96-well ELISA plates, 2 μ g/mL, 30 μ L/well, 4 degrees overnight, respectively. The following day, the well plates were washed 3 times with PBST and then blocked with 5% skim milk for 2h, after washing 3 times with PBST, the candidate antibody and positive control antibody (GSK2857916) in a gradient dilution were added and incubated for 1 h. Thereafter, after washing 3 times with PBST, secondary antibodies (anti-human-IgG-Kappa-HRP, abcam, ab79115) were added and incubated for 1 h. After incubation was complete, the plates were washed six times with PBST and developed with TMB (SurModics, TMBS-1000-01). Based on the results of the color development, the reaction was stopped by adding 2M HCl, and the plates were read by a microplate reader (Molecular Devices, SpectreMax 190) at OD450, and the results are shown in FIGS. 4(A) to 4(B), indicating that the candidate antibodies possessed effects close to or even superior to those of the affinity human BCMA and monkey BCMA of the GSK2857916 antibody.

6.2 detection of candidate antibodies blocking the binding of BCMA and BAFF based on ELISA

In this embodiment, the blocking system includes a process of early development, and parameters of the blocking system with both sensitivity and stability are determined according to a development result.

For blocking of BAFF, human BAFF protein was plated at 1. mu.g/mL, 30. mu.L/well overnight at 4 ℃. The following day, the well plates were washed 3 times with PBST and then blocked with 5% skim milk for 2 h. Candidate or positive control antibody (GSK2857916) was then serially diluted and pre-mixed with Biotin-labeled human BCMA (0.6 μ g/mL) for 0.5h, added to 96-well ELISA plates after blocking was complete and plate washing was complete, and incubated for 1 h. Thereafter, after 3 washes with PBST, a secondary antibody (NeutrAvidin-HRP, thermolasher, 31001) was added and incubated for 1 h. After incubation, the plates were washed six times with PBST, developed with TMB (SurModics, TMBS-1000-01), stopped by adding 2M HCl based on the results of the development, and read at OD450 with a microplate reader (Molecular Devices, SpectreMax 190), the results are shown in FIG. 5(A), which indicates that the candidate antibodies possess blocking effects on human BCMA and BAFF that are close to or even superior to GSK 2857916.

6.3 detection of candidate antibodies based on ELISA to block the binding of BCMA and APRIL

For blockade of APRIL, human APRIL protein was plated, 2 μ g/mL, 30 μ L/well, overnight at 4 ℃. The following day, the well plates were washed 3 times with PBST and then blocked with 5% skim milk for 1 h. Candidate or positive control antibody (GSK2857916) was then serially diluted and premixed with Biotin-labeled human BCMA (0.6 μ g/mL) for half an hour in advance, added to a 96-well flat bottom plate after blocking was complete and plate washing was complete, and incubated for 1 h. Thereafter, after 3 washes with PBST, a secondary antibody NeutrAvidin-HRP (thermolasher, 31001) was added and incubated for 1 h. After incubation, the plates were washed six times with PBST, developed with TMB (SurModics, TMBS-1000-01), stopped by adding 2M HCl based on the results of the development, and read at OD450 with a microplate reader (Molecular Devices, SpectreMax 190), the results are shown in FIG. 5(B), which indicates that the candidate antibodies possess a blocking effect on human BCMA and APRIL that is close to or even superior to that of the GSK2857916 antibody.

Example 7

Detection of affinity effect of candidate antibody FACS level and human and monkey BCMA expression cell strain

