TW202128769A - Antibodies binding bcma and uses thereof - Google Patents

Abstract

An isolated monoclonal antibody that specifically binds human BCMA, or the antigen-binding portion thereof. A nucleic acid molecule encoding the antibody or the antigen-binding portion thereof, an expression vector, a host cell and a method for expressing the antibody or the antigen-binding portion thereof are also provided. The present disclosure further provides an immunoconjugate, a bispecific molecule, a chimeric antigen receptor, an oncolytic virus and a pharmaceutical composition comprising the antibody or the antigen-binding portion thereof, as well as a treatment method using an anti-BCMA antibody or the antigen-binding portion thereof of the disclosure.

Description

Antibodies that bind BCMA and their uses

The present invention relates to isolated monoclonal antibodies (especially mouse, chimeric or humanized monoclonal antibodies) or antigen-binding fragments thereof, which specifically bind to humans with high affinity and functional activity BCMA. The present invention also provides a nucleic acid molecule encoding the antibody, an expression vector, host cell and method for expressing the antibody. The present invention further provides immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses and pharmaceutical compositions comprising the antibody or antigen-binding fragments thereof, as well as diagnosis and treatment using the anti-BCMA antibody of the present invention method.

B cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRS17), is a transmembrane protein of the extracellular domain rich in cysteine (Madry C et al., (1998) Int Immunol. 10( 11): 1693-702; Laabi Y et al. (1994) Nucleic Acids Res 22(7): 1147-54; Laabi Y et al. (1992) EMBO J 11(11): 3897-904). It works with the two members of the TNFR superfamily, the B cell activating factor receptor (BAFF-R) and the transmembrane promoter-calmodulin ligand interactor (TACI) to regulate humoral immunity, B cell development and homeostasis , Especially the proliferation, survival, maturation and differentiation of B cells into plasma cells (PC) (Mackay F et al., (2003) Annu Rev Immunol 21:231-64; Marsters SA et al., (2000) Curr Biol 10(13): 785-8; Gross JA et al. (2000), Nature 404(6781): 995-9; Thompson JS et al. (2000) J Exp Med 192(1): 129-35). BCMA is also important for the long-term survival of PCs.

BCMA is specifically expressed on the surface of plasmablasts and differentiated PCs, and its expression on malignant PCs is higher than that of normal PCs (Carpenter RO et al. (2013) Clin Cancer Res 19(8): 2048-60; O' Connor BP et al. (2004) J Exp Med 199(1): 91-8; Benson MJ et al. (2008) J Immunol 180(6): 3655-9; Yang M et al. (2005) J Immunol 175(5) : 2814-24; Avery DT et al. (2003) J Clin Invest 112(2): 286-97; Novak AJ et al. (2004) Blood 103(2): 689-94; Chiu A et al. (2007) Blood 109 (2): 729-39; Lee L et al., (2018) Blood 131(7): 746-58; Carpenter RO et al., (2013) ibid; Tai YT et al., (2014) Blood 123(20): 3128- 38; Claudio JO et al. (2002) Blood 100(6): 2175-86; Seckinger A et al. (2017) Cancer Cell 31(3): 396-410). It is related to leukemia, lymphoma and multiple myeloma. For example, elevated plasma BCMA levels associated with clinical conditions have been observed in patients with chronic lymphocytic leukemia (Kyle A Udd et al., (2015) Blood 126(23):2931). In addition, it was also found that BCMA is highly expressed in multiple myeloma tumor cells, and the overexpression of BCMA in these cells or the combination of APRIL and BCMA significantly promoted cell growth and survival in the body (Tai YT et al., (2016) Blood 127 ( 25): 3225-36; Matthes T et al. (2011) Blood 118(7): 1838-44). Furthermore, by binding to BCMA and TACI, APRIL and BAFF further initiate the NFκB pathway and up-regulate anti-apoptotic proteins (Mcl-1, Bcl-2, Bcl-xL) to protect multiple myeloma tumor cells from dexamethasone And serum deprivation-induced cell death (Moreaux J et al., (2004) Blood 103(8): 3148-57; Neri P et al., (2007) Clin Cancer Res 13(19): 5903-9; Shen X et al., (2016) ) Cell Biochem Funct 34(2): 104-10).

As the second most prevalent malignant tumor of the hematopoietic system, multiple myeloma is a clonal B-cell malignant tumor that appears at multiple sites in the bone marrow before spreading to the circulatory system, or a monoclonal immunoglobulin disease of unknown significance (MGUS) The course of the disease developed. The disease is usually characterized by increased abnormal proteins and osteoclast activity, as well as hypercalcemia, cytopenias, renal dysfunction, hyperviscosity, and peripheral neuropathy; and a decrease in normal antibody levels and the number of neutrophils This leads to life-threatening infections. Two monoclonal antibodies targeting CD38 and SLAMF7 were approved at the end of 2015 for the treatment of relapsed and refractory multiple myeloma, but the wide expression of the two antigens in normal tissues limits the long-term clinical application of monoclonal antibodies . In contrast, BCMA has become a potential therapeutic target due to its restricted expression pattern and transmembrane structure. The first therapeutic anti-BCMA antibody-drug conjugate (ADC) GSK2857916 can quickly clear multiple myeloma cells in two mouse models and significantly prolong the survival rate of mice (Tai YT et al., (2014) , Ibid). Another anti-BCMA ADC (HDP-101) has been shown to use picomolar doses to produce in vitro cytotoxicity to multiple myeloma cells, causing significant tumor regression and good tolerance. A variety of anti-BCMA CAR-T cell therapies also showed impressive clinical results (Shih-Feng Cho et al. (2018) Frontiers in Immunology 9: 1821).

Efforts are being made to find more effective and safer BCMA binding fragments, including antibodies.

The present invention provides isolated monoclonal antibodies, such as mouse, human, chimeric or humanized monoclonal antibodies, which bind to BCMA proteins (such as human BCMA and monkey BCMA) and are compatible with the prior art anti-BCMA Compared with antibodies (such as GSK2857916), this antibody has comparable or higher BCMA binding activity or affinity.

The antibody of the present invention has a wide range of applications, including the detection of BCMA protein and the treatment and prevention of BCMA-related diseases, such as autoimmune diseases, cancer and inflammation.

In one aspect, the present invention relates to an isolated monoclonal antibody (e.g., a mouse, chimeric or humanized antibody) or antigen-binding fragment thereof, which antibody or antigen-binding fragment thereof binds to BCMA, which has a region comprising CDR1, The heavy chain variable regions of the CDR2 and CDR3 regions, wherein the CDR1, CDR2 and CDR3 regions comprise: (1) SEQ ID NO: 1 (X1=H or F), 3 and 5, or (2) respectively As shown in SEQ ID NOs: 2, 4 and 6, with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 % Identical amino acid sequence.

In one aspect, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region comprising SEQ ID NO: 13, 14 (X1=1, X2=A, X3 =T, X4=C, X5=F; X1=V, X2=A, X3=T, X4=C, X5=F; X1=I, X2=V, X3=T, X4=C, X5=F ; X1=I, X2=A, X3=S, X4=C, X5=F; X1=I, X2=A, X3=Y, X4=C, X5=Y; X1=V, X2=V, X3 =S, X4=S, X5=Y; or X1=V, X2=V, X3=S, X4=C, X5=Y), 15, 16 (X1=I, X2=N, X3=F; X1 =V, X2=N, X3=F; X1=I, X2=T, X3=F; X1=I, X2=N, X3=Y; or X1=V, X2=T, X3=Y) or 17 Shown have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence, wherein the antibody or Its antigen-binding fragment binds to BCMA.

In one aspect, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, CDR2 region, and CDR3 region Containing: (1) with SEQ ID NO: 7 (X1=H or F), 9 (X1=N or T) and 11 respectively, or (2) with SEQ ID NO: 8, 10 and 12 respectively At least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence.

In one aspect, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region comprising SEQ ID NO: 18, 19 (X1=L, X2=P, X3 =P, X4=W, X5=Y; X1=M, X2=P, X3=P, X4=W, X5=Y; X1=L, X2=A, X3=P, X4=W, X5=Y ; X1=L, X2=P, X3=L, X4=W, X5=Y; X1=L, X2=P, X3=P, X4=L, X5=Y; X1=L, X2=P, X3 =P, X4=W, X5=F; or X1=M, X2=A, X3=P, X4=L, X5=F), 20, 21 (X1=S, X2=P, X3=W, X4 =Y, X5=F; X1=A, X2=P, X3=W, X4=Y, X5=F; X1=S, X2=L, X3=W, X4=Y, X5=F; X1=S , X2=P, X3=L, X4=Y, X5=F; X1=S, X2=P, X3=W, X4=F, X5=F; X1=S, X2=P, X3=W, X4 =Y, X5=Y; X1=A, X2=P, X3=L, X4=F, X5=Y) or 22 has at least 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence, wherein the antibody or antigen-binding fragment thereof binds to BCMA.

In one aspect, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region each comprising a CDR1 region, a CDR2 region and CDR3 region, wherein the CDR1, CDR2, and CDR3 of the heavy chain variable region and the CDR1, CDR2, and CDR3 of the light chain variable region include: (1) SEQ ID NO: 1 (X1=H), 3, 5 respectively , 7 (X1=H), 9 (X1=N) and 11, (2) are respectively SEQ ID NO: 1 (X1=F), 3, 5, 7 (X1=F), 9 (X1=T) And 11, or (3) at least 85%, 90%, 91%, 92%, 93%, 94%, 95% shown in SEQ ID NO: 2, 4, 6, 8, 10 and 12, respectively, 96%, 97%, 98%, 99% or 100% identical amino acid sequence, wherein the antibody or antigen-binding fragment thereof binds to BCMA.

In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region comprise: (1) respectively Take SEQ ID NO: 13 and 18, (2) take SEQ ID NO: 14 (X1=I, X2=A, X3=T, X4=C, X5=F; X1=V, X2=A, X3= T, X4=C, X5=F; X1=I, X2=V, X3=T, X4=C, X5=F; X1=I, X2=A, X3=S, X4=C, X5=F; X1=I, X2=A, X3=Y, X4=C, X5=Y; X1=V, X2=V, X3=S, X4=S, X5=Y; or X1=V, X2=V, X3 =S, X4=C, X5=Y) and 19 (X1=L, X2=P, X3=P, X4=W, X5=Y), (3) SEQ ID NO: 14 (X1=I, X2=A, X3=T, X4=C, X5=F) and 19 (X1=M, X2=P, X3=P, X4=W, X5=Y; X1=L, X2=A, X3=P , X4=W, X5=Y; X1=L, X2=P, X3=L, X4=W, X5=Y; X1=L, X2=P, X3=P, X4=L, X5=Y; or X1=L, X2=P, X3=P, X4=W, X5=F), (4) respectively as SEQ ID NO: 14 (X1=I, X2=A, X3=T, X4=C, X5= F; or X1=V, X2=V, X3=S, X4=C, X5=Y) and 19 (X1=M, X2=A, X3=P, X4=L, X5=F), (5) Take SEQ ID NO: 15 and 20 respectively, (6) Take SEQ ID NO: 16 (X1=I, X2=N, X3=F; X1=V, X2=N, X3=F; X1=I, X2 =T, X3=F; X1=I, X2=N, X3=Y; or X1=V, X2=T, X3=Y) and 21 (X1=S, X2=P, X3=W, X4=Y , X5=F), and (7) are based on SEQ ID NO: 16 (X1=I, X2=N, X3=F) and 21 (X1=A, X2=P, X3=W, X4=Y, X5= F; X1=S, X2=L, X3=W, X4=Y, X5=F; X1=S, X2=P, X3=L, X4=Y, X5=F; X1=S, X2=P, X3=W, X4=F, X5=F; or X1=S, X2=P, X3=W, X4=Y, X5=Y), (8) SEQ ID NO: 16 (X1=I, X2 =N, X3=F; or X1=V, X2=T, X3=Y) and 21 (X1=A, X2=P, X3=L, X4=F, X5=Y), or (9) respectively SEQ ID NOs: 1 7 and 22 have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.

In one embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region And light chain constant region, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region, wherein the The heavy chain constant region may have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% shown in SEQ ID No: 23 Or a human IgG1 constant region with 100% identical amino acid sequence, and the light chain constant region may have at least 85%, 90%, 91%, 92%, 93%, 94% shown in SEQ ID No: 24 , 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence of human kappa constant region, and the heavy chain variable region and light chain variable region comprise the amino acid sequence described above, Wherein, the antibody or antigen-binding fragment thereof binds to BCMA.

In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof binds human or monkey BCMA; and (b) blocks the interaction of human BCMA-human BAFF or APRIL.

In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is murine, human, chimeric or humanized monoclonal antibody or antigen-binding fragment thereof.

In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is a full-length antibody of IgG1, IgG2, or IgG4 isotype or an antigen-binding fragment thereof.

The present invention further provides a pharmaceutical composition, which contains the aforementioned isolated monoclonal antibody or antigen-binding fragment thereof, and more than one pharmaceutically acceptable carrier.

In some embodiments, it also contains more than one anti-tumor drug and/or cytokine.

The present invention further provides a method for treating cancer diseases related to increased expression of BCMA in a subject, comprising administering to the subject a therapeutically effective amount of the aforementioned isolated monoclonal antibody or antigen-binding portion thereof, or Pharmaceutical composition.

In some embodiments, the cancer disease is a non-solid tumor or a solid tumor.

In some embodiments, the cancer disease includes leukemia, lymphoma or multiple myeloma.

In some embodiments, it further comprises administering immune checkpoint antibodies, co-stimulatory antibodies and/or cytokines.

In some embodiments, the immune checkpoint antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, or a combination thereof.

In some embodiments, the costimulatory antibody is an anti-CD137 antibody, an anti-OX40 antibody, or an anti-GITR antibody, or a combination thereof.

In some embodiments, the cytokine is IL-2, IL-21, IFN-γ, TNF-α and/or IL-4, or a combination thereof.

In some embodiments, the antibody of the present invention comprises two heavy chains and two light chains or consists of two heavy chains and two light chains, wherein each heavy chain comprises the above-mentioned heavy chain constant region, and the heavy chain may Variable region or CDR sequence, and each light chain includes the above-mentioned light chain constant region, light chain variable region or CDR sequence, wherein the antibody binds to BCMA. The antibody of the present invention may be, for example, a full-length antibody of IgG1, IgG2, or IgG4 isotype. In other embodiments, the antibodies of the present invention may be single-chain variable fragment (scFv) antibodies, or antibody fragments (for example, Fab or F(ab') ₂ fragments).

