Antibodies against CD3 and BCMA, and bispecific binding proteins derived from them.

Abstract

This invention provides a novel antibody that binds to CD3 with high affinity and a novel antibody that binds to BCMA with high affinity. [Solution] High-affinity antibodies that recognize CD3 and B-cell maturation factor protein (BCMA) are provided. Binding sites derived from humanized anti-CD3 and anti-BCMA antibodies are incorporated into a Fabs-in-Tandem immunoglobulin format without significant loss of binding affinity, and the resulting bispecific polyvalent binding protein can simultaneously bind to both CD3 and BCMA. Such antibodies, their antigen-binding moieties, and bispecific FIT-Ig binding proteins are useful in the treatment of cancer.

Description

[Technical Field]

[0001] This invention relates to a novel antibody that recognizes CD3, and a B cell maturation antigen. This paper relates to novel antibodies that recognize BCMA, and to bispecific BCMA / CD3-binding proteins such as FIT-Ig-binding proteins and MAT-Fab-binding proteins produced using these antibodies. The antibodies and bispecific binding proteins are useful in the treatment of immune diseases and hematological cancers. [Background technology]

[0002] Cluster of Differentiation 3 (CD3) T cell receptors (TCRs) bind to antigens (Ags) presented by the major histocompatibility complex (MHC) and play a crucial role in T cell function. However, TCRs themselves do not participate in intracellular signaling. Instead, TCRs non-covalently associate with the differentiation antigen group 3 (CD3) complex and transmit intracellular signaling via the CD3 immunoreceptor tyrosine-based activation motifs (ITAMs). The CD3 T cell coreceptor helps activate both cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). It is a protein complex composed of four distinct chains. In mammals, this complex contains the CD3γ chain, CD3δ chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and the CD3δ chain (zeta chain) to generate an activation signal to T lymphocytes. The TCR, ζ chain, and CD3γ, δ, and ε chains together constitute the TCR complex. The four-chain complex of CD3 then forms the CD3εγ, CD3εδ, and ζζ dimers in a 1:1:1 stoichiometric ratio.

[0003] CD3 is initially expressed in the cytoplasm of prothymocytes, which are stem cells from which T cells originate in the thymus. Prothymocytes differentiate into general thymocytes and then into medullary thymocytes, but it is at this latter stage that the CD3 antigen begins to migrate to the cell membrane. The antigen is found bound to the membrane of all mature T cells and is virtually absent from other cell types, although it appears to be present in small amounts in Purkinje cells.

[0004] This high specificity, coupled with the presence of CD3 at all stages of T cell development, makes it a useful immunohistochemical marker for T cells in tissue sections. Because the antigen persists in almost all T-cell lymphomas and leukemias, it can be used to distinguish them from superficially similar B-cell and myeloid neoplasms. Several antibodies against the CD3ε chain have been shown to activate the TCR-CD3 complex, possibly by clustering the CD3 complex on T cells. Furthermore, bispecific antibodies targeting both CD3 and tumor-specific antigens are being studied for redirection of tumor eradication by T cells. Since CD3 is required for T cell activation, drugs targeting CD3 (often monoclonal antibodies) are being investigated as immunosuppressive therapies for type 1 diabetes and other autoimmune diseases (e.g., otelixizumab).

[0005] B cell maturation antigen (BCMA) B cell maturation antigens (BCMA, TNFRSF17, CD269) are members of the TNF receptor superfamily. BCMA expression is limited to the B cell lineage, primarily expressed in plasma cells and plasmablasts, and absent in naive B cells. BCMA has two ligands: proliferation-inducing ligands (APRIL, TNFSF13, TALL-2, CD256) and B cell activators (BAFF, BLYS, TNFSF13B, TALL-1, CD256). BCMA binds to APRIL (257). BCMA has a higher binding affinity to APRIL than BAFF. Multiple myeloma (MM) cells express high levels of BCMA. BCMA-targeted antibodies with ligand-blocking activity, both as naked IgG and as drug conjugates, can promote cytotoxicity of MM cells. Also, by restricted expression of BCMA in late-mature B cells, this makes it an ideal co-target for chimeric antigen receptor T cells (CAR-T cells), which are T cells that have been genetically engineered to express chimeric antigen receptors to target specific cellular proteins. CAR-T cells offer a promising immunotherapy for B-cell cancer, but their mechanisms of action are not well understood, and the side effects of CAR-T cell therapy are often severe, including cytokine release syndrome (cytokine storm) and neurotoxicity.

[0006] Understanding the roles of CD3 and BCMA has also led to related immunotherapies known as bispecific T-cell redirecting antibodies. Bispecific antibodies targeting both CD3 and BCMA may be useful in treating multiple myeloma via redirected T-cell cytotoxicity (RTCC). [Overview of the project]

[0007] The present invention provides novel antibodies that bind to CD3 with high affinity and novel antibodies that bind to BCMA with high affinity. The present invention also provides a BCMA / CD3 bispecific Fabs-in-Tandem immunoglobulin (FIT-Ig) that is reactive with both CD3 and BCMA. The present invention also provides a BCMA / CD3 bispecific monovalent asymmetric tandem Fab antibody (MAT-Fab) that is reactive with both CD3 and BCMA. The antibodies and bispecific binding proteins of the present invention can activate the TCR-CD3 complex. The bispecific multivalent binding proteins described herein are useful as CMA / CD3 bispecific inhibitors to provide a synergistic combination effect in the treatment of multiple myeloma (MM) cells by redirected T cell cytotoxicity.

[0008] The present invention also provides methods for producing and using anti-CD3 antibodies, anti-BCMA antibodies, and BCMA / CD3 bispecific binding proteins described herein, as well as methods for producing various compositions that can be used in detecting CD3 and / or BCMA in a sample or in treating or preventing disorders related to CD3 activity and / or BCMA activity in an individual.

[0009] In further embodiments, the present invention comprises a first, second, and third polypeptide chain, The first polypeptide chain is (i)VL from the amino terminus to the carboxyl terminus. A -CL-VH B -CH1-Fc(where CL is VH B (i) Directly fused, or (ii) VH B -CH1-VL A -CL-Fc(where CH1 is VL A (Includes being directly fused with) The second polypeptide chain is VH from the amino terminus to the carboxyl terminus. A -Contains CH1 and The third polypeptide chain is VL from the amino terminus to the carboxyl terminus. B -Includes CL; The present invention provides a bispecific Fabs-in-Tandem immunoglobulin (FIT-Ig) binding protein in which VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is a heavy chain constant domain, Fc is an immunoglobulin Fc region, A is an epitope of CD3 or BCMA, and B is an epitope of CD3 or BCMA, provided that A and B are different. In the present invention, such a FIT-Ig binding protein binds to both CD3 and BCMA.