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

7.1 FACS-based detection of the affinity Effect of candidate antibodies on human BCMA-HEK293 cells

Human BCMA-HEK293 cells in exponential growth phase were collected, centrifuged at 300g to remove supernatant, the cells were resuspended in prepared FACS buffer, counted and the cell suspension density adjusted to 2X 10⁶and/mL. Subsequently, BCMA-HEK293 cells were added to a 96-well round bottom plate at 100. mu.L per well and centrifuged at 300g to remove the supernatant. The candidate antibody and the positive control antibody diluted in the gradient are added into the corresponding holes, the cells are evenly blown by a discharging gun and are placed at 4 ℃ for incubation for 30 min. Centrifuging 300g of the incubated cell mixture to remove the supernatant, adding 200. mu.L of FACS buffer to the corresponding wells and resuspending the cells using a discharging gun; repeating twice, centrifuging at 300g to remove supernatant; PE-labeled anti-human-IgG-Fc flow antibody (Abcam,98596) was added, the cells were blown up with a calandria and incubated at 4 ℃ for 30min, and the supernatant was centrifuged at 300 g. Subsequently, FACS buffer was added and the cells were resuspended, after repeating twice the addition of FACS buffer to the wells, 200 μ L per well, and the cells were resuspended. Finally, detection was performed on a flow cytometer (Beckman, CytoFLEX AOO-1-1102). In this example, the results are shown in FIG. 3(A), and the results indicate that the affinity effect of antibody SY14-3rd-5-6-7 for human BCMA-HEK293 cells is superior to that of the positive control antibody (GSK 2857916).

7.2 FACS-based detection of affinity Effect of candidate antibodies on monkey BCMA-CHO cells

In this example, the procedure was completely identical to that of 7.1, except that the cells were monkey BCMA-CHO cells, and the results are shown in FIG. 3(B), which indicates that the affinity of antibody SY14-3rd-5-6-7 for monkey BCMA-CHO cells was superior to that of the positive control antibody (GSK 2857916). In addition, the difference in signal between human and monkey was presumed to be caused by the difference in BCMA expression level in BCMA-HEK293 or BCMA-CHO.

Example 8

Candidate antibody endocytosis effect detection

In this example, a method system for detecting the endocytosis of the antibody on the human myeloma cell line H929 was developed and the endocytosis effect of the candidate antibody was correspondingly detected, the basic principle is to detect the endocytosis effect of the antibody by cytotoxicity. Fab-ZAP (Atsbio, IT-51-100) is a Fab fragment linked to saporin, a ribosome inhibitor that inhibits protein synthesis and cell death. In this example, Fab-ZAP is a Fab fragment capable of binding to Fc of a human antibody, and the Fab-ZAP and an anti-BCMA antibody are incubated together to make the antibody carry a toxin, and when the anti-BCMA antibody is endocytosed by H929 cells, the toxin enters the cells along with the antibody and causes the cells to die, so that the endocytosis effect of the antibody can be detected by detecting the activity of the cells using MTS kit (Promega, G3580).

The specific detection method comprises the following steps: the H929 cells are revived in advance for one week and are passaged every 3 days after revived, and the inoculation density of each cell is 2 multiplied by 10⁵mL, cells were passaged for no more than 3 weeks. Sucking the cells in logarithmic growth phase, mixing the cells, counting and determining the survival rate. 1 96-well flat bottom plate was used and cell density was adjusted to 4X 10⁵mL, 50 μ L of cells were added to each well, gently tapped and mixed, and the cell culture plate was incubated in a 37 ℃ cell incubator for 16 hours. Fab-ZAP was then first prepared as a 27nM (2.16. mu.g/ml) dilution in 1640+ 10% FBS + 1% PS + 50. mu.M beta. -mercaptoethanol complete medium and added to the gradient diluted antibody to give a final incubated Fab-ZAP concentration of 13.5nM (1.08. mu.g/ml). According to the designed plate distribution, 50 mu L of diluent is taken out from the dilution plate by a 300 mu L discharging gun and added into the cell plate, the cell plate is gently flapped and evenly mixed, and the cell plate is put into a cell culture box for culture for 3 days. Thereafter, the MTS was thawed at room temperature in advance, the cell plate was taken out of the incubator, 20. mu.L of MTS was added to each well using a 100. mu.L 12-channel pipette, gently tapped and incubated in the incubator for 2 hours, and finally, the cell plate was placed in a microplate reader, read and stored at a wavelength of 492 nm.

In this example, the results are shown in FIGS. 6(A) and 6(B), and the results indicate that the endocytosis effects of the candidate antibodies SY14-3rd-5-6-7 and SY14-3rd-5-6-32 are superior to those of the positive control antibody (GSK 2857916).

Thus far, the results of all the tests for the candidate antibodies are summarized in FIG. 7.

Example 9

Antibody humanization engineering

In this example, the framework of the heavy and light chain V regions of murine antibodies were point mutated in amino acids to more closely resemble human Germinine. Wherein, the modified candidate antibodies comprise SY14-3rd-5-6-7 (also known as 5-6-7 or 5-6-7-WT) and SY14-3rd-5-6-32 (also known as 5-6-32 or 5-6-32-WT), wherein the modified preferred candidate antibodies are respectively known as 5-6-7-hu-2 and 5-6-32-hu-2.