Compared with the prior art anti-BCMA antibody such as GSK2857916, the antibody or antigen-binding fragment of the present invention has comparable (if not higher) binding activity and affinity to human BCMA and monkey BCMA, and inhibits the binding of BAFF and BCMA .

The present invention also provides an immunoconjugate comprising the antibody of the present invention or an antigen-binding fragment thereof linked to a therapeutic agent (eg, a cytotoxin). The present invention also provides a bispecific molecule, which comprises the antibody or antigen-binding fragment thereof of the present invention, and is linked to a second function having a binding specificity different from that of the antibody or antigen-binding fragment thereof of the present invention Part (for example, secondary antibody). In another aspect, the antibody or antigen-binding fragment thereof of the present invention can be made as part of a chimeric antigen receptor (CAR). Immune cells containing chimeric antigen receptors, such as T cells, are also provided. The antibody or antigen-binding fragment thereof of the present invention can also be encoded by an oncolytic virus or used in combination with an oncolytic virus.

The present invention also provides a composition comprising the antibody or antigen-binding fragment thereof, or immunoconjugate, bispecific molecule, oncolytic virus, CAR or CAR-T cell and a pharmaceutically acceptable carrier of the present invention.

The present invention also includes nucleic acid molecules encoding the antibodies of the present invention or antigen-binding fragments thereof, as well as expression vectors containing such nucleic acids and host cells containing such expression vectors. Also provided is a method for preparing an anti-BCMA antibody using a host cell containing an expression vector, the method comprising the following steps: (i) expressing the antibody in the host cell, and (ii) isolating the antibody from the host cell or its cell culture.

In yet another aspect, the present invention provides a method for treating tumors associated with increased BCMA expression, the method comprising administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention. The tumor may be leukemia, lymphoma or multiple myeloma. In some embodiments, the method includes administering a composition of the invention, a bispecific molecule, an immunoconjugate (such as an antibody-drug conjugate), CAR-T cell, or an antibody-encoding or antibody-carrying oncolytic virus, Or alternatively, a nucleic acid molecule capable of expressing the above-mentioned substances in a subject. In some embodiments, at least one additional anti-tumor antibody can be administered in combination with the antibody of the present invention or an antigen-binding fragment thereof, and the at least one additional anti-tumor antibody is selected from the group consisting of anti-PD-1 antibody and anti-PD-L1 antibody. , Anti-LAG-3 antibody, anti-CTLA-4 antibody and/or anti-TIM-3 antibody. In another embodiment, the antibody or antigen-binding fragment thereof of the present invention is combined with cytokines (such as IL-2, IL-21, TNF-α, IFN-γ and/or IL-4), or a costimulatory antibody ( For example, anti-CD137 antibody, anti-OX40 antibody and/or anti-GITR antibody) are administered together. The antibodies of the present invention can be mouse, human, chimeric or humanized antibodies.

Through the following detailed description and examples, other features and advantages disclosed in the present invention will become apparent, which should not be construed as restrictive. The contents of all references, Genbank entries, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

To ensure that the present invention can be more easily understood, first define certain terms. Additional definitions are set forth throughout the detailed description.

The term "BCMA" refers to a member of the B cell maturation antigen or tumor necrosis factor receptor superfamily 17. The term "BCMA" includes variants, isoforms, homologs, orthologs and paralogues. For example, in some cases, antibodies that specifically target human BCMA proteins can cross-react with BCMA proteins from species other than humans (such as monkeys). In other embodiments, antibodies that specifically target human BCMA protein may be completely specific to human BCMA protein, and will not cross-react or other types of cross-reactions with BCMA of other species, or can interact with certain types of BCMA. But not all BCMA proteins of other species cross-react.

The term "human BCMA" refers to a BCMA protein derived from a human amino acid sequence, for example, the amino acid sequence of human BCMA with Genbank accession number NP_001183.2. The terms "monkey or rhesus BCMA" and "mouse BCMA" refer to the amino acid sequences of monkey and mouse BCMA, respectively, for example, the BCMA sequence having the amino acid sequences of Genbank accession numbers XP_001106892.1 and NP_035738.1, respectively.

The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragments thereof (ie, "antigen-binding portions") or single chains thereof. Intact antibodies are glycoproteins containing two heavy (H) chains and two light (L) chains connected between chains by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as V _H herein) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, C _H1, C _H2, and C _H3 composition. Each light chain is comprised of a light chain variable region (abbreviated herein as V _L) and a light chain constant region. The light chain constant region consists of a domain CL. And V _L, V _H regions regions can be further subdivided into regions of hypervariability (termed complementarity determining regions (CDRs of)), the interval between the regions that are more conserved, termed framework regions (FR). Each V _H and V _L, the four FR and three CDR composition, from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (for example, effector cells) and the first component (C1q) of the classical complement system.

As used herein, the term "antigen-binding fragment" (or simply "antibody portion") of an antibody refers to one or more fragments of an antibody that specifically binds to an antigen (eg, BCMA protein). It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments covered by the term "antigen-binding fragment" of the antibody include: (i) Fab fragments, _{monovalent fragments composed of VL} , _VH , CL and _CH1 domains; (ii) F(ab') ₂ Fragment, a bivalent fragment consisting of two Fab fragments connected by disulfide bonds in the hinge region; (iii) _{Fd fragment composed of V H} and _CH1 domains; (iv) V _L and V _{H of a single arm of an antibody} Fv fragments composed of domains; (v) _{dAb fragments composed of V H} domains (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDR); and (viii) Nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains V _L and V _H FV fragment of a gene encoding a spaced, they may be connected using recombinant methods, by a linker, so that they become a single protein chain in which, V _L and V _H regions Region pairing to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879 -5883). Such single chain antibodies are included in the term "antigen-binding fragment" of antibodies. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and can be obtained by screening in a manner that has the same efficacy as intact antibodies.

As used herein, "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities, for example, an isolated antibody that specifically binds to BCMA protein is substantially free of specific binding to other antibodies other than BCMA protein. Antigen antibody. However, isolated antibodies that specifically bind to human BCMA protein may have cross-reactivity with other antigens, such as BCMA proteins from other species. In addition, the isolated antibody may be substantially free of other cellular materials and/or chemical substances.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules having a single molecular composition. The monoclonal antibody composition shows a single binding specificity and affinity for a specific epitope.

As used herein, the term "murine antibody" is intended to include antibodies with variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from mouse germline immunoglobulin sequences. The murine antibody of the present invention may include amino acid residues not encoded by the mouse germline immunoglobulin sequence, for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. However, as used herein, the term "murine antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammal have been grafted onto mouse framework sequences.

The term "chimeric antibody" refers to an antibody made by combining genetic material from a non-human source and genetic material from a human. Or more generally, chimeric antibodies are antibodies that have genetic material from one species and genetic material from another species.

As used herein, the term "humanized antibody" refers to an antibody from a non-human species whose protein sequence has been modified to increase similarity to antibody variants naturally produced in humans.

The term "isotype" refers to the antibody class (eg, IgM or IgG1) encoded by heavy chain constant region genes.

The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably with the term "antibody that specifically binds to an antigen" herein.

As used herein, an antibody that "specifically binds to human BCMA" is intended to refer to an antibody that binds to a human BCMA protein (and possibly from one or more non-human BCMA proteins) but does not substantially bind to a non-BCMA protein. Preferably, the antibody binds to the human BCMA protein with "high affinity" (ie, a K _D of 5.0×10 ^-8 M or less, more preferably 1.0×10 ^-8 M or less, and more preferably 7.0×10 ^{-9 M or less).}

As used herein, the term "substantially not binding" to a protein or cell means not binding to a protein or cell or not binding to a protein or cell with high affinity, that is, at 1.0×10 ^-6 M or more, more preferably 1.0 ×10 ^-5 M or more, more preferably 1.0 × 10 ^-4 M or more, more preferably 1.0 × 10 ^-3 M or more, even more preferably 1.0 × 10 ^-2 M or more K _D binds to proteins or cells.

The term "high affinity" for an IgG antibody refers to a target antigen having 1.0×10-6 M or less, more preferably 5.0×10 ^-8 M or less, even more preferably 1.0×10 ^-8 M or less, even more preferably 7.0 ×10 ^-9 M or less, and even more preferably an antibody with a K _D ^{of 1.0 × 10 -9 M or less.} However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for the IgM isotype refers to antibodies that have _{a K D} of ^{10 -6} M or less, more preferably 10 ^-7 M or less, even more preferably 10 ^-8 M or less.

As used herein, the term "K _assoc" or "K _a" is intended to refer to the association rate of a particular antibody antigen interaction, but as used herein, the term "K _dis" or "K _d" is intended to refer The dissociation rate of a specific antibody interacting with an antigen. As used herein, the term "K _D" is intended to refer to the dissociation constant, which is obtained from K _d and K _a ratio (i.e., K _d / K _a), and the molar concentration (M) is represented. The K _D value of the antibody can be determined using methods commonly used in the art. The preferred method for determining the K _{D of} an antibody is surface plasmon resonance, preferably using a biosensor system, such as the Biacore™ system.

The term "EC _50", also known as half maximal effective concentration, refers to a particular time after exposure, it can reach 50% of the maximum (i.e., 50% between the base and maximum values) corresponding to the biological effects of antibody concentration.

The term "IC ₅₀ ", also known as the half-maximal inhibitory concentration, refers to the concentration of the antibody corresponding to 50% inhibition of a specific biological or biochemical function

The term "subject" includes any human or non-human animal. The term "non-human animals" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals such as Non-human primates, sheep, dogs, cats, cows and horses are preferred.

The term "therapeutically effective amount" refers to an amount of the antibody of the present invention that is sufficient to prevent or ameliorate the symptoms associated with a disease or condition (for example, cancer) and/or reduce the severity of the disease or condition. The therapeutically effective amount should be understood in the context of the condition being treated, where those skilled in the art can easily recognize the actual effective amount.

Various aspects of the present invention will be described in more detail in the following subsections.

Anti-BCMA antibody has increased binding affinity to human BCMA and better anti-tumor effect

Compared with the previously described anti-BCMA antibody (eg GSK2857916), the antibody or antigen-binding fragment thereof of the present invention specifically binds to human BCMA with comparable (if not better) binding activity or affinity.

Other functional properties include the ability to block the interaction between BCMA and BAFF.

The preferred antibodies of the present invention are humanized monoclonal antibodies. Additionally or alternatively, for example, the antibody may be a chimeric monoclonal antibody.

Anti-BCMA monoclonal antibody

The preferred antibodies of the invention are the structurally and chemically characterized monoclonal antibodies as described below and in the Examples below. ID amino acid sequence of a heavy chain / light chain variable region of the antibody are summarized in Table 1, some antibodies share the same V _H or V _L. The heavy chain constant region of an antibody may be a human IgG1 heavy chain constant region having, for example, the amino acid sequence shown in SEQ ID NO: 23, and the light chain constant region of an antibody may have, for example, the amino acid sequence shown in SEQ ID NO: 24. Sequence of human kappa constant region. These antibodies may also contain a mouse IgG1 heavy chain constant region and a mouse kappa constant region.

The heavy chain variable region CDRs and light chain variable region CDRs in Table 1 are defined by the Kabat numbering system. However, as is well known in the art, CDR regions can also be defined based on other heavy chain and light chain variable region sequences such as Chothia and IMGT, AbM or Contact numbering systems/methods.

Table 1 Amino acid numbers of the variable region of the heavy chain and the variable region of the light chain Antibody V _H CDR1 V _H CDR2 V _H CDR3 V _H V _L CDR1 V _L CDR2 V _L CDR3 V _L Mouse B1G3 SEQ ID NO.: 1, X1=H SEQ ID NO.: 3 SEQ ID NO.: 5 SEQ ID NO.: 13 SEQ ID NO.: 7, X1=H SEQ ID NO.: 9, X1=N SEQ ID NO.: 11 SEQ ID NO.: 18 huB1G3-V1 SEQ ID NO.: 14, X1=I, X2=A, X3=T, X4=C, X5=F SEQ ID NO.: 19, X1=L, X2=P, X3=P, X4=W, X5=Y huB1G3-V2 SEQ ID NO.: 14, X1=V, X2=A, X3=T, X4=C, X5=F huB1G3-V3 SEQ ID NO.: 14, X1=I, X2=V, X3=T, X4=C, X5=F huB1G3-V4 SEQ ID NO.: 14, X1=I, X2=A, X3=S, X4=C, X5=F huB1G3-V5 SEQ ID NO.: 14, X1=I, X2=A, X3=Y, X4=C, X5=Y huB1G3-V6 SEQ ID NO.: 14, X1=I, X2=A, X3=T, X4=C, X5=F SEQ ID NO.: 19, X1=M, X2=P, X3=P, X4=W, X5=Y huB1G3-V7 SEQ ID NO.: 19, X1=L, X2=A, X3=P, X4=W, X5=Y huB1G3-V8 SEQ ID NO.: 19, X1=L, X2=P, X3=L, X4=W, X5=Y huB1G3-V9 SEQ ID NO.: 19, X1=L, X2=P, X3=P, X4=L, X5=Y huB1G3-V10 SEQ ID NO.: 19, X1=L, X2=P, X3=P, X4=W, X5=F huB1G3-V11 SEQ ID NO.: 14, X1=V, X2=V, X3=S, X4=S, X5=Y SEQ ID NO.: 19, X1=L, X2=P, X3=P, X4=W, X5=Y huB1G3-V12 SEQ ID NO.: 14, X1=I, X2=A, X3=T, X4=C, X5=F SEQ ID NO.: 19, X1=M, X2=A, X3=P, X4=L, X5=F huB1G3-V13 SEQ ID NO.: 14, X1=V, X2=V, X3=S, X4=S, X5=Y huB1G3-V14 SEQ ID NO.: 14, X1=V, X2=V, X3=S, X4=C, X5=Y SEQ ID NO.: 19, X1=L, X2=P, X3=P, X4=W, X5=Y huB1G3-V15 SEQ ID NO.: 19, X1=M, X2=A, X3=P, X4=L, X5=F Mouse B1C1 SEQ ID NO.: 1, X1=F SEQ ID NO.: 3 SEQ ID NO.: 5 SEQ ID NO.: 15 SEQ ID NO.: 7, X1=F SEQ ID NO.: 9, X1=T SEQ ID NO.: 11 SEQ ID NO.: 20 huB1C1-V1 SEQ ID NO.: 16, X1=I, X2=N, X3=F SEQ ID NO.: 21, X1=S, X2=P, X3=W, X4=Y, X5=F huB1C1-V2 SEQ ID NO.: 16, X1=V, X2=N, X3=F huB1C1-V3 SEQ ID NO.: 16, X1=I, X2=T, X3=F huB1C1-V4 SEQ ID NO.: 16, X1=I, X2=N, X3=Y huB1C1-V5 SEQ ID NO.: 16, X1=I, X2=N, X3=F SEQ ID NO.: 21, X1=A, X2=P, X3=W, X4=Y, X5=F huB1C1-V6 SEQ ID NO.: 21, X1=S, X2=L, X3=W, X4=Y, X5=F huB1C1-V7 SEQ ID NO.: 21, X1=S, X2=P, X3=L, X4=Y, X5=F huB1C1-V8 SEQ ID NO.: 21, X1=S, X2=P, X3=W, X4=F, X5=F huB1C1-V9 SEQ ID NO.: 21, X1=S, X2=P, X3=W, X4=Y, X5=Y huB1C1-V11 SEQ ID NO.: 16, X1=V, X2=T, X3=Y SEQ ID NO.: 21, X1=S, X2=P, X3=W, X4=Y, X5=F huB1C1-V12 SEQ ID NO.: 16, X1=I, X2=N, X3=F SEQ ID NO.: 21, X1=A, X2=P, X3=L, X4=F, X5=Y huB1C1-V13 SEQ ID NO.: 16, X1=V, X2=T, X3=Y Mouse B1A1 SEQ ID NO.: 2 SEQ ID NO.: 4 SEQ ID NO.: 6 SEQ ID NO.: 17 SEQ ID NO.: 8 SEQ ID NO.: 10 SEQ ID NO.: 12 SEQ ID NO.: 22