[0010] In one further embodiment, the Fab fragment of such a FIT-Ig binding protein contains a VL derived from a parent antibody that binds to either the antigen target CD3 or BCMA.A Domain and VH A A VL derived from a different parental antibody in which the domain is incorporated and binds to the other of the antigen targets CD3 and BCMA B Domain and VH B The VH of the first, second, and third polypeptide chains is such that the domain is incorporated A -CH1 / VL A -CL and VH B -CH1 / VL B From the pairing of -CL, a tandem Fab portion that recognizes CD3 and BCMA is obtained

[0011] In the present invention, the BCMA / CD3 FIT-Ig binding protein advantageously comprises first, second, and third polypeptide chains, wherein the first polypeptide chain, from amino terminus to carboxyl terminus, is VL CD3 -CL-VH BCMA -CH1-Fc (where CL is directly fused to VH BCMA ), the second polypeptide chain, from amino terminus to carboxyl terminus, comprises VH CD3 -CH1; and the third polypeptide chain, from amino terminus to carboxyl terminus, comprises VL BCMA -CL; VL CD3 is the light chain variable domain of an anti-CD3 antibody, CL is the light chain constant domain, VH CD3 is the heavy chain variable domain of an anti-CD3 antibody, CH1 is the heavy chain constant domain, VL BCMA is the light chain variable domain of an anti-BCMA antibody, VH BCMA is the heavy chain variable domain of an anti-BCMA antibody, and Fc is the immunoglobulin Fc region. Advantageously, in the first polypeptide chain, the domain VL CD3 -CL is the same as the light chain of the anti-CD3 parental antibody, the domain VH CD3 -CH1 is the same as the heavy chain variable domain and heavy chain constant domain of the anti-CD3 parental antibody, the domain VL BCMA -CL is the same as the light chain of the anti-BCMA parental antibody, and the domain VH BCMA-CH1 is the same as the heavy chain variable domain and heavy chain constant domain of the anti-BCMA parent antibody.

[0012] In another embodiment, the BCMA / CD3 FIT-Ig binding protein may advantageously comprise first, second, and third polypeptide chains, the first polypeptide chain being VL from the amino terminus to the carboxyl terminus. BCMA -CL-VH CD3 -CH1-Fc(where CL is VH CD3 The second polypeptide chain comprises (directly fused to), and the second polypeptide chain is VH from the amino terminus to the carboxyl terminus. BCMA - Containing CH1; and the third polypeptide chain is VL from the amino terminus to the carboxyl terminus. CD3 -Includes CL; VL CD3 is the light chain variable domain of the anti-CD3 antibody, CL is the light chain constant domain, and VH CD3 CH1 is the heavy chain variable domain of the anti-CD3 antibody, and VL is the heavy chain constant domain. BCMA This is the light chain variable domain of the anti-BCMA antibody, VH BCMA is the heavy chain variable domain of the anti-BCMA antibody, and Fc is the immunoglobulin Fc region. Advantageously, in the first polypeptide chain, domain VL BCMA -CL is the same as the light chain of the anti-BCMA parent antibody, and domain VH BCMA -CH1 is the same as the heavy chain variable and heavy chain constant domains of the anti-BCMA parent antibody, and domain VL CD3 -CL is the same as the light chain of the anti-CD3 parent antibody, and the domain VH CD3 -CH1 is the same as the heavy chain variable domain and heavy chain constant domain of the anti-CD3 parental antibody.

[0013] In another embodiment, the BCMA / CD3 FIT-Ig binding protein may advantageously comprise first, second, and third polypeptide chains, wherein the first polypeptide chain is VH from the amino terminus to the carboxyl terminus. BCMA -CH1-VL CD3 -CL-Fc(where CH1 is VL CD3The second polypeptide chain comprises (directly fused to) and the second polypeptide chain is VL from the amino terminus to the carboxyl terminus. BCMA - Containing CL; and the third polypeptide chain is VH from the amino terminus to the carboxyl terminus. CD3 -Includes CH1; VL CD3 is the light chain variable domain of the anti-CD3 antibody, CL is the light chain constant domain, and VH CD3 CH1 is the heavy chain variable domain of the anti-CD3 antibody, and VL is the heavy chain constant domain. BCMA This is the light chain variable domain of the anti-BCMA antibody, VH BCMA is the heavy chain variable domain of the anti-BCMA antibody, and Fc is This is the immunoglobulin Fc region. Advantageously, in the first polypeptide chain, the domain VL BCMA -CL is the same as the light chain of the anti-BCMA parent antibody, and domain VH BCMA -CH1 is the same as the heavy chain variable domain and heavy chain constant domain of the anti-BCMA parent antibody, and domain VL CD3 -CL is the same as the light chain of the anti-CD3 parent antibody, and the domain VH CD3 -CH1 is the same as the heavy chain variable domain and heavy chain constant domain of the anti-CD3 parental antibody.

[0014] In another embodiment, the BCMA / CD3 FIT-Ig binding protein may advantageously comprise first, second, and third polypeptide chains, wherein the first polypeptide chain is VH from the amino terminus to the carboxyl terminus. CD3 -CH1-VL BCMA -CL-Fc(where CH1 is VL BCMA The second polypeptide chain comprises (directly fused to) and the second polypeptide chain is VL from the amino terminus to the carboxyl terminus. CD3 - Containing CL; and the third polypeptide chain is VH from the amino terminus to the carboxyl terminus. BCMA -Includes CH1; VL CD3 is the light chain variable domain of the anti-CD3 antibody, CL is the light chain constant domain, and VH CD3CH1 is the heavy chain variable domain of the anti-CD3 antibody, and VL is the heavy chain constant domain. BCMA This is the light chain variable domain of the anti-BCMA antibody, VH BCMA is the heavy chain variable domain of the anti-BCMA antibody, and Fc is the immunoglobulin Fc region. Advantageously, in the first polypeptide chain, domain VL BCMA -CL is the same as the light chain of the anti-BCMA parent antibody, and domain VH BCMA -CH1 is the same as the heavy chain variable domain and heavy chain constant domain of the anti-BCMA parent antibody, and domain VL CD3 -CL is the same as the light chain of the anti-CD3 parent antibody, and the domain VH CD3 -CH1 is the same as the heavy chain variable domain and heavy chain constant domain of the anti-CD3 parental antibody.