Example 10

Humanized antibody functional validation

10.1 detection of affinity Effect of two candidate antibodies on human and monkey BCMA before and after humanization based on ELISA

See example 6 for specific procedures and results shown in fig. 8(a), 8(B), 9(a) and 9(B), which indicate that the antibodies before and after humanization were substantially identical to human and monkey BCMA binding activity.

10.2 detection of the Effect of two candidate antibodies on blocking BCMA and BAFF before and after humanization based on ELISA

For specific operation, see example 6, the results are shown in fig. 10(a) and 11(a), which indicate that the humanized engineered antibody blocks BCMA and BAFF binding slightly less effectively than the positive antibody.

10.3 detection of the Effect of two candidate antibodies on blocking BCMA and APRIL before and after humanization based on ELISA

For specific operation, see example 6, results are shown in 10(B) and 11(B), which indicate that the humanized engineered antibody blocks BCMA and APRIL binding slightly less effectively than the positive antibody.

10.4 FACS-based detection of the affinity Effect of candidate antibodies on human BCMA-HEK293 cells

For specific operation, see example 7, the results are shown in fig. 12(a), which shows that the binding effect of the antibody and human BCMA-HEK293 cells before and after humanization was substantially identical and superior to that of the positive control antibody (GSK 2857916).

10.5 FACS-based detection of affinity Effect of candidate antibodies on monkey BCMA-CHO cells

For specific operation, see example 7, and the results are shown in fig. 12(B), which shows that the binding effect of the antibody and the monkey BCMA-CHO cell before and after humanization was substantially identical and superior to that of the positive control antibody (GSK 2857916).

10.6 FACS-based detection of the affinity Effect of candidate antibodies to human myeloma cell line H929 cells

For specific operation, see example 7, results are shown in fig. 12(C), which indicate that the binding effect of the antibody and H929 cells before and after humanization was substantially identical and superior to that of the positive control antibody (GSK 2857916).

10.7 detection of endocytosis Effect of the antibody before and after humanization

In this example, the endocytic effect on human myeloma cell line H929 cells before and after humanization of two candidate antibodies was also examined. Referring to example 8, the results are shown in FIGS. 13-A and 13-B, and indicate that the internalization of the humanized antibody 5-6-7-huV2 is partially improved compared to that before humanization (5-6-7-WT), and that the internalization of the antibody on human myeloma cell line H929 before and after humanization is substantially the same as that of the antibody before and after humanization, and the effect is superior to that of the positive control antibody (GSK 2857916).

Thus far, all the results of the detection of the two candidate antibodies before and after humanization are summarized in fig. 14.

Example 11

Detection of candidate antibody DSF before and after humanization

In this example, thermostability data for two candidate antibodies before and after humanization as well as for a positive control antibody (GSK2857916) were examined. The specific process is as follows: antibody solutions were prepared at 0.25mg/mL, 19. mu.L/well, with three parallel wells per test article, and PBS and IPI as references. Then 1. mu.L of 100 XSYPRO orange dye was added to each well and the machine was prepared. The method comprises the steps of carrying out testing by using an ABI 7500FAST RT-PCR instrument, selecting a melting curve according to the test type, adopting a continuous mode, scanning the temperature range of 25-95 ℃, the heating rate of 1%, balancing for 5min at 25 ℃, acquiring data in the heating process, selecting ROX for a reporting group, selecting None for a quenching group, determining the reaction volume of 20 mu L, and determining the temperature corresponding to the first peak valley of the first-order derivative of the melting curve as the denaturation temperature of a candidate antibody, wherein the results are detailed in tables 1-3.

TABLE 1-3 detection results of thermal stability of humanized antibodies

Example 12

Candidate antibody affinity detection before and after humanization

In this example, candidate antibodies before and after humanization of 5-6-7 and 5-6-32 were tested for affinity to human and monkey BCMA using a Fortebio Octet RED96 instrument.