It may be combined with other anti-BCMA antibody V _H and V _L, a sequence human BCMA (or CDR sequences) with an anti-BCMA antibody V _H and V _L, the sequences of the invention (or CDR sequences) "mixed and matched." Preferably, when V _H and V _L, the chain (or chains within these CDR) mixed and matched, from a particular V _H / V _L V _H sequence is replaced with a structure similar V _H sequence. Likewise, preferably from a particular V _H / V _L V _L sequence pairs are replaced by similar structures V _L sequence.

Therefore, in one embodiment, the antibody or antigen-binding fragment thereof of the present invention includes:

(A) The heavy chain variable region, which comprises the amino acid sequence listed in Table 1 above; and

(B) a light chain variable region, the light chain variable region comprising the amino acid sequence as set forth in Table 1 above, or another anti-BCMA antibody V _L, wherein the antibody specifically binds to human BCMA.

In another embodiment, the antibody or antigen-binding fragment thereof of the present invention includes:

(A) The CDR1, CDR2, and CDR3 regions of the heavy chain variable regions listed in Table 1 above; and

(B) The CDR1, CDR2, and CDR3 regions of the light chain variable region listed in Table 1 above, or the CDR of another anti-BCMA antibody, wherein the antibody specifically binds to human BCMA.

In another embodiment, the antibody or antigen-binding fragment thereof includes the CDRs of other antibodies that bind to human BCMA (for example, CDR1, CDR2, and/or CDR3 from the light chain variable region of a different anti-BCMA antibody, and/or from CDR1 and/or CDR3 of the heavy chain variable region) is the heavy chain variable CDR2 region of an anti-BCMA antibody combined.

In addition, it is well known in the art that independent of the CDR1 domain and/or CDR2 domain, the CDR3 domain alone can determine the binding specificity of an antibody to a homologous antigen, and it is well known that it can be expected based on a common CDR3 sequence Generate multiple antibodies with the same binding specificity. See, for example, Klimka et al., British J. of Cancer 83(2):252-260 (2000); Beiboer et al., J. Mol. Biol. 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915 (1998); Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994); Barbas et al., Proc. Natl. Acad. Sci. USA 92: 2529-2533 ( 1995); Ditzel et al., J. Immunol. 157:739-749 (1996); Berezov et al., BIAjournal 8: Scientific Review 8 (2001); Igarashi et al., J. Biochem (Tokyo) 117:452-7 (1995); Bourgeois et al., J. Virol 72: 807-10 (1998); Levi et al., Proc. Natl. Acad. Sci. USA 90: 4374-8 (1993); Polymenis and Stoller, J. Immunol. 152: 5218-5329 ( 1994) and Xu and Davis, Immunity 13: 37-45 (2000). See also, U.S. Patent Nos.: 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185. Each of these references is incorporated herein by reference as a separate whole.

Therefore, in another embodiment, the antibody of the present invention comprises CDR2 of the heavy chain variable region of an anti-BCMA antibody and at least CDR3 of the heavy chain and/or light chain variable region of an anti-BCMA antibody or another anti-BCMA antibody. The CDR3 of the variable region of the heavy chain and/or light chain, wherein the antibody can specifically bind to human BCMA. These antibodies preferably (a) compete for binding to BCMA; (b) retain functional characteristics; (c) bind to the same epitope; and/or (d) have binding affinity similar to the anti-BCMA antibody of the present invention. In another embodiment, the antibody may further comprise CDR2 of the light chain variable region of an anti-BCMA antibody or CDR2 of the light chain variable region of another anti-BCMA antibody, wherein the antibody can specifically bind to human BCMA. In another embodiment, the antibody of the present invention may include the CDR1 of the heavy chain and/or light chain variable region of an anti-BCMA antibody or the CDR1 of the heavy chain and/or light chain variable region of another anti-BCMA antibody, wherein , The antibody can specifically bind to human BCMA.

Conservative modification

In another embodiment, the antibody of the present invention comprises a heavy chain and/or light chain variable region sequence having a CDR1 sequence, a CDR2 sequence and a CDR3 sequence, and the sequence differs by one or more from the amino acid sequence of the anti-BCMA antibody of the present invention. Multiple conservative modifications. It is understood in the art that certain conservative sequence modifications can be made without losing the antigen binding ability of the antibody. See, for example, Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272: 26864-26870 ; Hall et al., (1992) J. Immunol. 149: 1605-12; Kelley and O'Connell, (1993) Biochem. 32: 6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10: 341- 6; and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

Therefore, in one embodiment, the antibody comprises a heavy chain variable region containing CDR1 sequence, CDR2 sequence and CDR3 sequence and/or a light chain variable region containing CDR1 sequence, CDR2 sequence and CDR3 sequence, wherein:

(A) The CDR1 sequence of the heavy chain variable region contains the sequences listed in Table 1 above and/or conservative modifications thereof; and/or

(B) The CDR2 sequence of the heavy chain variable region contains the sequence listed in Table 1 above and/or conservative modifications thereof; and/or

(C) The CDR3 sequence of the heavy chain variable region includes the sequences listed in Table 1 above and their conservative modifications; and/or

(D) The CDR1 sequence and/or CDR2 sequence and/or CDR3 sequence of the light chain variable region include the sequences listed in Table 1 above and/or conservative modifications thereof; and

(E) The antibody specifically binds to human BCMA.

The antibody of the present invention has one or more of the following functional properties, such as high-affinity binding to human BCMA, and the ability to induce ADCC or CDC in cells expressing BCMA.

In various embodiments, the antibody can be, for example, a mouse, human, humanized or chimeric antibody.

As used herein, the term "conservative sequence modification" is intended to refer to an amino acid modification that does not significantly affect or change the binding properties of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. The modification can be introduced into the antibody of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are the use of amino acid residues with similar side chains to make amino acid substitutions. The categories of amino acid residues with similar side chains have been defined in the art. These amino acid categories include: amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), and uncharged Amino acids with polar side chains (such as glycine, aspartamide, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine Acid, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine) Amino acids) and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of the present invention can be replaced with other amino acid residues with the same side chain properties, and the functional assay method described herein can be used to test the changes in the antibody. Reserved functions (ie, the functions clarified above).

Engineered and modified antibodies

Having anti BCMA antibody may be used according to the present invention, one or more antibody V _H / V _L sequences disclosed herein as starting material for the preparation of antibodies of the present invention is a modified engineered antibodies. By modifying one or both variable regions (i.e., V _H and / or V _L), for example, a one or more CDR regions and / or one or more framework regions of one or more amino acid residues engineered Antibody. Additionally or alternatively, an antibody can be engineered by modifying amino acid residues in the constant region, for example, an engineered antibody that alters the effector function of the antibody.

In certain embodiments, CDR grafting can be used to engineer antibody variable regions. The antibody mainly interacts with the target antigen through six complementarity determining region (CDR) amino acid residues located in the heavy and light chains. Therefore, the amino acid sequence within the CDR between each antibody is more diverse than the sequence outside the CDR. Because CDR sequences are responsible for the interaction between most antibodies and antigens, recombinant antibodies can be expressed by constructing expression vectors that can mimic specific naturally-occurring antibodies. The expression vector contains The CDR sequences of antibodies are grafted to the framework region sequences from different antibodies with different properties (see, for example, Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., ( 1989) Proc. Natl. Acad. USA 86: 10029-10033. See also, U.S. Patent Nos.: 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Therefore, another embodiment of the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain variable region comprises The light chain variable region contains the CDR1, CDR2 and CDR3 sequences of the present invention as described above, and the light chain variable region contains the CDR1, CDR2 and CDR3 sequences of the present invention as described above. V _H and V _L CDR sequences of the present invention although these antibodies comprise monoclonal antibodies, but they may contain different framework region sequences.

Such framework region sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase ); And, Kabat et al., (1991), ibid; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836 ; The respective contents are expressly incorporated into this article by reference. As another example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in HCo7 HuMAb mice can be obtained through Genbank accession numbers: 1-69 (NG-0010109, NT-024637 & BC070333), 3-33 (NG-0010109 & NT --024637) and 3-7 (NG--0010109 & NT--024637). As another example, the following heavy chain germline sequences found in HCo12 HuMAb mice can be obtained through Genbank accession numbers: 1-69 (NG-0010109, NT-024637 & BC070333), 5-51 (NG- -0010109 & NT--024637), 4-34 (NG--0010109 & NT--024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678).

Using one of the sequence similarity search methods known to those skilled in the art called Gapped BLAST, antibody protein sequences are compared based on a compiled protein sequence database (Altschul et al., (1997), supra).

The preferred framework sequence used in the antibody of the present invention is a sequence that is structurally similar to the framework sequence used in the antibody of the present invention. The _VH CDR1 sequence, CDR2 sequence and CDR3 sequence can be transplanted to the same framework region as the sequence found in the germline immunoglobulin gene, which is derived from the framework sequence; or the CDR sequence can be transplanted to the same species. The line sequence is compared to the framework region containing one or more mutations. For example, it has been found that in some cases, amino acid residues in the framework region are mutated to maintain or enhance the antigen binding ability of the antibody (see, for example, U.S. Patent Nos.: 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Another type of variable region modification is to make the V _H and / or V _L CDR1 region, a CDR2 region amino acid residue is mutated and / or CDR3 regions to thereby improve one or more binding properties of the antibody (e.g. affinity). Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, and the influence of the mutations on antibody binding ability or other functional properties can be evaluated by in vitro or in vivo detection methods known in the art. Preferably, conservative modifications (as known in the art) are introduced. The mutation may be an amino acid substitution, addition or deletion, preferably an amino acid substitution. In particular, no more than 1, 2, 3, 4, or 5 amino acid residues in the CDR region are mutated.

Therefore, in another embodiment, the present invention provides an isolated anti-BCMA monoclonal antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment thereof comprises a variable region of a heavy chain and a variable region of a light chain. The variable region includes: (a) V _H CDR1 region, which includes the sequence of the present invention or an amino acid sequence with 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; (b) V _H CDR2 region, which contains the sequence of the present invention or an amino acid sequence with 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; (c) _VH CDR3 region, which contains the sequence of the present invention or having one, two, three, four or five amino acid substitutions, amino acid deletions or additions; (d) V _L CDR1 region comprising the sequence of the invention or having 1, 2, 3, four or five amino acid substitutions, amino acid deletions or additions; (e) V _L CDR2 region comprising the sequence of the invention or having one, two, three, four or five amino acid substitutions, deletions or Add the amino acid sequence; and (f) V _L CDR3 region comprising the sequence of the invention or having 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions of amino acid sequence.

Engineered antibodies of the invention include antibodies have amino acid residues V _H and / or V _L framework region is modified (e.g., for improving the properties of the antibody) is. In particular, modification of such framework amino acid residues can reduce the immunogenicity of the antibody. For example, one strategy is to "backmutate" one or more framework region amino acid residues to the corresponding germline sequence. More specifically, the antibody produced by somatic mutation may contain a framework region different from the germline sequence from which the antibody is derived. Such amino acid residues can be distinguished by comparing the sequence of the antibody framework region with the germline sequence from which the antibody is derived.

Another type of framework region amino acid residue modification is included in the framework region or one or more amino acid residue mutations in one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody . This strategy is also called "deimmunization" and is described in further detail in US Patent Publication No. 20030153043.

In addition, in addition to modifying the amino acid residues in the framework or CDR regions, the Fc region of the antibody of the present invention can also be engineered to change one or more functional properties of the antibody, such as serum half-life, complement fixation, and Fc receptors. Binding and/or antigen-dependent cytotoxicity. In addition, the antibody of the present invention can also be chemically modified (for example, one or more chemical groups can be attached to the antibody), or modified to change its glycosylation, again changing one or more of the antibody. A variety of functional characteristics.

In one embodiment, the C _H1 hinge region is modified such that the hinge region of changing the number of cysteine residues, e.g. increased or decreased. This strategy is further described in U.S. Patent No. 5,677,425. For example, the number of hinge regions C _H1 in the cysteine residues are altered may facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, with respect to the native Fc- hinge domain of staphylococcal protein A (SpA) binding, the one or more amino acid mutations are introduced into the Fc- hinge fragment C _H2 -C _H3 domain interface region of the antibody will destroy SpA Combine. This strategy is described in further detail in US Patent No. 6,165,745.