[0015] In the above formula relating to the first polypeptide chain of the FIT-Ig binding protein, the Fc region may be a native or mutant Fc region. In certain embodiments, the Fc region is a human Fc region derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. In certain embodiments, Fc is a human Fc derived from IgG1, such as those shown below.

[0016] [ka]

[0017] In one embodiment of the present invention, the FIT-Ig binding protein of the present invention retains one or more properties of a parental antibody from which the sequence of a Fab fragment is utilized and incorporated into the FIT-Ig structure. In one further embodiment, the FIT-Ig appears to retain a binding affinity for target antigens (i.e., CD3 and BCMA) comparable to that of the parental antibody, meaning that the binding affinity of the FIT-Ig binding protein to CD3 and BCMA antigen targets, as measured by surface plasmon resonance or biolayer interferometry, does not differ by more than 10-fold compared to the binding affinity of the parental antibody to those individual target antigens.

[0018] In one embodiment, the BCMA / CD3 FIT-Ig binding protein of the present invention binds to CD3 and BCMA and comprises a first polypeptide chain consisting of, or comprising the amino acid sequence of SEQ ID NO: 50; a second polypeptide chain consisting of, or comprising the amino acid sequence of SEQ ID NO: 51; and a third polypeptide chain consisting of, or comprising the amino acid sequence of SEQ ID NO: 52. It is composed.

[0019] In another embodiment, the BCMA / CD3 FIT-Ig binding protein of the present invention binds to CD3 and BCMA and comprises a first polypeptide chain consisting of, or comprising, the amino acid sequence of SEQ ID NO: 53; a second polypeptide chain consisting of, or comprising, the amino acid sequence of SEQ ID NO: 54; and a third polypeptide chain consisting of, or comprising, the amino acid sequence of SEQ ID NO: 55.

[0020] In another embodiment, the BCMA / CD3 FIT-Ig binding protein of the present invention binds to CD3 and BCMA and comprises a first polypeptide chain consisting of, or comprising, the amino acid sequence of SEQ ID NO: 80; a second polypeptide chain consisting of, or comprising, the amino acid sequence of SEQ ID NO: 81; and a third polypeptide chain consisting of, or comprising, the amino acid sequence of SEQ ID NO: 82.

[0021] In another embodiment, the BCMA / CD3 FIT-Ig binding protein of the present invention binds to CD3 and BCMA and comprises a first polypeptide chain consisting of, or comprising, the amino acid sequence of SEQ ID NO: 83; a second polypeptide chain consisting of, or comprising, the amino acid sequence of SEQ ID NO: 84; and a third polypeptide chain consisting of, or comprising, the amino acid sequence of SEQ ID NO: 85.

[0022] In another embodiment, the BCMA / CD3 FIT-Ig binding protein of the present invention binds to CD3 and BCMA and comprises a first polypeptide chain consisting of, or comprising the amino acid sequence of SEQ ID NO: 86; a second polypeptide chain consisting of, or comprising the amino acid sequence of SEQ ID NO: 87; and a third polypeptide chain consisting of, or comprising the amino acid sequence of SEQ ID NO: 88.

[0023] The present invention also provides a novel antibody capable of binding to human CD3, wherein the antigen-binding domain of this antibody comprises a set of six CDRs selected from the group of CDR sets defined below, namely CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

[0024] [Table 1]

[0025] The present invention also provides a novel antibody capable of binding to human BCMA, wherein the antigen-binding domain of this antibody comprises a set of six CDRs selected from the group of CDR sets defined below, namely CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

[0026] [Table 2]

[0027] In one embodiment, the binding protein according to the present invention is a bispecific polyvalent immunoglobulin-binding protein comprising two or more antigen-binding sites, wherein at least one antigen-binding site comprises a CDR set selected from the above-mentioned CDR sets 1 and 2, and at least one antigen-binding site comprises a CDR set selected from the above-mentioned CDR sets 3 and 4.

[0028] In one embodiment, the anti-CD3 antibody according to the present invention comprises a VH domain and a VL domain, and these two variable domains comprise an amino acid sequence selected from the following VH / VL pairs.

[0029] [Table 3]

[0030] In a further embodiment, the anti-BCMA antibody according to the present invention comprises a VH domain and a VL domain, the two variable domains comprising an amino acid sequence selected from the following VH / VL pairs.

[0031] [Table 4]

[0032] In another embodiment, an anti-CD3 antibody or an anti-BCMA antibody can be used by techniques well established in the art to produce derivative-binding proteins that recognize the same target antigen. Such derivatives may be, for example, single-chain antibodies (scFv), Fab fragments (Fab), Fab' fragments, F(ab')2, Fv, and disulfide-bonded Fv.

[0033] Immunoglobulin Fab fragments consist of two components covalently associated to form an antibody binding site. Since these two components are each a variable domain-constant domain chain (VH-CH1 or VL-CL), each VC chain of the Fab is sometimes described as one "half" of the Fab binding unit.

[0034] In another aspect of the present invention, the antibodies or bispecific binding proteins described herein can modulate the biological function of CD3, BCMA, or both. In another aspect, the anti-CD3 antibodies described herein can inhibit CD3 signaling. In another aspect, the anti-BCMA antibodies described herein can inhibit the interaction of BCMA with its ligand APRIL and / or BAFF, and optionally, the anti-BCMA antibodies according to the present invention can inhibit BCMA-mediated cellular signaling pathways.

[0035] In one embodiment, the anti-CD3 antibody or its antigen-binding fragment described herein has a binding rate constant (k) to human CD3 when measured by surface plasmon resonance or biolayer interferometry. on ) is at least 1 × 10 5 M -1 s -1 For example, at least 3.3 × 10 5 M -1 s -1 That's all.

[0036] In another embodiment, the anti-CD3 antibody or its antigen-binding fragment described herein has a dissociation rate constant (k) for human CD3 when measured by surface plasmon resonance or biolayer interferometry. off ) is 5 x 10 -3 It is less than.

[0037] In another embodiment, the anti-CD3 antibody or its antigen-binding fragment described herein has a dissociation constant (K) for human CD3. D ) is 2 × 10 -8 Less than M, for example, 1.5 × 10 -8 It is less than M.