12.1 Material preparation

1g of BSA was weighed, 500. mu.L of Tween 20 was weighed, and 1000mL of 1 XPBS was added and mixed. Filtering, packaging and storing. 0.1mL of a 0.1M glycine solution having a pH of 2.0 was added to 0.9mL of ultrapure water, followed by mixing. The antibody was diluted to 10. mu.g/mL with KB buffer, and the antigen was diluted to a series of concentration gradients of 200, 50, 12.5, 0nM in sequence with KB buffer.

12.2 Experimental procedure

The test sample plates (GreinierBio, PN655209) were started at least 10min after light-shielding and pre-wetting the sensors (Anti-Human Fba-CH 12 nd Generation, FAB2G) and were run according to the pre-set program after the test was correct. Among them, 200. mu.L/well of KB buffer was added to columns 1, 10 and 12 of sample plate 1, 0.01M pH2.0 of glycine solution was added to column 11, the prepared sample solutions were added to columns 2 to 8 (one sample plus 4 wells, i.e., one column plus 2 samples), human BCMA-Fc was added to column ninth in order from high to low in concentration, i.e., 200nM antigen solution was added to wells 1, 5, 50nM antigen solution was added to wells 2, 6, 12.5nM antigen solution was added to wells 3, 7, and 0nM antigen solution was added to wells 4, 8. Sample plate 2 was prepared with the exception that the antigen in column nine was replaced with monkey BCMA protein, and the remainder was kept unchanged. Data results are detailed in tables 1-4.

TABLE 1-4 results of affinity assay of humanized antibody with human and monkey BCMA

Example 13

Based on the above examples, 5-6-7-hu-2 and 5-6-32-hu-2 were selected, analyzed and sequenced. The sequences of the light and heavy chain variable regions of the antibody of the invention (SEQ ID NOS: 13-16) were determined by defining the variable regions of the human antibody sequences based on the IMGT database (http:// www.imgt.org /), and the complementarity determining region sequences of the heavy and light chains of the antibody (SEQ ID NOS: 1-12) were determined by analyzing the variable region sequences in such a manner that the CDRs were defined by AbM.

The sequence protected by the invention is specifically as follows:

SEQ ID NO:1

GHIFTNFHFH

SEQ ID NO:2

GYIFTNYHMH

SEQ ID NO:3

GIYPGNGDTF

SEQ ID NO:4

GIYPGNGDIF

SEQ ID NO:5

GSYYGYIDAMDY

SEQ ID NO:6

GSYYGYIDAMDY

SEQ ID NO:7

RASQDISNYLN

SEQ ID NO:8

RASQDISNDLN

SEQ ID NO:9

YTSRLHS

SEQ ID NO:10

YTSRLPS

SEQ ID NO:11

QQGNTLPWT

SEQ ID NO:12

QQGHTLPWT

SEQ ID NO:13

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGNTLPWTFGQGTKLEIK

SEQ ID NO:14

DIQMTQSPSSLSASVGDRVTITCRASQDISNDLNWYQQKPGKAPKLLIYYTSRLPSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQGHTLPWTFGQGTKLEIK

SEQ ID NO:15

QVQLVQSGAEVKKPGSSVKISCKASGHIFTNFHFHWVRQAPGQGLEWIGGIYPGNGDTFYNQKFQGRATITADKSTSTAYMELSSLRSEDTAVYYCVRGSYYGYIDAMDYWGQGTSVTVSS

SEQ ID NO:16

QVQLVQSGAEVKKPGASVKMSCKASGYIFTNYHMHWVRQAPGQGLEWIGGIYPGNGDIFYAQKFQGRATITADKSTSTAYIELSSMRSEDTAVYYCARGSYYGYIDAMDYWGQGTSVTVSS

SEQ ID NO:17

DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIK

SEQ ID NO:18

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS

the above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Suzhou Qinhuaye pharmaceutical Co., Ltd

<120> BCMA-targeting humanized monoclonal antibody with human monkey crossover

<130> TPD01188

<141> 2020-09-25

<150> 201910960313.4

<151> 2019-10-10

<160> 18

<170> SIPOSequenceListing 1.0

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<212> PRT

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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

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<212> PRT

<213> Artificial Sequence

<400> 3

Gly Ile Tyr Pro Gly Asn Gly Asp Thr Phe

1 5 10

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<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

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<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

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BCMA-targeted humanized monoclonal antibodies with human monkey crossover

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