In another embodiment, the antibody is glycosylated. For example, an aglycosylated antibody can be prepared (ie, the antibody lacks glycosylation). Glycosylation can be changed, for example, by changing one or more glycosylation sites within the antibody sequence to increase the affinity of the antibody for the antigen. Alternatively, one or more amino acid substitutions can be made to cause the clearance of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody to the antigen. See U.S. Patent Nos. 5,714,350 and 6,350,861.

Additionally or alternatively, antibodies with altered glycosylation types can be prepared, such as hypofucosylated antibodies with reduced fucosyl residue content or antibodies with increased bisecting GlcNac structure. It has been demonstrated that this type of altered glycosylation increases the ADCC ability of antibodies. Such carbohydrate modification can also be achieved by expressing antibodies in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art and can be used in host cells expressing recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705 and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), so that the antibodies expressed in the Ms704, Ms705 and Ms709 cell lines lack rock Alcose. Ms704, Ms705 and Ms709 FUT8-/- cell lines were constructed by using two alternative vectors to target the destruction of the FUT8 gene in CHO/DG44 cells (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes a cell line with a loss of function of the FUT8 gene, which encodes a fucosyltransferase, and the loss of function of this gene reduces the expression of α-1,6 bond-related enzymes in the cell line. As a result, the antibody expressed by this cell line is hypofucosylated. EP 1,176,195 also describes cell lines with low or no enzyme activity, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662), for adding fucose to N that binds to the Fc region of an antibody. -Acetyl Glucosamine). PCT publication WO 03/035835 describes a variant of the CHO cell line, Lec13 cells, which can reduce the link between fucose and Asn297, and can also enable the host cell to express low-fucosylated antibodies (see Shields et al., ( 2002) J. Biol. Chem. 277: 26733-26740). As described in PCT publication WO 06/089231, modified glycosylated antibodies can also be produced in eggs. Alternatively, modified glycosylated antibodies can be produced in plant cells such as duckweed. A method of producing antibodies in a plant system is disclosed in a U.S. patent application filed on August 11, 2006 at Alston & Bird LLP attorney file number 040989/314911. PCT publication WO 99/54342 describes a cell line engineered to express glycoprotein modified glycosyltransferases (for example, β(1,4)-N-acetylglucosamine aminotransferase III (GnTIII)). This engineering The antibody expressed by the modified cell line has an increased bisected GlcNac structure, which can increase the ADCC activity of the antibody (see Umana et al. (1999) Nat. Biotech. 17: 176-180). Alternatively, fucosidase can be used to cleave off the fucose residues of the antibody. For example, the fucosidase α-L-fucosidase removes the fucosyl group of antibodies (Tarentino et al. (1975) Biochem. 14: 5516-23).

Another modification of the antibodies of the invention is pegylation. For example, the antibody can be pegylated to increase the biological (eg serum) half-life of the antibody. In order to pegylate an antibody, that is, the antibody or fragment thereof is reacted with polyethylene glycol (PEG) (for example, a reactive ester or aldehyde derivative of PEG), so that one or more PEG groups Linked to the antibody or antibody fragment. Preferably, the PEGylation is an acylation reaction or an alkylation reaction of a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to cover any form of PEG that has been used to derive other proteins, such as mono(C ₁ -C ₁₀ )alkoxy- or aryloxy-polyethylene glycol Or polyethylene glycol-maleimide. In certain embodiments, the pegylated antibody is an aglycosylated antibody. The method of protein PEGylation is known in the art and can be applied to the antibody of the present invention. See, EP 0 154 316 and EP 0 401 384.

Physical properties of antibodies

The antibody of the present invention can be characterized by various physical properties to detect and/or distinguish its different classes.

For example, an antibody can include one or more glycosylation sites in the light chain or heavy chain variable region. Due to changes in antigen binding, such glycosylation sites can lead to increased immunogenicity of antibodies or changes in antibody pK (Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172 : 5489-94; Wallick et al. (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al. (1985) Nature 316: 452-7; Mimura et al. (2000) Mol Immunol 37: 697-706). It is known that glycosylation occurs at motifs containing N-X-S/T sequences. In certain embodiments, it is preferable to obtain an anti-BCMA antibody that does not contain variable region glycosylation. This can be done by selecting antibodies that do not contain glycosylation motifs in the variable region or mutant glycosylated amino acid residues.

In a preferred embodiment, the antibody does not contain asparagine isomerization sites. The deamidation of asparagine can occur on the NG or DG sequence, resulting in isoaspartic acid residues. Because the amino acid residues introduce the linkage into the polypeptide chain, the stability of the polypeptide chain is reduced. (Isoaspartic acid effect).

Each antibody has a unique isoelectric point (pI), which is usually between pH 6 and 9.5. The pi of IgG1 antibody is usually at pH 7-9.5, while the pi of IgG4 antibody is usually at pH 6-8. Antibodies with pI values outside the normal range may have partial unfolding and instability in vivo. Therefore, it is preferable to obtain an anti-BCMA antibody with a pI value within the normal range. This can be achieved by selecting antibodies with a pi value within the normal range or by mutating charged amino acid residues.

Nucleic acid molecules encoding antibodies of the invention

In another aspect, the present invention provides nucleic acid molecules encoding the heavy and/or light chain variable regions or CDR regions of the antibodies of the invention. The nucleic acid may be present in intact cells, or cell lysates, or in a partially purified or substantially pure form. When purified from other cellular components or other contaminants (such as other cellular nucleic acids or proteins) by standard techniques, the nucleic acid is "isolated" or "substantially pure." The nucleic acid of the present invention may be DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

The nucleic acid of the present invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (for example, hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), cDNA can be obtained by standard PCR amplification or cDNA cloning techniques. The cDNA encodes the light chain and heavy chain of the antibody produced by the hybridoma. For antibodies obtained from an immunoglobulin gene library (for example, using phage display technology), nucleic acids encoding such antibodies can be recovered from the gene library.

Preferred nucleic acid molecules of the invention include those that encode BCMA monoclonal antibody V _H and V _L, a CDR sequence or nucleic acid molecule. Once DNA fragments encoding V _H and V _L, the segment is obtained, these can be further recombinant DNA fragments by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, Fab fragment genes or to a scFv gene. In these recombination, this DNA fragment encoding the V _L or V _H connected to the encoding another protein (e.g., an antibody constant region or a flexible linker) of another DNA segment. As used in the present invention, the term "operably linked" is intended to join two DNA fragments so that the amino acid sequence encoded by the two DNA fragments remains in frame.

V _H encoded by a DNA operatively linked to another DNA molecule encoding heavy chain constant region (C _H1, C _H2, and C _H3), and can convert the isolated DNA encoding the V _H region of the full length heavy chain to encode Gene. The sequences of human heavy chain constant region genes are known in the art, and DNA fragments covering these regions can be obtained by standard PCR amplification techniques. The heavy chain constant region may be the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, but is most preferably the constant region of IgG1 or IgG4. For a Fab fragment heavy chain gene, the V _H coding DNA can be operatively linked to another DNA molecule encoding only the heavy chain constant regions C _H1.

By a DNA encoding the V _L operatively linked to another DNA molecule encoding the light chain constant region, CL, can convert the isolated DNA encoding the V _L region to the gene encoding a full-length light chain (as well as a Fab light chain gene). The sequences of human light chain constant region genes are known in the art, and DNA fragments covering these regions can be obtained by standard PCR amplification techniques. In a preferred embodiment, the light chain constant region may be a kappa or lambda constant region.

To generate scFv gene, encoding the V _H and V _L, the DNA fragment is operatively linked to a flexible linker coding (e.g., encoding the amino acid sequence (Gly4-Ser) 3) of another DNA segment, which can be V _H and V _L, sequence expressed as a contiguous single-chain protein, i.e., V _L and V _H regions joined by the flexible linker (see e.g., Bird et, (1988) Science 242: 423-426 ; Huston et, (1988) Proc Natl Acad. .. Sci. USA 85: 5879-5883; McCafferty et al. (1990) Nature 348:552-554).

Production of monoclonal antibodies of the present invention

The well-known somatic cell hybridization (hybridoma) technology of Kohler and Milstein (1975) Nature 256:495 can be used to produce the monoclonal antibody (mAb) of the present invention. Other embodiments for producing monoclonal antibodies include viral infection of B lymphocytes or immortalization of B lymphocytes, and phage display technology. Chimeric or humanized antibodies are also well known in the art. See U.S. Patent Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370, the contents of which are fully incorporated herein in their entirety by reference.

Preparation of hybridoma expressing monoclonal antibody of the present invention

The antibodies of the present invention can also be produced in host cells using recombinant DNA technology and gene transfection methods known in the art (for example, Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors, so that genes are operatively linked to transcription and translation control sequences . In the present invention, the term "operably linked" is intended to mean linking the gene encoding the antibody to the vector, so that the transcription and translation control elements in the vector can perform their functions of regulating the transcription and translation of the antibody gene.

The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that regulate the transcription or translation of antibody genes. Such regulatory sequences are described in, for example, Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Regulatory sequences for expression in mammalian host cells include viral elements that initiate high-level protein expression in mammalian cells, such as promoters derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus and polyoma virus or Enhancers, such as the adenovirus major late promoter (AdMLP). Alternatively, non-viral regulatory sequences can be used, such as ubiquitin promoters or β-globin promoters. Furthermore, the regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which contains elements from the SV40 early promoter and human T-cell leukemia virus type 1 long terminal repeat sequence (Takebe et al., (1988) Mol . Cell. Biol. 8: 466-472). The expression vector and expression control elements are chosen to be compatible with the expression host cell used.

The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or spaced expression vector. In a preferred embodiment, the gene encoding the variable region is inserted into an expression vector encoding the constant region of the heavy chain and the constant region of the light chain of the isotype antibody, so that _{the gene encoding the VH} region is operatively linked to the CH vector encoding the gene region and a gene encoding the V _L region is operably linked to a gene encoding a CL region within the vector, thereby constructing a gene encoding the variable region in a full-length chain gene may encode any antibody isotype. Additionally or alternatively, the recombinant expression vector also contains a gene encoding a signal peptide that promotes the secretion of the antibody from the host cell. The antibody gene can be cloned into a vector, so that the signal peptide gene is linked in frame to the gene encoding the amino terminal of the antibody. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin).

In addition to antibody chain genes and regulatory sequences, the recombinant expression vector of the present invention may also carry additional elements, such as elements that regulate the replication of the host cell vector (such as an origin of replication) and selectable marker genes. Selectable marker genes facilitate the selected host cell (see, for example, U.S. Patent Nos. 4,399,216; 4,634,665 and 5,179,017). For example, the marker gene introduced into the vector is usually resistant to drugs (such as G418, hygromycin, or methotrexate). Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (used in dhfr host cells with methotrexate selection/amplification) and neo gene (used in G418 selection).

In order to express the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to cover various techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible for both prokaryotic and eukaryotic host cells to express the antibodies of the present invention, the antibodies of the present invention are expressed in eukaryotic cells, and most preferably mammalian host cells, because such eukaryotic cells, especially mammalian cells, are more likely to express the antibodies of the present invention. Prokaryotic cells are more suitable for assembling and secreting antibodies that are properly folded and immunologically active.

Preferably, the mammalian host cells used to express the antibody of the present invention include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220; used with the selectable marker DHFR, for example, as described in RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cell. In particular, for use in NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When a recombinant expression vector encoding an antibody gene is transfected into a mammalian host cell, the host cell is cultured for a period of time to express the antibody, and more preferably, the expressed antibody can be secreted into the culture medium in which the host cell is grown. The antibody is recovered from the culture medium using standard protein purification methods.

Immunoconjugate

The antibody of the present invention can be conjugated with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerism Enzyme I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents. In ADC, the linker for coupling the antibody and the therapeutic agent is preferably a cleavable linker, such as a peptidyl linker, a disulfide bond or a hydrazone linker. More preferably, the linker is a peptidyl linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu -Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu. It can be as in U.S. Patent Nos. 7,087,600, 6,989,452 and 7,129,261; PCT publications WO 02/096910, WO 07/038,658, WO 07/051,081, WO 07/059,404, WO 08/083,312 and WO 08/103,693; U.S. Patent Publication 20060024317, 20060004081 The ADC was prepared by the method described in 20060247295 and 20060247295, the disclosure of which is incorporated herein by reference.

Bispecific molecule

In another aspect, the bispecific molecule disclosed in the present invention comprises one or more antibodies of the present invention linked to at least one other functional molecule, and the other functional molecule can select another polypeptide or protein, for example, another An antibody or ligand. The bispecific molecule can be combined with at least two different binding sites or targeting molecules. Therefore, as used in the present invention, a "bispecific molecule" includes two or more specific molecules.

In one embodiment, in addition to the anti-Fc binding specificity and the anti-BCMA binding specificity, the bispecific molecule also has a third specificity. The third specificity can be for the anti-enhancement factor (EF), and the molecule can optionally bind to surface proteins involved in cytotoxic activity and thereby increase the immune response to target cells. For example, the anti-enhancement factor can be combined with cytotoxic T cells (for example, via CD2, CD3, CD8, CD28, CD4, or ICAM-1) or other immune cells, thereby enhancing the immune response to the target cells.

Bispecific molecules can have many different forms and particle sizes. At one end of the particle size spectrum, the bispecific molecule retains the traditional antibody form. The difference is that it does not have two binding arms with the same specificity, but two binding arms with different specificities. At the other end is a bispecific molecule composed of two single-chain antibody fragments (scFv) connected by a peptide chain, the so-called Bs(scFv) 2 construct. The mid-size bispecific molecule includes two different F(ab) fragments connected by a peptidyl linker. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic cell hybridization, or chemical synthesis. See, for example, Kufer et al., supra; Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al., Immunology Today, 21(8), 391-397 (2000), and Cited references.

Antibody-encoding or antibody-carrying oncolytic virus

Oncolytic viruses preferentially infect and kill cancer cells. The antibody of the present invention can be used in combination with an oncolytic virus. Alternatively, the oncolytic virus encoding the antibody of the present invention can be infected into the human body.

Chimeric antigen receptor

The present invention also provides a chimeric antigen receptor (CAR) comprising anti-BCMA scFv, said anti-BCMA scFv comprising the CDR region and the heavy chain/light chain variable region of the present invention.

The anti-BCMA CAR may comprise: (a) the extracellular antigen binding domain comprises an anti-BCMA scFv; (b) a transmembrane domain; and (c) an intracellular signal transduction domain.