[0038] In one embodiment, the anti-BCMA antibody or its antigen-binding fragment described herein has a binding rate constant (k) to human BCMA, as measured by surface plasmon resonance or biolayer interferometry. on) is at least 4 × 10 4 M -1 s -1 , for example, at least 1 × 10 5 M -1 s -1 , at least 2 × 10 5 M -1 s -1 or more.

[0039] In another embodiment, the anti-BCMA antibody or antigen-binding fragment thereof described herein has a dissociation rate constant (k off ) with respect to human BCMA of less than 5 × 10 -3 s -1 , less than 1 × 10 -3 s -1 , less than 5 × 10 -4 s -1 , less than 2 × 10 -5 s -1 , or less than 1 × 10 -5 s -1 when measured by surface plasmon resonance or biolayer interferometry.

[0040] In another embodiment, the anti-BCMA antibody or antigen-binding fragment thereof described herein has a dissociation constant (K D ) with respect to BCMA of less than 2 × 10 -8 M, less than 1 × 10 -9 M, or less than 5 × 10 -10 M.

[0041] In one embodiment, the bispecific BCMA / CD3 FIT-Ig binding protein capable of binding to CD3 and BCMA according to the invention has an association rate constant (k on ) with respect to human CD3 of at least 1 × 10 5 M -1 s -1 , for example, at least 2 × 10 5 M -1 s -1 , or at least 3 × 10 5 M -1 s -1The above is the binding rate constant (k) of the same binding protein to human BCMA. on ) is at least 5 × 10 4 M -1 s -1 For example, at least 6 × 10 4 M -1 s -1 , or at least 8 × 10 4 M -1 s -1 That concludes the description. In further embodiments, a bispecific BCMA / CD3 FIT-Ig binding protein capable of binding to CD3 and BCMA as described herein has a binding rate constant (k) to human CD3 from which the anti-CD3 and anti-BCMA specificity of the FIT-Ig binding protein, respectively, is derived. on ) is the parental anti-CD3 antibody against CD3 on The decrease is not more than 10 times, and the k of the parental anti-BCMA antibody against BCMA on This is a decrease not exceeding 10 times. In other words, the binding rate constant for each antigen (CD3 or BCMA) held by the FIT-Ig binding protein is the binding rate constant (k) shown by the reactivity of the parent antibody with each CD3 antigen or BCMA antigen. on ) is higher, the same, or a decrease of less than one order of magnitude. As disclosed herein, the BCMA / CD3 FIT-Ig binding protein has a k against each antigen indicated by the parent antibody. on Compared to k for one or both antigens on Improvement may be observed, or the response to one or both antigens may be impaired. on These are substantially the same as those indicated by the parent antibody, or the k for one or both antigens indicated by the FIT-Ig binding protein. on If the antibody level is lower compared to the parent antibody level, the decrease may not exceed a 10-fold decrease. For example, the antibody level of FIT-Ig against a specific antigen. on The parent antibody against that antigen on The decrease compared to is less than 50% and less than 25%. In bispecific FIT-Ig, the parent antibody k on Compared to this high kon The fact that the value is retained is a remarkable achievement in this field.

[0042] In one embodiment, the bispecific FIT-Ig binding protein according to the present invention, which can bind to CD3 and BCMA, has a dissociation rate constant (k) for human CD3 when measured by surface plasmon resonance or biolayer interferometry. off ) is 1 × 10 -2 s -1 less than , 8×10 -3 s -1 Less than 7 x 10 -3 s -1 Less than, for example, 6 × 10 -3 s -1 The dissociation rate constant (k) for the same binding protein to human BCMA is less than the same value. off ) is 5 x 10 -5 s -1 Less than 4 x 10 -5 s -1 Less than 3 x 10 -5 s -1 Less than, or 5 x 10 -6 s -1 It is less than.

[0043] In another embodiment, the bispecific BCMA / CD3 FIT-Ig binding protein according to the present invention, which can bind to CD3 and BCMA, has a dissociation constant (K) for CD3. D ) is 5 x 10 -8 Less than M, 3 x 10 -8 Less than M, 2 x 10 -8 Less than M, or 1.75 × 10 -8 The dissociation constant (K) for the same binding protein to human BCMA is less than M. D ) is 1 × 10 -9 Less than M, 6 x 10 -10 Less than M, 3 x 10 -10 Less than M, 1 x 10 -10 Less than M, 8 x 10 -11 Less than M, or 6 × 10 -11The value is less than M. In further embodiments, the bispecific FIT-Ig binding proteins described herein that can bind to CD3 and BCMA have a dissociation constant (K) for human CD3. D ) However, the anti-CD3 and anti-BCMA specificity of the FIT-Ig binding protein are derived from the K of the parental anti-CD3 antibody against CD3. D There is no difference of more than 10 times, and the K against BCMA of parental anti-BCMA antibody i D There is no difference of more than 10 times. In other words, the dissociation constant (K) held by the FIT-Ig binding protein D The binding affinity of the parent antibody to each antigen (CD3 or BCMA), as shown by ), is determined by the reactivity of the parent antibody with the CD3 antigen or BCMA antigen, respectively, as shown by K D It is within a single digit.

[0044] As disclosed herein, the BCMA / CD3 FIT-Ig binding protein is K for each antigen indicated by the parent antibody. D Compared to K for one or both antigens D It shows improvement (i.e., lower K) D It may have a value (which binds more strongly) or K for one or both antigens. D These are either the same as those indicated by the parent antibody, or the K for one or both antigens indicated by the FIT-Ig binding protein. D This is the K of the parent antibody. D It shows a decrease compared to (i.e., a higher K D There may be cases where the K value is present, resulting in weaker binding, but between the FIT-Ig binding protein and the parent antibody, D If there is a difference, that difference is within 10 times. For example, the BCMA / CD3 FIT-Ig binding protein has a K for each antigen shown by one or both of its parent antibodies against one or both antigens. D Lower K D This indicates (a stronger bond). K D The retention of parental anti-CD3 antibody and anti-BCMA antibody binding affinity of ±10 times in this study is a remarkable achievement in the field of technology.

[0045] The present invention also provides a pharmaceutical composition comprising at least one anti-CD3 antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier, as described herein. The present invention also provides a pharmaceutical composition comprising at least one anti-BCMA antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition comprising a combination of an anti-CD3 antibody and an anti-BCMA antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier, as described herein. The present invention also provides a bispecific polyvalent immunoglobulin-binding protein reactive with both CD3 and BCMA, the binding protein incorporating a VH / VL binding site derived from the anti-CD3 antibody and anti-BCMA antibody described herein. In particular, the present invention provides a pharmaceutical composition comprising at least one FIT-Ig-binding protein or at least one MAT-Fab-binding protein capable of binding to CD3 and BCMA and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention may further comprise at least one additional active ingredient. In some embodiments, such additional components include, but are not limited to, therapeutic agents, contrast agents, cytotoxic agents, angiogenesis inhibitors, kinase inhibitors, co-stimulant blockers, adhesion blockers, antibodies of different specificities or their functional fragments, detectable labels or reporters; agonists or antagonists for specific cytokines, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, and nerve agents. Examples include muscle-blocking agents, antibiotics, corticosteroids, anabolic steroids, erythropoietin, immunogens, immunosuppressants, growth hormone, hormone replacement therapy, radiopharmaceuticals, antidepressants, antipsychotics, stimulants (e.g., amphetamines, caffeine, etc.), β-agonists, inhaled steroids, epinephrine or its analogues, and cytokines.

[0046] In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent for treating a disorder in which CD3-mediated signaling activity and / or BCMA-mediated signaling activity is detrimental.

[0047] In further embodiments, the present invention provides isolated nucleic acids or antigen-binding fragments thereof encoding one or more amino acid sequences of the anti-CD3 antibody of the present invention; isolated nucleic acids or antigen-binding fragments thereof encoding one or more amino acid sequences of the anti-BCMA antibody of the present invention; and isolated nucleic acids encoding one or more amino acid sequences of a bispecific Fabs-in-Tandem immunoglobulin (FIT-Ig) binding protein capable of binding to both CD3 and BCMA. Such nucleic acids may be inserted into vectors for performing various genetic analyses or for expressing, characterizing, or improving one or more properties of the antibodies or binding proteins described herein. A vector may comprise one or more nucleic acid molecules encoding one or more amino acid sequences of the antibodies or binding proteins described herein, wherein one or more nucleic acid molecules are functionally linked to a suitable transcription and / or translation sequence that enables the expression of the antibodies or binding proteins in a specific host cell harboring the vector. Examples of vectors for cloning or expressing nucleic acids encoding the amino acid sequences of the binding proteins described herein include, but are not limited to, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ, and their derivatives.

[0048] The present invention also provides a host cell comprising a vector comprising nucleic acids encoding one or more amino acid sequences of antibodies or binding proteins described herein. Useful host cells in the present invention may be prokaryotic or eukaryotic cells. An exemplary prokaryotic host cell is Escherichia coli. Eukaryotic cells useful as host cells in the present invention include protist cells, animal cells, plant cells, and fungal cells. An exemplary fungal cell is yeast cell, including Saccharomyces cerevisiae. Exemplary animal cells useful as host cells according to the present invention include, but are not limited to, mammalian cells, avian cells, and insect cells. Exemplary mammalian cells include, but are not limited to, CHO cells, HEK cells, and COS cells. An insect cell useful as a host cell according to the present invention is the insect Sf9 cell.

[0049] In another aspect, the present invention provides a method for producing an anti-CD3 antibody or a functional fragment thereof, comprising culturing a host cell comprising an expression vector encoding the antibody or functional fragment in a culture medium under conditions sufficient to induce the host cell to express the antibody or fragment capable of binding to CD3. In another aspect, the present invention provides a method for producing an anti-BCMA antibody or a functional fragment thereof, comprising culturing a host cell comprising an expression vector encoding the antibody or functional fragment in a culture medium under conditions sufficient to induce the host cell to express the antibody or fragment capable of binding to BCMA. In another aspect, the present invention provides a method for producing a bispecific polyvalent binding protein capable of binding to CD3 and BCMA, specifically a FIT-Ig binding protein, comprising culturing a host cell comprising an expression vector encoding the binding protein in a culture medium under conditions sufficient to induce the host cell to express the binding protein capable of binding to CD3 and BCMA. The proteins thus produced can be isolated and used in the various compositions and methods described herein.

[0050] In one embodiment, the present invention provides a method for treating cancer in a subject of interest, comprising administering to the subject an anti-CD3 antibody or CD3-binding fragment as described herein, the antibody or binding fragment can bind to CD3 and inhibit CD3-mediated signaling in CD3-expressing cells. In another embodiment, the present invention provides a method for treating cancer in a subject of interest, comprising administering to the subject an anti-BCMA antibody or BCMA-binding fragment as described herein, the antibody or binding fragment can bind to BCMA and inhibit BCMA-mediated signaling in BCMA-expressing cells. In yet another embodiment, the present invention provides a method for treating cancer in a subject of interest, comprising administering to the subject a bispecific FIT-Ig-binding protein capable of binding to both CD3 and BCMA as described herein, the binding protein can bind to CD3 and BCMA and inhibit CD3-mediated signaling in CD3-expressing cells and BCMA-mediated signaling in BCMA-expressing cells.

[0051] In another embodiment, the present invention provides a method for treating an autoimmune disease or cancer in a target of interest, wherein the binding protein can bind to CD3 and BCMA, and the autoimmune disease or cancer is an autoimmune disease or a cancer that typically responds to immunotherapy. In another embodiment, the cancer is a cancer not associated with immunotherapy. In another embodiment, the cancer is a cancer that is refractory or recurrent. In another embodiment, the binding protein inhibits the proliferation or survival of tumor cells. In another embodiment, the cancer is selected from the group consisting of melanoma (e.g., metastatic melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies.