CAR can include a signal peptide at the N-terminus of the extracellular antigen-binding domain, which guides neonatal receptors into the endoplasmic reticulum; and can include a hinge peptide at the N-terminus of the extracellular antigen-binding domain to make the receptor Easier to use in combination. Preferably, the CAR includes an initial intracellular signaling domain and one or more costimulatory signaling domains at the intracellular signaling domain. Among them, the main and most effective initial intracellular signaling domain includes the CD3-ζ cytoplasmic domain of ITAM, whose phosphorylation can activate T cells; the costimulatory signaling domain can be selected from costimulatory proteins, such as CD28, CD137 or OX40.

Further, CAR can add factors that enhance T cell expansion and persistence and anti-tumor activity, such as cytokine and costimulatory ligands.

The present invention also provides engineered immune effector cells, including the CAR. In some embodiments, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMC), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In some embodiments, the immune effector cells are T cells.

Pharmaceutical composition

In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more antibodies of the present invention, or antigen-binding fragments thereof, or bispecific molecules, CAR-T cells, oncolytic Viruses, immunoconjugates. When the composition contains more than one antibody (or its antigen-binding fragment, or bispecific molecule, CAR-T cell, oncolytic virus, immunoconjugate), the antibody (or its antigen-binding fragment, or bispecific molecule) can be administered separately , CAR-T cells, oncolytic viruses, immunoconjugates). The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug, such as an anti-tumor drug.

The pharmaceutical composition may contain any number of excipients. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending aids, solubilizers, coloring agents, flavoring agents, coating agents, disintegrating agents, lubricants , Sweeteners, preservatives, isotonic agents or combinations thereof. The selection and use of suitable excipients are taught in the following, edited by Gennaro, Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (for example, by injection or infusion). In view of the different routes of administration, the active ingredient can be coated in the material to protect it from acid and other natural conditions that may inactivate it. As used in the present invention, the term "parenteral administration" refers to non-intestinal and local administration methods, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intrasaccular, intraorbital, intracardiac, skin Intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injection and infusion. Alternatively, the antibodies of the present invention can be administered by non-parenteral routes (for example, topical, epidermal or mucosal administration routes), such as intranasal, oral, vaginal, rectal, sublingual or topical administration.

The pharmaceutical composition can be a sterile aqueous solution or dispersion. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

The amount of the active ingredient can be combined with the carrier material to form a single dosage form. The amount of the active ingredient is determined according to the subject and the mode of administration, and is usually the amount of the composition capable of producing a therapeutic effect. Generally, a composition formed with a pharmaceutically acceptable carrier contains about 0.01% to about 99% active ingredient, preferably about 0.1% to about 70%, most preferably about 1% to about 30% active ingredient.

Adjust the dosage regimen to provide the best response (eg, therapeutic response). For example, it can be administered in a single large dose, in several times over time, or the dose can be reduced or increased proportionally according to the treatment situation. It is particularly advantageous to formulate compositions for parenteral administration in dosage unit form for ease of administration and uniformity of dosage. As used in the present invention, a dosage unit form refers to a physically discrete unit, which is suitable as a unit dose for treating a subject; each unit dose contains a predetermined amount of active ingredient, and the predetermined amount of the active ingredient is calculated by It is obtained by administering the active ingredient together with the required pharmaceutical carrier to produce the desired therapeutic effect. Alternatively, when the antibody is administered as a sustained-release preparation, the required frequency of administration can be reduced.

The dosage of the composition may be in the range of about 0.0001 mg/kg to 100 mg/kg, more usually 0.01 mg/kg to 5 mg/kg. For example, the dosage may be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg body weight. An exemplary treatment regimen involves dosing once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every 3 to 6 months. once. The preferred dosing regimen of the anti-BCMA antibody of the present invention includes intravenous administration of 1 mg/kg body weight or 3 mg/kg body weight according to one of the following dosing schedules: (i) six doses every four weeks, and then every Administer for three months; (ii) administer every three weeks; (iii) administer 3 mg/kg body weight once, and then administer 1 mg/kg body weight every three weeks. In some methods, the dose is adjusted so that the plasma antibody concentration can reach about 1-1000 µg/mL; in some methods, the plasma antibody concentration is about 25-300 µg/mL.

The "therapeutically effective dose" of the anti-BCMA antibody or its antigen-binding fragment, or bispecific molecule, CAR-T cell, oncolytic virus, or immunoconjugate of the present invention is preferably one with reduced severity of disease symptoms and no progression of disease symptoms Increase in frequency and duration, or prevent injury or disability caused by disease torment. For example, relative to an untreated subject, the "therapeutically effective dose" of a tumor-bearing subject preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and more preferably at least About 80%. A therapeutically effective amount of a therapeutic antibody can reduce the size of a tumor or improve the symptoms of a subject, which is usually a human or other mammal.

The pharmaceutical composition can be selected as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by J.R. Robinson, Marcel Dekker, Inc., New York, 1978.

The therapeutic pharmaceutical composition can be delivered by a medical device selected from: (1) a needle-free hypodermic injection device (for example, US Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 and 4,596,556); (2) Micro infusion pump (U.S. Patent No. 4,487,603); (3) Transdermal device (U.S. Patent No. 4,486,194); (4) Infusion equipment (U.S. Patent Nos. 4,447,233 and 4,447,224); and (5) Infiltration device (U.S. Patent No. 4,439,196 and 4,475,196); the disclosure of which is incorporated herein by reference.

In certain embodiments, the monoclonal antibodies of the invention can be formulated to ensure biodistribution in the body. For example, in order to ensure that the therapeutic antibody of the present invention crosses the blood-brain barrier, it can be formulated into liposomes, which can contain targeting moieties to enhance selective delivery to specific cells or organs. See, for example, U.S. Patent Nos. 4,522,811, 5,374,548, 5,416,016, and 5,399,331; VV Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038; Bloeman et al., (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180; Briscoe et al. (1995) Am. J. Physiol. 1233: 134; Schreier et al. (1994) J Chem. 269:9090; Keinanen and Laukkanen, (1994) FEBS Lett. 346:123; and Killion and Fidler, (1994) Immunomethods 4:273.

Uses and methods of the present invention

The composition comprising the antibody or antigen-binding fragment thereof, or bispecific molecule, CAR-T cell, oncolytic virus, and immunoconjugate of the present invention has a variety of in vitro and in vivo effects, and is used to treat diseases related to elevated BCMA expression Tumor.

In view of the fact that the anti-BCMA antibody of the present invention can inhibit the proliferation and survival of tumor cells expressing BCMA, the present invention provides a method for inhibiting the growth of tumor cells in a subject. Tumor growth in the subjects was suppressed. Non-limiting examples of tumors that the antibodies of the present invention can be used to treat include, but are not limited to, leukemia, lymphoma, and multiple myeloma. In addition, the use of the antibody of the present invention can inhibit the growth of refractory or relapsed malignant tumors.

Generally speaking, the antibodies of the present invention can be used to enhance the immune response ability of a subject.

Combination therapy

In another aspect, the present invention provides a method of combination therapy, wherein the anti-BCMA antibody or antigen-binding fragment thereof, or bispecific molecule, CAR-T cell, oncolytic virus, immunoconjugate and effective One or more additional antibodies that inhibit tumor growth in the subject are co-administered. In one embodiment, the present invention provides a method for inhibiting tumor growth in a subject, the method comprising administering to the subject an anti-BCMA antibody (or antigen-binding fragment thereof, or CAR-T cell, oncolytic virus, immunoconjugate物) and one or more additional antibodies selected from the group consisting of anti-OX40 antibody, anti-TIM-3 antibody, anti-CD137 antibody, anti-GITR antibody, anti-LAG-3 antibody, anti-PD-L1 antibody and anti-PD-1 Antibodies and/or anti-CTLA-4 antibodies. In some embodiments, the subject is a human.

The initiation of BCMA signal transduction can be further combined with standard cancer treatments. For example, BCMA signal transduction initiation can be combined with CTLA-4 and/or LAG-3 and/or PD-1 blockade and chemotherapy regimens. For example, a chemotherapeutic agent (which may be a cytotoxic agent) can be administered with an anti-BCMA antibody. For example, patients receiving anti-BCMA therapy are given epirubicin, oxaliplatin, and 5-FU.

Optionally, the combination of anti-BCMA antibody and one or more additional antibodies (for example, anti-CTLA-4 antibody and/or anti-LAG-3 antibody and/or anti-PD-1 antibody) can be further used in combination with immunogenic reagents, The immunogenic reagent is selected from cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules) and cells transfected with genes encoding immunostimulatory cytokines (He et al., (2004) J. Immunol. 173 : 4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigen, Trp-2, MART1, and/or tyrosinase, or tumor cells expressing cytokine GM-CSF.

Other treatments in combination with anti-BCMA antibodies include, but are not limited to, interleukin 2 (IL-2) therapy, radiation therapy, surgical therapy, or hormone deprivation therapy.

The combination therapy of the therapeutic agents of the present invention can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or simultaneously administered as a separate composition and each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents may be administered sequentially.

In addition, if the combination therapy of more than one agent is administered sequentially, the order of administration may be reversed or kept in the same order at each time point of administration, and sequential administration may be combined with simultaneous administration or any combination thereof .

The present invention is further illustrated by the following examples, which should not be construed as further limitations. The contents of all drawings and all references, Genbank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Specific embodiment

Example 1 Preparation of mouse-derived anti-BCMA monoclonal antibody using hybridoma technology

immunity

The mice were immunized according to the method described in E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998. Recombinant human BCMA protein (Cat#BC7-H5254, Acro biosystems) with a human IgG1 Fc tag at the C-terminus was used as the immunogen. Human BCMA-his protein (Cat#BCA-H522Y, Acro biosystems) was used to determine the titer of antiserum and to screen hybridomas that secrete antigen-specific antibodies. For the initial immunization and booster immunization, the immunization dose is 25 µg human BCMA-Fc protein per injection per mouse. In order to enhance the immune response, complete Freund's adjuvant and incomplete Freund's adjuvant (Sigma, St. Louis, USA) were used for the primary and booster immunizations, respectively. In short, the adjuvant-antigen mixture is prepared by the following method, first gently vortexing to mix the adjuvant in the vial. Transfer the adjuvant to an autoclaved 1.5 mL microcentrifuge tube. Prepare the antigen with a concentration of 0.5-1.0 mg/mL with PBS or saline. The calculated amount of antigen was then added to the microcentrifuge tube containing the adjuvant, and the mixture was gently vortexed for 2 minutes to generate a water-in-oil emulsion. The resulting adjuvant-antigen emulsion is then sucked into a syringe for immunization of animals. Mice were injected at a dose containing a total of 25 µg of antigen in a volume of 50-100 µL. After immunization, the injected mice were boosted 2-3 times according to the titer of antiserum. Before the hybridoma cells are fused, the adjuvant-antigen emulsion is injected intraperitoneally into the high-titer animals for the final booster immunization.

Hybridoma cell fusion and screening

Before cell fusion, the cells of the murine myeloma cell line (SP2/0-Ag14, ATCC#CRL-1581) were cultured to the logarithmic growth phase. According to the method described in Kohler G and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity", Nature, 256:495-497 (1975), the immunized mice were sacrificed, and the immunization was taken out under aseptic conditions. From the mouse spleen, a spleen cell suspension was prepared, and splenic B cells and murine myeloma cells in the logarithmic growth phase were fused using PEG chemistry. Then, the fused hybridoma cells were plated into a 96-well plate and cultured in DMEM medium containing 20% FCS/HAT. Usually, viable hybridoma clones can be observed under the microscope after 7 to 10 days. Two weeks after the cells were plated, the hybridoma culture supernatant of each well was collected, and the hybridomas expressing anti-BCMA antibody were screened by recombinant human BCMA-his protein using the indirect ELISA method. In short, 60 µL of recombinant human BCMA-his protein (Cat#BCA-H522Y, Acro biosystems) at a concentration of 2.0 µg/mL was used to coat the ELISA plate at 4°C overnight. Wash the plate 4 times with PBST, add 200 µL of PBST solution containing 5% w/v skimmed milk to each well to block. Add 60 µL of the diluted hybridoma culture supernatant to each well and incubate at 37°C for 40 minutes. Then wash the plate 4 times, add 100µL of diluted horseradish peroxidase labeled F(ab') ₂ fragmented goat anti-mouse-IgG secondary antibody (Jackson Immuno research, Cat#115-036-071) to each well Incubate at 37°C for 40 minutes. After washing the plate, add 100μL of TMB color developing solution to each well. After incubating for 3-15 minutes in the dark at room temperature, add H ₂ SO ₄ solution to each well to stop the reaction, and detect the OD value at 450 nm. Then, the screened positive hybridoma cells that can secrete antibodies that bind to human BCMA-his protein are transferred to a 24-well plate and continued to be cultured. Re-screening was performed by ELISA to screen out positive hybridoma cells that specifically recognize human BCMA and block the binding of BCMA to BAFF ligand, and subcloned by limiting dilution method to ensure the positive rate of hybridoma cells, and finally obtain stable secretion targets. Hybridoma cell line with antibodies. . The monoclonal antibody is then purified. In short, use 5 to 10 column volumes of PBS buffer to equilibrate the ProteinA Sepharose column (from Bergeron (Shanghai) Biotechnology Co., Ltd., Cat#AA0273). Load the supernatant of the hybridoma cell line, and wash it with PBS buffer until the protein absorbance reaches the baseline. The antibody was eluted with elution buffer (0.1M glycine-HCl, pH 2.7), and antibody peaks were collected immediately. The eluted antibody was immediately neutralized with neutralization buffer (1M Tris-HCl, pH 9.0) and placed at 1.5 mL tube. The precipitate fractions containing IgG were combined and dialyzed against PBS buffer at 4°C overnight. Subsequently, the purified monoclonal antibody was subjected to in vitro functional activity characterization.

Example 2 Using BIACORE surface plasmon resonance technology to detect the affinity of anti-mouse BCMA monoclonal antibodies

The binding affinity dynamics of the anti-mouse BCMA monoclonal antibody (mAb) prepared in Example 1 was characterized by the Biacore T200 system (GE Healthcare, Pittsburgh, PA, USA).