[0052] The therapeutic methods described herein may further comprise administering an immunostimulatory adjuvant, such as a CpG oligodeoxynucleotide (CpG ODN) comprising a complete or partial phosphodiester or phosphorothioate skeleton, to a subject requiring treatment. For example, in the therapeutic methods of the present invention, an immunostimulatory adjuvant may be incorporated into a composition comprising the antibody or FIT-Ig binding protein of the present invention, and the composition administered to a subject requiring treatment. In another embodiment, the therapeutic methods of the present invention may comprise a step of administering the antibody or FIT-Ig binding protein described herein to a subject requiring treatment, and another step of administering an immunostimulatory adjuvant to the subject before, simultaneously with, or after the step of administering the antibody or FIT-Ig binding protein of the present invention to the subject. [Brief explanation of the drawing]

[0053] [Figure 1]Figure 1 is a collection of plots showing the binding of test antibodies to human CD3ε / γ heterodimer-Fc fusion protein targets immobilized on microplates. The binding activity of newly isolated mAbCD3-001 and mAbCD3-002 is compared to a reference anti-CD3 monoclonal antibody ("control α-CD3 mAb") and an unrelated mouse antibody ("mIgG") as a negative control. [Figure 2] Figure 2 is a collection of plots showing the binding of test antibodies to cynomolgus monkey CD3ε / γ heterodimer-Fc fusion protein targets immobilized on microplates. The binding activity of newly isolated mAbCD3-001 and mAbCD3-002 is compared to a reference anti-CD3 monoclonal antibody ("control α-CD3 mAb") and an unrelated mouse antibody ("mIgG") as a negative control. [Figure 3] Figure 3 is a bar graph showing that the proliferation of in vitro cultured human T cells was stimulated by the anti-CD3 antibody mAbCD3-001 (the present invention) and OKT3 (positive control). [Figure 4] Figure 4 is a bar graph showing that the secretion of interferon-gamma (IFN-g) from in vitro cultured human T cells was stimulated by the anti-CD3 antibody mAbCD3-001 (the present invention) and OKT3 (positive control). [Figure 5A-5H] Figures 5A–5H are fluorescence plots comparing the binding activity of humanized anti-CD3 antibody constructs using various humanized VH variants of mAbCD3-001 and one of two VK variants (EM0006-01VK.1 or EM0006-01VK.1A, see Table 2). These plots indicate that the VH variant is important for CD3 binding activity, and that VL modification had little effect on binding to Jurkat cells. The classification of some plots in panels 5A–5H of the graphs allowed for the identification of extremely high-affinity humanized antibodies such as HuEM0006-01-8 and HuEM0006-01-17 by contrasting them with intermediate and low-affinity binders. [Figure 6]Figure 6 is a graph showing the ability of various anti-BCMA antibodies to inhibit NF-κB phosphorylation induced by the BCMA ligand BAFF in the BCMA-expressing tumor cell line NCI-H929. Anti-BAFF mAbs and unrelated anti-RAC1 mouse IgG were used as positive and negative controls, respectively. The novel anti-BCMA antibodies mAbBCMA-002 and mAbBCMA-003 described herein were comparable to or better than the reference antibodies described in the patent literature (indicated as TAB1 and TAB2 in Figure 6). See Example 3.3 below for details. [Figure 7] Figure 7 is a graph showing the ability of various anti-BCMA antibodies to inhibit NF-κB phosphorylation induced by the BCMA ligand BAFF in the HEK293-transfected BCMA-expressing cell line HEK293F-BCMA-NF-κB-luc. Anti-BCMA reference antibodies TAB1 and TAB2 were used as positive controls for comparison. Unrelated anti-Ro1 mouse IgG was used as a control. [Figure 8] Figure 8 is a graph showing the ability of anti-BCMA antibodies to inhibit NF-κB-luciferase signaling induced by BCMA ligand APRIL (TNFSF13) in BCMA-transfected HEK293 cells. [Figure 9] Figure 9 is a graph showing the binding of BCMA / CD3 bispecific FIT-Ig Fab fragments to BCMA-expressing NCI-H929 cells. The three FIT-Fab fragments tested all have the same BCMA-binding domain. See Examples 4.1, 4.2, and 4.3. [Figure 10] Figure 10 is a graph showing the binding of BCMA / CD3 FIT-Ig binding proteins to CHO cells (CHOK1 / CD3 / TCR) transfected to express the human T cell receptor complex (see Example 1.1). The three FIT-Ig binding proteins tested have different CD3 binding sites derived from three different humanized anti-CD3 antibodies. See Example 4.3. [Figure 11]Figure 11 is a graph showing the ability of BCMA / CD3 bispecific FIT-Igs and BCMA / CD3 FIT-Fab to redirect activation of Jurkat-NFAT cells co-cultured with NCI-H929 cells. For comparison, monospecific anti-CD3 IgG (HuEM1006-01-24) and its Fab fragment (HuEM1006-01-24-Fab) were tested, with unrelated human IgG used as a negative control. [Figure 12] Figure 12 is a graph showing the ability of the BCMA / CD3 bispecific FIT-Fab-binding protein to redirect activation of Jurkat-NFAT cells co-cultured with NCI-H929. Combinations of anti-BCMA monoclonal antibodies and anti-CD3 monoclonal antibodies, as well as an unrelated Fab (FIT1002-5a-Fab), were used as controls. [Figure 13] Figure 13 is a graph showing the ability of various BCMA / CD3 bispecific FIT-Fabs to redirect T cell cytotoxicity against NCI-H929 cells. Unrelated FIT-Fab (FIT1002-5a-Fab), a combination of anti-CD3 Fab mAb and anti-BCMA mAb (combo), a reference anti-BCMA mAb (TAB1) alone, anti-CD3 Fab alone (HuEM1006-01-24-Fab), and unrelated human IgG were used as controls. [Figure 14] Figure 14 is a graph showing that humanized BCMA / CD3 FIT-Ig and BCMA / CD3 FIT-Fab exhibit limited activation of non-targeted, redirected Jurkat-NFAT cells when assays are performed without using BCMA-expressing NCI-H929 target cells. Anti-CD3 IgG (HuEM1006-01-24), its Fab fragment (HuEM1006-01-24-Fab), unrelated FIT-Ig (FIT1002-5a), and unrelated human IgG (hIgG) were used as controls. [Figure 15]Figure 15 is a graph showing that the humanized BCMA / CD3 FIT-Ig according to the present invention was able to redirect T cell cytotoxicity against NCI-H929 tumor cells. Mouse-human chimeric FIT-Ig (FIT1006-4b) and unrelated FIT-Ig (FIT1002-5a) were used as controls. [Figure 16] Figure 16 is a graph showing the binding activity of the two BCMA / CD3 humanized bispecific FIT-Ig binding proteins described above to BCMA-expressing NCI-H929 cells. An unrelated human IgG antibody (hIgG) was used as a control. [Figure 17] Figure 17 is a graph showing the binding activity of the two BCMA / CD3 humanized bispecific FIT-Ig binding proteins described above to CD3-expressing Jurkat cells. An unrelated human IgG antibody (hIgG) was used as a control. [Figure 18-19] Figures 18 and 19 show the binding activity to BCMA-expressing target cells (Figure 18) and CD3-expressing target cells (Figure 19), confirming that both target bispecific constructs of two alternative configurations. [Figure 20] Figure 20 shows the inhibition of tumor growth in human PBMC-grafted NPSG mice achieved by treatment with BCMA × CD3 FIT-Ig. [Figure 21] Figure 21 shows B cell depletion induced by treatment with BCMA × CD3 FIT-Ig. [Figure 22] Figure 22 shows the transient absence of circulating T cells in FIT-Ig treated cynomolgus monkeys. Detailed description of the invention