In short, using the standard amine coupling kit provided by Biacore (GE Healthcare, Pittsburgh, PA, USA), the goat anti-mouse IgG antibody (GE Healthcare, Cat#BR100838, Mouse Antibody Capture Kit) was covalently covalently coupled with primary amine. Connected to CM5 biosensor chip (chip coated with carboxymethyl dextran). The unreacted part on the surface of the biosensor is blocked with ethanolamine. Then, the 80 nM anti-mouse BCMA antibody and reference antibody GSK2857916 (also called BCMA-mab1 or BM) purified by the previous steps at a concentration of 80 nM were loaded onto the wafer at a flow rate of 10 µL/min. Then, use HBS EP buffer (provided by Biacore) to configure different concentrations of recombinant human BCMA-his (Acro biosystems, Cat#BCA-H522Y, MW: 7.8kDa) or cynomolgus monkey BCMA-Fc recombinant protein (Acro biosystems, Cat #BCA-C5253, MW: 32.1kDa), and load the sample onto the wafer at a flow rate of 30μL/min. Track the antigen-antibody binding kinetics for 2 minutes, and track the dissociation kinetics for 10 minutes. The BIA evaluation software was used to fit the binding and dissociation curves to the 1:1 Langmuir binding model. K _D values for, K _a values and _d values is calculated on K and are summarized in Table 2 below.

Table 2 Binding affinity of mouse anti-BCMA antibody Biacore Kinetic Test Human BCMA-his protein Cynomolgus BCMA-Fc protein K _a K _d K _D K _a K _d K _D Mouse monoclonal antibody (M ^-1 s ^-1 ) (s ^-1 ) (M) (M ^-1 s ^-1 ) (s ^-1 ) (M) B1A1 2.45E+06 0.001613 6.57E-10 1.63E+06 0.01476 9.06E-09 B1G3 5.88E+05 4.01E-04 6.83E-10 2.87E+05 5.77E-04 2.01E-09 B1C1 5.75E+05 5.06E-04 8.79E-10 3.54E+05 6.59E-04 1.86E-09 BM 3.30E+05 2.45E-04 7.42E-10 / / /

Experimental results show that the antibody of the present invention specifically binds to human BCMA protein, and its binding affinity is equivalent to or slightly better than the reference antibody. Antibodies B1A1, B1G3 and B1C1 can also specifically bind to monkey BCMA protein.

Example 3 Binding activity of mouse anti-BCMA monoclonal antibody

The binding activity of mouse anti-BCMA antibody was determined by capture ELISA and flow cytometry (FACS).

Capture ELISA method: use horseradish peroxidase labeled F(ab') ₂ fragmented goat anti-mouse secondary antibody (Jackson Immuno Research, Cat#115- 005-071) Coated 96-well plate to detect the binding activity of mouse anti-BCMA antibody, or use F(ab') ₂ fragmented goat anti-human secondary antibody (Jackson Immuno Research, Cat#109-005-097, 100 µL/well) was coated on a 96-well plate to detect the binding activity of the reference antibody BM, and incubated overnight at 4°C. Wash the plate 4 times with PBS solution containing 0.05% Tween 20, then add 200 µL of PBST buffer containing 5% w/v skimmed milk powder to each well, and block at 37°C for 2 hours. Wash the plate again and add 100μL diluted (ie 5-fold dilution with PBST buffer containing 2.5% skimmed milk powder, with 10000 ng/mL or 66.67nM as the starting concentration) anti-mouse BCMA antibody (B1C1, B1G3 or B1A1) Or reference antibody BM, and hIgG (human immunoglobulin for intravenous injection (pH 4), Hualan Bioengineering Co., Ltd.) as a negative control, 100μL per well, incubate at 37°C for 40 minutes, and then wash the plate 4 times. Add 100 µL of 60nM biotin-labeled human BCMA-his protein solution (in PBST containing 2.5% skimmed milk powder) to each well, incubate at 37°C for 40 minutes, wash the plate 4 times, add 100 µL, PBST to each well Solution 1: 10000 diluted horseradish peroxidase-labeled streptavidin (Jackson Immuno Research, Cat#016-030-084), incubate at 37°C for 40 minutes. After washing the plate, add 100 µL of TMB chromogenic solution (Innoreagents, Cat#TMB-S-002) to each well, incubate at room temperature and dark for 15 minutes, add 50 µL of 1M H ₂ SO ₄ solution to each well to stop the reaction, and Read the absorbance at 450-630 nm and plot the antibody concentration. Use Graphpad Prism software to analyze the test data and calculate the EC ₅₀ value.

Use flow cytometry (FACS) to detect the activity of anti-mouse BCMA antibody binding to the BCMA antigen on the surface of U266 cells. The specific method: collect the human myeloma cell line U266 expressing human BCMA on the membrane surface in the logarithmic growth phase, wash twice and use 2 Resuspend the cells in %v/v fetal calf serum in PBS buffer (FACS buffer). Plate 2×10 ⁵ cells per well in a 96-well plate, centrifuge to remove the supernatant, add various concentrations of anti-mouse BCMA antibody or reference antibody diluted in FACS buffer, and incubate on ice for 40 minutes. Wash twice with FACS buffer, then add phycoerythrin (PE) fluorescently labeled F(ab') ₂ fragmented goat anti-mouse IgG secondary antibody (diluted 1:1000 in FACS buffer, Jackson Immunoresearch, Cat#115-116-072). After incubating at 4°C for 40 minutes in the dark, the cells were washed 3 times and resuspended in FACS buffer. The fluorescence intensity of the cells in each well was measured using the FACS Canto II-HTS flow cytometer from Becton Dickinson. Use Graphpad Prism software to analyze the data, and calculate the EC ₅₀ value of antibody binding to cell surface antigen (ie, the antibody concentration corresponding to 50% of the maximum fluorescence intensity of BCMA antibody binding to U266 cells).

The results are summarized in Table 3 below and shown in Figures 1A and 1B.

Table 3 Binding activity of mouse anti-BCMA antibody Mouse monoclonal antibody FACS cell binding (EC ₅₀ , nM) Combination capture ELISA (EC ₅₀ , nM) B1A1 2.66 1.06 B1C1 5.84 0.84 BM / 1.27 B1G3 3.95 0.67 BM / 1.11

The results show that the antibody of the present invention specifically binds to human BCMA and has a similar or slightly lower EC ₅₀ value compared with the reference antibody. In addition, it can be seen from FIG. 1A and FIG. 1B that the maximum absorbance value of the B1A1/B1G3/B1C1 group is similar to or slightly lower than the reference antibody.

Example 4 Use of competitive ELISA for functional assay

4.1 Ligand blocking ELISA

A competitive ELISA method was used to measure the ability of anti-BCMA antibodies to block the BCMA-BAFF interaction. In short, human BCMA-his protein (Acro biosystems, Cat#BCA-H522Y) was prepared with PBS, coated with a 96-well plate at a final concentration of 2 µg/mL, and incubated overnight at 4°C. The next day, wash the plate with PBS solution containing 0.05% Tween 20 (ie PBST), and add PBST containing 5% w/v skimmed milk powder to each well, and block at 37°C for 2 hours. Then wash the plate again with PBST washing buffer.

Dilute the anti-BCMA antibody or reference antibody with PBST containing 2.5% w/v skimmed milk powder (with 200 nM or 30000ng/mL as the starting concentration, 4-fold dilution), and add to 96 coated with good human BCMA-his protein Incubate the plate at 37°C for 40 minutes. Wash the plate 4 times with PBST, then add 100μL/well, 10nM biotin-labeled human BAFF-Fc protein (Sino biological inc., Cat#10056-H01H), and incubate at 37°C for 40 minutes. Wash the plate, add 100 µL/well of horseradish peroxidase-labeled streptavidin, and incubate at 37°C for 40 minutes. Wash the plates. Finally, TMB was added, and the _{reaction was terminated with 1M H 2} SO ₄ , and the absorbance was read at 450-630 nm. Use Graphpad Prism software to analyze the data and calculate the IC ₅₀ value.

4.2 Reference antibody blocking ELISA

A competitive ELISA method was used to measure the ability of the anti-BCMA antibody of the present invention to block the binding of the reference antibody BM to human BCMA. In short, the reference antibody BM was prepared with PBS, coated with a 96-well plate at a final concentration of 2 µg/mL, and incubated overnight at 4°C. On the second day, the plate was washed with PBST, and a PBST solution containing 5% w/v skimmed milk powder was added and blocked at 37°C for 2 hours. When blocking, the anti-BCMA antibody or reference antibody (with 100 nM or 15000 ng/mL as the starting concentration, 4-fold dilution) and the final concentration of 10 nM prepared with PBST containing 2.5% skimmed milk powder The mixture of protein-labeled human BCMA-his protein was incubated at 25°C for 40 minutes. After washing the plate, add the mixture of antibody and biotin-labeled human BCMA-his protein to a 96-well plate coated with the reference antibody. After incubating at 37°C for 40 minutes, the plate was washed with PBST. Then 100 μL/well of horseradish peroxidase-labeled streptavidin was added and incubated at 37°C for 40 minutes. The plate was washed again with PBST. Finally, TMB was added, and the _{reaction was terminated with 1M H 2} SO ₄ , and the absorbance was read at 450-630 nm. Use Graphpad Prism software to analyze the data and calculate the IC ₅₀ value.

The results of the above three detection methods are summarized in Table 4 below and shown in Figure 2A, Figure 2B, Figure 3A and Figure 3B.

Table 4 Functional activity test results of anti-BCMA antibodies Mouse monoclonal antibody BCMA-BAFF blocking ELISA (IC ₅₀ , nM) Reference antibody blocking ELISA (IC ₅₀ , nM) B1A1 7.00 1.91 B1C1 8.04 2.69 BM 9.61 1.93 B1G3 5.84 3.12 BM 10.67 2.64

It can be seen from Table 4 that compared with the reference antibody, the anti-BCMA antibody of the present invention can block the human BCMA-BAFF interaction _{with a lower IC 50 value.} In addition, as shown in Figure 2A and Figure 2B, there is no significant difference in absorbance between the antibodies B1A1 and B1C1 and the reference antibody, and B1G3 has a better blocking ability than the reference antibody.

The experimental results also show that the antibodies of the present invention can block the interaction between human BCMA and the reference antibody, indicating that they have the same or similar binding epitopes as the reference antibody.

Example 5 Preparation and Characterization of Chimeric Antibodies

The heavy chain variable region and light chain variable region of the anti-mouse BCMA monoclonal antibody were sequenced and summarized in Table 1.

The heavy chain variable region and light chain variable region of the anti-mouse BCMA monoclonal antibody were cloned into the human IgG1 heavy chain constant region (SEQ ID NO: 23) and human kappa light chain constant region (SEQ ID NO: 24), respectively, namely , Connect the C-terminus of the variable region of the anti-mouse BCMA monoclonal antibody to the N-terminus of the constant region of the human IgG1 heavy chain and human kappa light chain, respectively.

The activity of the obtained chimeric antibody was detected using the same method as the capture ELISA method, the competitive ELISA method, and the flow cytometry in the foregoing Examples.

As shown in Table 5 below, the experimental results show that the chimeric antibody and mouse antibody have similar binding capabilities.

Table 5 Binding and functional activities of chimeric antibodies Antibody Capture ELISA (EC ₅₀ , nM) BCMA-BAFF blocking ELISA (IC ₅₀ , nM) Reference antibody blocking ELISA (IC ₅₀ , nM) Mouse B1G3 0.54 3.03 3.91 Mating B1G3 1.47 9.04 9.89 Mouse B1C1 0.73 2.61 3.61 Mosaic B1C1 1.25 2.29 3.01

Example 6 Humanization of anti-BCMA mouse monoclonal antibodies B1G3 and B1C1

The anti-BCMA mouse antibodies B1G3 and B1C1 were selected for humanization and further research. As described in detail below, the mouse monoclonal antibody is humanized using mature CDR grafting methods.

The light chain variable region and heavy chain variable region sequences of the mouse antibodies B1H2, B1G3, and B1C1 were blast aligned with the human immunoglobulin gene database to select the mouse antibodies B1G3 and B1C1 humanized Receptor framework region. The human germline with the highest homology to the mouse antibodies B1G3 and B1C1 was selected as the acceptor framework for humanization. Transplant the mouse antibody heavy chain variable region or light chain variable region CDR into the selected human germline framework region, and back-mutate the amino acid residues in the framework region to obtain more candidate humanized antibodies Heavy heavy chain variable chain or area variable Finally, a total of 15 humanized B1G3 antibodies (ie, huB1G3-V1 to huB1G3-V15) and 12 humanized B1C1 antibodies (huB1C1-V1 to huB1C1-V9 and huB1C1-V11 to huB1C1-V13) were obtained, The sequences of the variable region of the heavy chain and the variable region of the light chain are shown in Table 1.

Will contain the nucleotide sequence encoding the humanized B1G3 or B1C1 heavy chain variable region and the human IgG1 heavy chain constant region (SEQ ID NO: 23) and the nucleotide sequence containing the light chain variable region and human kappa light chain constant region The nucleotide sequence was constructed as a heavy chain expression vector and a light chain expression vector, and added to 20 mL of 293F suspension cell culture medium at a ratio of 47.62% and 52.38%, and 60μg/mL PEI was added, mixed, and incubated at room temperature. Transient transfection into 293F cells in logarithmic growth phase can connect the C-terminus of the variable region to the N-terminus of the respective constant region. After culturing in a shake flask for six days, the cell supernatant was collected, centrifuged quickly to pellet the cells, and filtered through a 0.22 µm filter. The antibody was purified by protein A affinity chromatography. In short, the Protein A Sepharose column (from Bergeron (Shanghai) Biotechnology Co., Ltd., Cat#AA0273) was equilibrated with 5 to 10 column volumes using PBS buffer. The cell supernatant was loaded, and then the purification column was washed with PBS buffer until the absorbance of the protein reached the baseline. The antibody was eluted with elution buffer (0.1M glycine-HCl, pH 2.7), and antibody peaks were collected immediately. The eluted antibody was immediately neutralized with neutralization buffer (1M Tris-HCl, pH 9.0) and placed at 1.5 mL tube. The precipitate fractions containing IgG were combined and dialyzed in PBS at 4°C overnight.

Example 7 Characterization of humanized antibodies B1G3 and B1C1

According to the protocol in the foregoing examples, the affinity, binding activity and other functional activities of the humanized antibody B1G3 and the humanized antibody B1C1 to human BCMA protein and cyno BCMA protein were tested by Biacore, capture ELISA and competitive ELISA. In the capture ELISA, a 2 µg/mL F(ab' ₎₂ fragmented goat anti-human IgG antibody (Jackson Immunoresearch, Cat#109-005-097) was used to coat a 96-well plate, and the mouse antibody was used as a control.

The detection results of the capture ELISA are summarized in Table 6 below and shown in Figures 4A-4C and 5A-5C. It can be seen that huB1G3-V8 and huB1C1-V6 do not bind to human BCMA; the remaining humanized antibodies have comparable or slightly lower binding activity than their parent antibodies or reference antibodies.

Table 6 The binding activity of humanized antibody B1G3 and humanized antibody B1C1 to human BCMA mAbs Capture ELISA (EC ₅₀ ,ng/ml) mAbs Capture ELISA (EC ₅₀ ,ng/ml) mAbs Capture ELISA (EC ₅₀ ,ng/ml) Mouse B1G3 0.942 Mouse B1G3 1.089 Mouse B1G3 0.898 Mating B1G3 1.701 Chimeric B1G3 2.143 Mating B1G3 2.387 huB1G3-V1 1.880 huB1G3-V6 2.136 huB1G3-V11 1.937 huB1G3-V2 1.888 huB1G3-V7 1.949 huB1G3-V12 1.753 huB1G3-V3 2.265 huB1G3-V8 - huB1G3-V13 1.801 huB1G3-V4 1.745 huB1G3-V9 2.263 huB1G3-V14 1.977 huB1G3-V5 1.759 huB1G3-V10 1.841 huB1G3-V15 2.011 BM 1.088 BM 1.155 BM 1.033 Mouse IgG - Mouse IgG - Mouse IgG - Human IgG - Human IgG - Human IgG - Mouse B1C1 0.638 Mouse B1C1 0.663 Mouse B1C1 1.201 Mosaic B1C1 0.832 Chimeric B1C1 1.122 Chimeric B1C1 1.050 huB1C1-V1 0.675 huB1C1-V8 0.859 huB1C1-V11 1.331 huB1C1-V2 0.705 huB1C1-V9 0.769 huB1C1-V12 1.184 huB1C1-V3 0.670 BM 0.801 huB1C1-V13 1.140 huB1C1-V4 0.709 Mouse IgG - BM 1.228- huB1C1-V5 0.753 Human IgG - Mouse IgG - huB1C1-V6 - Human IgG - huB1C1-V7 0.573 BM 0.591 Mouse IgG - Human IgG -

The humanized antibodies huB1G3-V13 and huB1C1-V13 were further characterized. In brief, the binding activity of these two antibodies with human and monkey BCMA, and their ability to block the interaction of BCMA with BAFF or the reference antibody with BCMA were tested according to the protocol of the previous example.

The experimental results are summarized in Table 7 below and shown in Figures 6 to 11.

The capture ELISA test results showed that the antibody huB1G3-V13 has good binding activity to human BCMA, which is comparable to that of the reference antibody (Figure 6); the binding affinity of huB1G3-V13 to human or monkey BCMA is slightly lower than that of the reference antibody. In addition, the antibody huB1G3-V13 can effectively block the BCMA-BAFF interaction, which has almost the same blocking ability as the reference antibody (Figure 7); the antibody huB1G3-V13 can also block the interaction between the reference antibody and BCMA ( Figure 8).

The capture ELISA test results showed that the antibody huB1C1-V13 has good binding activity to human BCMA, which is slightly lower than the reference antibody (Figure 9); the binding affinity of huB1C1-V13 to human or monkey BCMA is also slightly lower than that of the reference antibody. In addition, huB1C1-V13 can effectively block the interaction between BCMA and BAFF, and has almost the same blocking ability as the reference antibody (Figure 10); huB1C1-V13 can also block the interaction between the reference antibody and BCMA (Figure 10). 11).

Table 7 Binding and functional activities of humanized antibodies huB1G3-V13 and huB1C1-V13 BCMA antibody Binding activity assay Functional activity determination People BCMA Crab-eating monkey BCMA Competitive ELISA (IC ₅₀ , nM) Capture ELISA (EC ₅₀ , nM) Biacore dynamics (K _D , M) Biacore dynamics (K _D , M) BCMA/BAFF-Fc ligand blocking ELISA Reference antibody blocking ELISA huB1G3-V13 1.33 2.03E-09 8.87E-09 3.86 6.41 Mating B1G3 1.47 2.22E-09 Not tested 9.04 9.89 Mouse B1G3 0.54 6.83E-10 2.01E-09 3.03 3.91 BM 0.71 7.42E-10 5.99E-10 3.64 2.23 huB1C1-V13 1.60 2.05E-09 5.11E-08 4.05 6.16 Mosaic B1C1 1.25 1.25E-09 / 2.29 3.01 Mouse B1C1 0.73 8.79E-10 1.86E-09 2.61 3.61 BM 0.93 7.42E-10 5.99E-10 3.59 1.64

Although the present invention has been described by one or more embodiments, it should be understood that the present invention is not limited to these embodiments, and the description of the present invention is intended to cover all alternatives falling within the spirit and broad scope of the appended claims. , Amendments and changes. All references cited in the present invention are incorporated into the present invention by reference in their entirety. Sequence summary table: Description/ Sequence/SEQ ID NO. VH CDR1 for mouse, chimeric and humanized B1G3 and B1C1 NX1GMN (SEQ ID NO.: 1) VH CDR1 for mouse, chimeric and humanized B1G3, X1=H, NHGMN (SEQ ID NO.: 30) VH CDR2 for mouse, chimeric and humanized B1G3 WINTYTGEPTYADDFKG (SEQ ID NO.: 3) VH CDR3 for mouse, chimeric and humanized B1G3 YSYYGYYHAMDY (SEQ ID NO.: 5) VL CDR1 for mouse, chimeric and humanized B1G3 and B1C1 RASSSVX1YMH (SEQ ID NO.: 7) VL CDR1 for mouse, chimeric and humanized B1G3, X1=H, RASSSVHYMH (SEQ ID NO.: 31) VL CDR2 for mouse, chimeric and humanized B1G3 and B1C1 ATSX1LAS (SEQ ID NO.: 9) VL CDR2 for mouse, chimeric and humanized B1G3, X1=N, ATSNLAS (SEQ ID NO.: 32) VL CDR3 for mouse, chimeric and humanized B1G3 QQWSSNPT (SEQ ID NO.: 11) VH for mouse and chimeric B1G3 QIQLVQSGPELKKPGETVKISCKASGYTFTNHGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSATTAYLQINNLRIEDTATYFCARYSYYGYYHAMDYWGQGTSVTVSS (SEQ ID NO.: 13) VH for huB1G3-V1, huB1G3-V6- huB1G3-V10, huB1G3-V12, VH for huB1G3-V2, VH for huB1G3-V3, VH for huB1G3-V4, VH for huB1G3-V5, VH for huB1G3-V11 V13, VH for huB1G3-V14 and huB1G3-V15 EX1QLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSX2X3TAYLQIX4SLKAEDTAVYX5CARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 14) VH for huB1G3-V1, huB1G3-V6- huB1G3-V10, huB1G3-V12, X1 = I, X2 = A, X3 = T, X4=C, X5=F EIQLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSATTAYLQICSLKAEDTAVYFCARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO.: 33) EX1QLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSX2X3TAYLQIX4SLKAEDTAVYX5CARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 14) VH for huB1G3-V2, X1 = V, X2 = A, X3 = T, X4 = C, X5 = F E V QLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSATTAYLQICSLKAEDTAVYFCARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 34) EX1QLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSX2X3TAYLQIX4SLKAEDTAVYX5CARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 14) VH for huB1G3-V3, X1 = I, X2 = V, X3 = T, X4 = C, X5 = F EIQLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTS V TTAYLQICSLKAEDTAVYFCARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 35) EX1QLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSX2X3TAYLQIX4SLKAEDTAVYX5CARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 14) VH for huB1G3-V4, X1 = I, X2 = A, X3 = S, X4 = C, X5 = F EIQLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSA S TAYLQICSLKAEDTAVYFCARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 36) EX1QLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSX2X3TAYLQIX4SLKAEDTAVYX5CARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 14) VH for huB1G3-V5, X1 = I, X2 = A, X3 = Y, X4 = C, X5 = Y EIQLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSATTAYLQICSLKAEDTAVY Y CARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 37) EX1QLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSX2X3TAYLQIX4SLKAEDTAVYX5CARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 14) VH for huB1G3-V11 and huB1G3-V13, X1 = V, X2 = V, X3 = S, X4 = S, X5 = Y E V QLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 38) EX1QLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSX2X3TAYLQIX4SLKAEDTAVYX5CARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 14) VH for huB1G3-V14 and huB1G3-V15, X1 = V, X2 = V, X3 = S, X4 = C, X5 = Y EVQLVQSGSELKKPGASVKVSCKASGYTFTNHGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQICSLKAEDTAVYYCARYSYYGYYHAMDYWGQGTLVTVSS (SEQ ID NO .: 39) VL for mouse and chimeric B1G3 QIVLSQSPAILSASPGEKVTMTCRASSSVHYMHWYQQKPGSPPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPTFGGGTKLEIK (SEQ ID NO.: 18) VL for huB1G3-V1- huB1G3-V5, huB1G3-V11, and huB1G3-V14, VL for huB1G3-V6, VL for huB1G3-V7, VL for huB1G3-V8, VL for huB1G3-V9, VL for huB1G3-V9, VL for huB1G3-V6 for huB1G3-V12, huB1G3-V13 and huB1G3-V15 DIQX1TQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKX2PKX3X4IYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 19) X1 = L, X2 = P, X3 = P, X4 = W, X5 = Y DIQLTQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKPPKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 40) DIQX1TQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKX2PKX3X4IYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 19) VL for huB1G3-V6, X1 = M, X2 = P, X3 = P, X4 = W, X5 = Y DIQMTQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKPPKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 41) DIQX1TQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKX2PKX3X4IYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 19) VL for huB1G3-V7, X1 = L, X2 = A, X3 = P, X4 = W, X5 = Y DIQLTQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKAPKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 42) DIQX1TQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKX2PKX3X4IYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 19) VL for huB1G3-V8, X1 = L, X2 = P, X3 = L, X4 = W, X5 = Y DIQLTQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKPPKLWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 43) DIQX1TQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKX2PKX3X4IYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 19) VL for huB1G3-V9, X1 = L, X2 = P, X3 = P, X4 = L, X5 = Y DIQLTQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKPPKPLIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 44) DIQX1TQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKX2PKX3X4IYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 19) VL for huB1G3-V10, X1 = L, X2 = P, X3 = P, X4 = W, X5 = F DIQLTQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKPPKPWIYATSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 45) DIQX1TQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKX2PKX3X4IYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 19) VL for huB1G3-V12, huB1G3-V13 and huB1G3-V15, X1 = M, X2 = A, X3 = P, X4 = L, X5 = F DIQMTQSPSSLSASVGDRVTITCRASSSVHYMHWYQQKPGKAPKPLIYATSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPTFGQGTKVEIK (SEQ ID NO .: 46 ) NX1GMN (SEQ ID NO.: 1) VH CDR1 for mouse, chimeric and humanized B1C1, X1=F NFGMN (SEQ ID NO.: 47) VH CDR2 for mouse, chimeric and humanized B1C1 WINTYTGEPTYADDFKG (SEQ ID NO.: 3) VH CDR3 for mouse, chimeric and humanized B1C1 YSYYGYYHAMDY (SEQ ID NO.: 5) RASSSVX1YMH (SEQ ID NO.: 7) VL CDR1 for mouse, chimeric and humanized B1C1, X1=F RASSSVFYMH (SEQ ID NO.: 48) ATSX1LAS (SEQ ID NO.: 9) VL CDR2 for mouse, chimeric and humanized B1C1, X1=T ATSTLAS (SEQ ID NO.: 49) VL CDR3 for mouse, chimeric and humanized B1C1 QQWSSNPT (SEQ ID NO.: 11) VH for mouse and chimeric B1C1 QIQLVQSGPELKKPGETVKISCKASGYIFTNFGMNWVKQAPGKDLKWMGWINTYTGEPTYADDFKGRFAFSLENSASTASLQIDNLKNEDTATYFCARYSYYGYYHAMDYWGQGASVTVSS (SEQ ID NO.: 15) VH for huB1C1-V1, huB1C1-V5- huB1C1-V9, and huB1C1-V12, VH for huB1C1-V2, VH for huB1C1-V3, VH for huB1C1-V4, VH for huB1C1-V11 and huB1C1-V13 EX1QLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDX2SVSTAYLQISSLKAEDTAVYX3CARYSYYGYYHAMDYWGQGTTVTVSS (SEQ ID NO.: 16) VH for huB1C1-V1, huB1C1-V5- huB1C1-V9, and huB1C1-V12 X1=I, X2=N, X3=F EIQLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVRQAPGQGLEWMGWINTYLTYVVRQAPGQGLEWMGWINTYLTYVVRQAPGQGLEWMGWINTYLTYVVRQAPGQGLEWMGTDYVSGVSGVSGVSSYYFKGY: EX1QLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDX2SVSTAYLQISSLKAEDTAVYX3CARYSYYGYYHAMDYWGQGTTVTVSS (SEQ ID NO .: 16) VH for huB1C1-V2, X1 = V, X2 = N, X3 = F EVQLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDNSVSTAYLQISSLKAEDTAVYFCARYSYYGYYHAMDYWGQGTTVTVSS (SEQ ID NO .: 51) EX1QLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDX2SVSTAYLQISSLKAEDTAVYX3CARYSYYGYYHAMDYWGQGTTVTVSS (SEQ ID NO .: 16) VH for huB1C1-V3, X1 = I, X2 = T, X3 = F EIQLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARYSYYGYYHAMDYWGQGTTVTVSS (SEQ ID NO .: 52) EX1QLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDX2SVSTAYLQISSLKAEDTAVYX3CARYSYYGYYHAMDYWGQGTTVTVSS (SEQ ID NO .: 16) VH for huB1C1-V4, X1 = I, X2 = N, X3 = Y EIQLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDNSVSTAYLQISSLKAEDTAVYYCARYSYYGYYHAMDYWGQGTTVTVSS (SEQ ID NO .: 53) EX1QLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDX2SVSTAYLQISSLKAEDTAVYX3CARYSYYGYYHAMDYWGQGTTVTVSS (SEQ ID NO .: 16) VH for huB1C1-V11 and huB1C1-V13, X1 = V, X2 = T, X3 = Y EVQLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARYSYYGYYHAMDYWGQGTTVTVSS (SEQ ID NO .: 54) VL for mouse and chimeric B1C1 QIVLSQSPTILSASPGEEVTMTCRASSSVFYMHWYQQKPGSSPKPWIYATSTLASGVPARFSGSGSGTSYSLSISRVEAEDAATYFCQQWSSNPTFGGGTKLEIK (SEQ ID NO.: 20) VL for huB1C1-V1- huB1C1-V4 and huB1C1-V11, VL for huB1C1-V5, VL for huB1C1-V6, VL for huB1C1-V7, VL for huB1C1-V8, VL for huB1C1-V9, VL for huB1C1 huB1C1-V13 DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKX1PKX2X3IYATSTLASGVPSRFSGSGSGTEX4TLTISSLQPEDFATYX5CQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 21) VL for huB1C1-V1- huB1C1-V4 and huB1C1-V11, X1 = S, X2 = P, X3 = W, X4 = Y, X5 = F DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKSPKPWIYATSTLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYFCQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 55) DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKX1PKX2X3IYATSTLASGVPSRFSGSGSGTEX4TLTISSLQPEDFATYX5CQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 21) VL for huB1C1-V5, X1 = A, X2 = P, X3 = W, X4 = Y, X5 = F DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKAPKPWIYATSTLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYFCQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 56) DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKX1PKX2X3IYATSTLASGVPSRFSGSGSGTEX4TLTISSLQPEDFATYX5CQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 21) VL for huB1C1-V6, X1 = S, X2 = L, X3 = W, X4 = Y, X5 = F DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKSPKLWIYATSTLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYFCQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 57) DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKX1PKX2X3IYATSTLASGVPSRFSGSGSGTEX4TLTISSLQPEDFATYX5CQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 21) VL for huB1C1-V7, X1 = S, X2 = P, X3 = L, X4 = Y, X5 = F DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKSPKPLIYATSTLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYFCQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 58) DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKX1PKX2X3IYATSTLASGVPSRFSGSGSGTEX4TLTISSLQPEDFATYX5CQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 21) VL for huB1C1-V8, X1 = S, X2 = P, X3 = W, X4 = F, X5 = F DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKSPKPWIYATSTLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 59) DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKX1PKX2X3IYATSTLASGVPSRFSGSGSGTEX4TLTISSLQPEDFATYX5CQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 21) VL for huB1C1-V9, X1 = S, X2 = P, X3 = W, X4 = Y, X5 = Y DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKSPKPWIYATSTLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 60) DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKX1PKX2X3IYATSTLASGVPSRFSGSGSGTEX4TLTISSLQPEDFATYX5CQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 21) VL for huB1C1-V12 and huB1C1-V13, X1 = A, X2 = P, X3 = L, X4 = F, X5 = Y DIQLTQSPSFLSASVGDRVTITCRASSSVFYMHWYQQKPGKAPKPLIYATSTLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPTFGGGTKVEIK (SEQ ID NO .: 61) VH CDR1 for mouse, chimeric and humanized B1A1 SAYYWN (SEQ ID NO.: 2) VH CDR2 for mouse, chimeric and humanized B1A1 YIRYDGSNNYNPSLKN (SEQ ID NO.: 4) VH CDR3 for mouse, chimeric and humanized B1A1 YDYDEVFAY (SEQ ID NO.: 6) VL CDR1 for mouse, chimeric and humanized B1A1 KASQEVSTAVA (SEQ ID NO.: 8) VL CDR2 for mouse, chimeric and humanized B1A1 WASMRHT (SEQ ID NO.: 10) VL CDR3 for mouse, chimeric and humanized B1A1 QQYYSTPLT (SEQ ID NO.: 12) VH for B1A1 DVQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGNKLEWMGYIRYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTAEDTATYYCAPYDYDEVFAYWGQGTLVTVSA (SEQ ID NO.: 17) VL for B1A1 DIVMTQSHKFMSTSVGDRVSITCKASQEVSTAVAWYQQKPGQSPKLLIYWASMRHTGVPDRFTGSGSGTDYTLIITSVQAEDLALYYCQQYYSTPLTFGAGTKLELK (SEQ ID NO.: 22) Heavy chain constant region for chimeric and humanized antibodies ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO .: 23) GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO.:28) Light chain constant region for chimeric and humanized antibodies RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO .: 24) CGTACGGTGGCGGCGCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA (SEQ ID NO .: 29) Human BCMA MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKNOCKEICKVDSDHCFPLPAMEEGATILVTTKNOCKS Heavy chain of Benchmark QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO .: 26) Light chain of Benchmark DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREATEKVQWKDSHQSLQTLACEKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREATEKVQWKDSHQVVTSLIDKASLID Note: In the nucleotide sequence list submitted, X1, X2, X3, X4, X5 are represented by Xaa