[0054] The present invention relates to novel anti-CD3 antibodies, novel anti-BCMA antibodies, their antigen-binding moieties, and polyvalent bispecific binding proteins, such as Fabs-in-Tandem immunoglobulin (FIT-Ig) and "monovalent asymmetric tandem Fab bispecific antibodies" or "MAT-Fab bispecific antibodies" or simply "MAT-Fab antibodies." Various aspects of the present invention relate to anti-CD3 antibodies and anti-BCMA antibodies and antibody fragments, FIT-Ig binding proteins that bind to human CD3 and human BCMA, and MAT-Fab binding proteins, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for producing such antibodies, functional antibody fragments, and binding proteins. The present invention also encompasses methods of using the antibodies, functional antibody fragments, and bispecific binding proteins of the present invention for detecting human CD3, human BCMA, or both; for inhibiting human CD3 and / or human BCMA activity in vitro or in vivo; and for treating diseases, particularly cancer, mediated by the binding of CD3 and / or BCMA to their ligands, namely the T cell receptor and proliferation-inducing ligand (APRIL), respectively.

[0055] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings generally understood by those skilled in the art. The scope of a term must be clear, but in the event of any potential ambiguity, the definitions provided herein shall take precedence over dictionary or external definitions. Furthermore, unless otherwise specified in the context, singular terms shall include plural forms, and plural terms shall include singular forms. In this specification, unless otherwise specified, the use of "or" means "and / or". Furthermore, the use of the term "including," as well as "includes," The use of other forms such as "included" is not restrictive. Furthermore, unless otherwise specified, terms such as "element" or "component" include both elements and components comprising one unit and elements and components comprising multiple subunits.

[0056] In general, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. The methods and techniques of the present invention are generally carried out in accordance with conventional methods well known in the art, and, unless otherwise indicated, as described in the various general and more specific references cited and described herein. Enzyme reactions and purification techniques are carried out according to the manufacturer's specifications, or as commonly achieved in the field, or as described herein. The nomenclature and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein, as well as their experimental procedures and techniques, are well known and commonly used by those skilled in the art. Standard techniques are used in chemical synthesis, chemical analysis, drug preparation, formulation, delivery, and patient treatment.

[0057] The terms selected are defined below to make the present invention easier to understand.

[0058] The term "polypeptide" refers to any polymer chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to polymer chains of amino acids. The term "polypeptide" includes natural or artificial proteins, protein fragments, and polypeptide analogs of protein amino acid sequences. The term "polypeptide" includes their fragments and variants (including variant fragments) unless otherwise inconsistent in context. In the case of antigenic polypeptides, a polypeptide fragment may include at least one consecutive or nonlinear epitope of the polypeptide. The precise boundaries of at least one epitope fragment can be determined using the usual art of the art. The fragment comprises at least about five consecutive amino acids, e.g., at least about ten consecutive amino acids, at least about fifteen consecutive amino acids, or at least about twenty consecutive amino acids. Variants of polypeptides are as described herein.

[0059] The terms “isolated protein” or “isolated polypeptide” refer to a protein or polypeptide that, for reasons of its origin or source of origin, is not associated with the naturally associated components that accompany it in its natural state, substantially contains no other proteins of the same species, is expressed by cells of a different species, or does not exist in nature. Thus, polypeptides that are chemically synthesized or synthesized in cell systems different from those of which they originate are “isolated” from their naturally associated components. Proteins can also be made substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

[0060] The term "recover" refers to the process of substantially eliminating naturally associated components from chemical species such as polypeptides, for example, by isolation using protein purification techniques well known in the art.

[0061] The term "biological activity" refers to anti-CD3 antibodies or anti-BCMA antibodies as described herein. This refers to all biological properties. Examples of biological properties of CD3 antibodies include, but are not limited to, binding to the CD3 protein; and examples of biological properties of anti-BCMA antibodies include, but are not limited to, binding to growth-inducing ligands (APRIL) and / or B-cell activator (BAFF) proteins.

[0062] With regard to interactions with a second chemical species of antibody, binding protein, or peptide, the terms “specific binding” or “specifically binding” mean that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the second chemical species. For example, antibodies generally recognize and bind to specific protein structures rather than proteins. If an antibody is specific to epitope “A”, the presence of molecules containing epitope A (or free, unlabeled A) in the reactant containing labeled “A” and the antibody will reduce the amount of labeled A that binds to the antibody.

[0063] The term "antibody," in a broad sense, refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains, namely two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains the essential epitope binding characteristics of the Ig molecule. Such mutant, variant, or derivative antibody forms are known in the art. Their non-limiting embodiments are discussed below.

[0064] In full-length antibodies, each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), into which more conserved regions called framework regions (FRs) are inserted. Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The first, second, and third CDRs of the VH domain are generally listed as CDR-H1, CDR-H2, and CDR-H3, and similarly, the first, second, and third CDRs of the VL domain are generally listed as CDR-L1, CDR-L2, and CDR-L3. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0065] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a mutant Fc region. The Fc region of immunoglobulins generally comprises two constant domains, namely the CH2 domain and the CH3 domain, and optionally, a CH4 domain, as is the case with the Fc regions of IgM and IgE antibodies. The Fc regions of IgG, IgA, and IgD antibodies comprise a hinge region, a CH2 domain, and an H3 domain. In contrast, the Fc regions of IgM and IgE antibodies lack a hinge region but comprise the CH2, CH3, and CH4 domains. Mutant Fc regions with amino acid residue substitutions in the Fc portion to alter antibody effector function are known in the art (see, for example, Winter et al., U.S. Patents 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and the half-life / clearance rate of antibody and antigen-antibody complexes. While these effector functions may be desirable for therapeutic antibodies, they may be unnecessary or even harmful depending on the therapeutic objective. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate A through binding to FcγRs and complement C1q, respectively. DCC and CDC are mediated. In yet another embodiment, at least one amino acid residue is substituted in the constant region of the antibody, e.g., the Fc region of the antibody, so that the effector function of the antibody is altered. Dimerization of two identical heavy chains of immunoglobulin is mediated by dimerization of the CH3 domain and stabilized by a disulfide bond in the hinge region connecting the CH1 constant domain to the Fc constant domains (e.g., CH2 and CH3). The anti-inflammatory activity of IgG is entirely dependent on the sialylation of the N-linked glycan of the IgG Fc fragment. Precise glycan requirements for anti-inflammatory activity have been determined so that suitable IgG1 Fc fragments can be produced, thereby generating fully recombinant sialylated IgG1 Fc with significantly enhanced potency (see Anthony et al., Science, 320:373-376 (2008)). ).