without.

[Figure 1A] and [Figure 1B] show the binding ability of murine antibodies B1A1 and B1C1 (A) and B1G3 (B) to human BCMA. [Figure 2 A] and [Figure 2B] show the ability of murine antibodies B1A1 and B1C1 (A) and B1G3 (B) to block the interaction between human BCMA and BAFF. [Figure 3A] and [Figure 3B] show the ability of murine antibodies B1A1 and B1C1 (A) and B1G3 (B) to block the binding of reference antibody BM to human BCMA. [Figure 4A], [Figure 4B] and [Figure 4C] show the humanized antibodies huB1G3-V1 to huB1G3-V5 (A), huB1G3-V6 to huB1G3-V10 (B) and huB1G3-V11 to huB1G3-V15 ( C) Ability to bind human BCMA. [Figure 5A], [Figure 5B] and [Figure 5C] show the humanized antibodies huB1C1-V1 to huB1C1-V7 (A), huB1C1-V8 to huB1C1-V9 (B) and huB1C1-V11 to huB1C1-V13 ( C) Ability to bind human BCMA. [Figure 6] shows the binding ability of the humanized antibody huB1G3-V13 to human BCMA. [Figure 7] shows the ability of the humanized antibody huB1G3-V13 to block the interaction between human BCMA and BAFF. [Figure 8] shows the ability of the humanized antibody huB1G3-V13 to block the interaction of the reference antibody BM-human BCMA. [Figure 9] shows the binding ability of the humanized antibody huB1C1-V13 to human BCMA. [Figure 10] shows the ability of the humanized antibody huB1C1-V13 to block the interaction between human BCMA and BAFF. [Figure 11] shows the ability of the humanized antibody huB1C1-V13 to block the interaction between the reference antibody BM and human BCMA.

Claims (19)

An isolated monoclonal antibody or antigen-binding fragment thereof that binds BCMA, comprising: The heavy chain variable region of a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region and the CDR3 region comprise: SEQ ID NO: 1 (X1=H or F), 3 and 5, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 as shown in SEQ ID NOs: 2, 4 and 6, respectively % Or 100% identical amino acid sequence. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the CDR1 region, the CDR2 region and the CDR3 region further comprise SEQ ID NO: 13, SEQ ID NO: 14 (X1=1, X2=A, X3=T, X4=C, X5=F; X1=V, X2=A, X3=T, X4=C, X5=F; X1=I, X2=V, X3=T, X4=C, X5= F; X1=I, X2=A, X3=S, X4=C, X5=F; X1=I, X2=A, X3=Y, X4=C, X5=Y; X1=V, X2=V, X3=S, X4=S, X5=Y; or X1=V, X2=V, X3=S, X4=C, X5=Y), SEQ ID NO: 15, SEQ ID NO: 16 (X1=I, X2=N, X3=F; X1=V, X2=N, X3=F; X1=I, X2=T, X3=F; X1=I, X2=N, X3=Y; or X1=V, X2 =T, X3=Y) or SEQ ID NO: 17 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or 100% identical amino acid sequence. According to claim 1, the isolated monoclonal antibody or antigen-binding fragment thereof, wherein the CDR1 region, the CDR2 region and the CDR3 region further include SEQ ID NO: 7 (X1 = H or F), SEQ ID NO: 9 (X1=N or T) and 11, or at least 85%, 90%, 91%, 92%, 93% shown in SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 respectively, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the CDR1 region, the CDR2 region and the CDR3 region further comprise SEQ ID NO: 18, SEQ ID NO: 19 (X1=L, X2=P, X3=P, X4=W, X5=Y; X1=M, X2=P, X3=P, X4=W, X5=Y; X1=L, X2=A, X3=P, X4=W, X5= Y; X1=L, X2=P, X3=L, X4=W, X5=Y; X1=L, X2=P, X3=P, X4=L, X5=Y; X1=L, X2=P, X3=P, X4=W, X5=F; or X1=M, X2=A, X3=P, X4=L, X5=F), SEQ ID NO: 20, SEQ ID NO: 21 (X1=S, X2=P, X3=W, X4=Y, X5=F; X1=A, X2=P, X3=W, X4=Y, X5=F; X1=S, X2=L, X3=W, X4= Y, X5=F; X1=S, X2=P, X3=L, X4=Y, X5=F; X1=S, X2=P, X3=W, X4=F, X5=F; X1=S, X2=P, X3=W, X4=Y, X5=Y; X1=A, X2=P, X3=L, X4=F, X5=Y) or SEQ ID NO: 22 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 3, wherein its heavy chain variable region and light chain variable region further comprise SEQ ID NO:1 (X1=H), SEQ ID NO: 3. SEQ ID NO: 5, SEQ ID NO: 7 (X1=H), SEQ ID NO: 9 (X1=N), and SEQ ID NO: 11, respectively as SEQ ID NO: 1 (X1=F), SEQ ID NO: ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 (X1=F), SEQ ID NO: 9 (X1=T) and SEQ ID NO: 11, or as SEQ ID NO: 2 and SEQ ID respectively NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein its heavy chain variable region and light chain variable region further comprise SEQ ID NO: 13 and SEQ ID NO: 18, respectively, and SEQ ID NO: :14 (X1=I, X2=A, X3=T, X4=C, X5=F; X1=V, X2=A, X3=T, X4=C, X5=F; X1=I, X2=V , X3=T, X4=C, X5=F; X1=I, X2=A, X3=S, X4=C, X5=F; X1=I, X2=A, X3=Y, X4=C, X5 =Y; X1=V, X2=V, X3=S, X4=S, X5=Y; or X1=V, X2=V, X3=S, X4=C, X5=Y) and SEQ ID NO: 19 (X1=L, X2=P, X3=P, X4=W, X5=Y), respectively as SEQ ID NO: 14 (X1=I, X2=A, X3=T, X4=C, X5=F) And SEQ ID NO: 19 (X1=M, X2=P, X3=P, X4=W, X5=Y; X1=L, X2=A, X3=P, X4=W, X5=Y; X1=L , X2=P, X3=L, X4=W, X5=Y; X1=L, X2=P, X3=P, X4=L, X5=Y; or X1=L, X2=P, X3=P, X4=W, X5=F), respectively with SEQ ID NO: 14 (X1=I, X2=A, X3=T, X4=C, X5=F; or X1=V, X2=V, X3=S, X4=C, X5=Y) and SEQ ID NO: 19 (X1=M, X2=A, X3=P, X4=L, X5=F) are respectively SEQ ID NO: 15 and SEQ ID NO: 20, respectively Take SEQ ID NO: 16 (X1=I, X2=N, X3=F; X1=V, X2=N, X3=F; X1=I, X2=T, X3=F; X1=I, X2=N , X3=Y; or X1=V, X2=T, X3=Y) and SEQ ID NO: 21 (X1=S, X2=P, X3=W, X4=Y, X5=F), respectively with SEQ ID NO: 16 (X1=I, X2=N, X3=F) and SEQ ID NO: 21 (X1=A, X2=P, X3=W, X4=Y, X5=F; X1=S, X2=L , X3=W, X4=Y, X5=F; X1=S, X2=P, X3=L, X4=Y, X5=F; X1=S, X2=P, X3=W, X4=F, X5 =F; or X1=S, X2=P, X3=W, X4=Y, X5=Y), respectively in SEQ ID NO: 16 (X1=I, X2=N, X3=F; or X1=V, X2=T, X3=Y) and SEQ ID NO: 21 (X1=A, X2=P, X3=L , X4=F, X5=Y), or at least 85%, 90%, 91%, 92%, 93%, 94%, 95% shown in SEQ ID NO: 17 and SEQ ID NO: 22, respectively, 96%, 97%, 98%, 99% or 100% identical amino acid sequence. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 6, wherein its heavy chain constant region further comprises at least 85%, 90%, 91%, 92%, 93%, 94 shown in SEQ ID NO: 23 %, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence, and the light chain constant region comprises at least 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein (a) binds to human or monkey BCMA; and (b) blocks the interaction of human BCMA-human BAFF or APRIL. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the isolated monoclonal antibody or antigen-binding fragment thereof is murine, human, chimeric or humanized monoclonal antibody or antigen-binding fragment thereof . The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that the isolated monoclonal antibody or antigen-binding fragment thereof is a full-length antibody of IgG1, IgG2, or IgG4 isotype or an antigen-binding fragment thereof. A pharmaceutical composition, characterized in that it contains the isolated monoclonal antibody or antigen-binding fragment thereof as described in claim 1, and one or more pharmaceutically acceptable carriers. Such as the pharmaceutical composition of claim 11, further comprising one or more anti-tumor drugs and/or cytokines. A method for treating cancer diseases related to increased expression of BCMA in a subject, which is characterized by comprising administering to the subject a therapeutically effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, Or the pharmaceutical composition of claim 11. According to the method of claim 13, the cancer disease is a non-solid tumor or a solid tumor. According to the method of claim 13, the cancer disease includes leukemia, lymphoma or multiple myeloma. The method of claim 13, further comprising administering immune checkpoint antibodies, co-stimulatory antibodies and/or cytokines. According to the method of claim 16, the immune checkpoint antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, or a combination thereof. According to the method of claim 16, the costimulatory antibody is an anti-CD137 antibody, an anti-OX40 antibody or an anti-GITR antibody, or a combination thereof. According to the method of claim 16, the cytokine is IL-2, IL-21, IFN-γ, TNF-α and/or IL-4, or a combination thereof.

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