[0066] The terms “antigen-binding moiety” and “antigen-binding fragment” or “functional fragment” of an antibody are used interchangeably and refer to one or more fragments of an antibody that retain the ability to specifically bind to the same antigen (e.g., CD3, BCMA) as the full-length antibody from which the moiety or fragment originates. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Embodiments of such antibodies may also be in a bispecific, bispecific, or multispecific form that specifically binds to two or more different antigens (e.g., CD3 and other different antigens such as BCMA). Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) the Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) the F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) the Fd fragment, consisting of a VH domain and a CH1 domain; (iv) the Fv fragment, consisting of the VL and VH domains of a single arm of the antibody; (v) the dAb fragment, containing a single variable domain (Ward et al., Nature, 341: 544-546 (1989); PCT Publication No. WO90 / 05144); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate isolated genes, they can be recombined to form a single protein (known as single-stranded Fv (scFv)) by pairing the VL and VH regions; for example, Bird et al. They can be linked by synthetic linkers that enable them to be produced as (see Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). Such single-chain antibodies are also included in the term "antigen-binding portion" and equivalent terms of the antibodies shown above. Other forms of single-chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but because a linker that is too short to allow pairing between these two domains on the same chain is used, pairing with a complementary domain on another chain is forced, creating two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)). The combined portion is publicly known in the art (Kontermann and Duebel, eds.) Antibody Engineering (Springer-Verlag, New York, 2001), p. 790 (ISBN 3-540-41354-5). Furthermore, single-chain antibodies also include "linear antibodies" comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with a complementary light chain polypeptide (Zapata et al., Protein Eng., 8(10): 1057-1062 (1995); and US Patent No. 5,641,870).

[0067] The immunoglobulin constant (C) domain refers to the heavy chain (CH) or light chain (CL) constant domain. The amino acid sequences of the mouse and human IgG heavy and light chain constant domains are known in the art.

[0068] The terms "monoclonal antibody" or "mAb" refer to a substantially homogeneous population of antibodies. The term "monoclonal" refers to antibodies obtained from a specific process; that is, individual antibodies forming a population are identical except for trace amounts of naturally occurring mutations. Monoclonal antibodies are highly specific and directed to a single antigenic determinant (epitope). Furthermore, in contrast to polyclonal antibody preparations, which generally contain various antibodies directed to various determinants (epitopes), each mAb is directed to a single determinant on an antigen. The modifier "monoclonal" should not be interpreted as requiring the production of the antibody by a specific method.

[0069] The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences in the CDR, particularly CDR3 (mutations introduced, for example, by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, are grafted onto a human framework sequence.

[0070] The term "recombinant human antibody" refers to antibodies expressed using a recombinant expression vector transfected into host cells, or antibodies isolated from a recombinant combinatorial human antibody library (Hoogenboom, HR, Trends Biotechnol., 15: 62-70 (1997); Azzazy and Highsmith, Clin. Biochem., 35: 425-445 (2002); Gavilondo and Larrick, BioTechniques, 29: 128-145 (2000); Hoogenboom and Chames, Immunol. Today, 21: 371-378 (2000)) , antibodies isolated from animals transgenic to human immunoglobulin genes (e.g., mice) (e.g., Taylor et al., Nucl. Acids Res., 20: 6287-6295 (1992); Kellermann and Green, Curr. Opin. Biotechnol., 13: 593-597 (2002); Little et al., See Immunol. Today, 21: 364-370 (2000); or prepared by any other means. This includes any human antibody prepared, expressed, produced, or isolated by recombinant means, such as antibodies involving splicing of human immunoglobulin gene sequences to other DNA sequences, which are expressed, produced, or isolated. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subject to in vitro mutagenesis (or in vivo somatic mutagenesis if transgenic animals for human Ig sequences are used), so the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences derived from and related to human germline VH and VL sequences, but which may not naturally exist within the human germline antibody repertoire in vivo.

[0071] The term "chimeric antibody" refers to an antibody that contains a heavy chain and light chain variable region sequence of one species and a constant region sequence of another species, for example, an antibody having a mouse heavy chain and light chain variable region linked to a human constant region.

[0072] The term "CDR graft antibody" refers to an antibody comprising heavy and light chain variable region sequences in which one or more CDR regions of VH and / or VL are replaced with CDR sequences of another species, for example, an antibody having human heavy and light chain variable regions in which one or more human CDRs are replaced with mouse CDR sequences.

[0073] The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH sequence and / or VL sequence has been modified to be more "human-like," i.e., more similar to the human germline variable sequence. One type of humanized antibody is a CDR graft antibody in which a CDR sequence derived from a non-human species (e.g., mouse) has been introduced into the human VH and VL framework sequences. Humanized antibodies bind immunospecifically to the target antigen and substantially possess the amino acid sequence of a human antibody. An antibody or a variant, derivative, analogue, or fragment thereof comprising a framework region and a constant region and a complementarity-determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term “substantially” with respect to a CDR means a CDR having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, generally two, variable domains (Fab, Fab', F(ab')2, Fv), where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin (i.e., a donor antibody), and all or substantially all of the framework region corresponds to that of a human immunoglobulin consensus sequence. In some embodiments, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), generally that of a human immunoglobulin. In some embodiments, a humanized antibody comprises at least variable domains of both the light chain and the heavy chain. The antibody may also contain the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only the humanized light chain. In some embodiments, the humanized antibody contains only the humanized heavy chain. In certain embodiments, the humanized antibody contains only the humanized variable domain of the light chain and / or the humanized heavy chain.

[0074] The humanized antibody may be selected from any class of immunoglobulin, including but not limited to IgM, IgG, IgD, IgA, and IgE, as well as any isotype, including IgG1, IgG2, IgG3, and IgG4. The humanized antibody may contain sequences derived from two or more classes or isotypes, and specific constant domains may be selected using techniques well known in the art to optimize the effector function of the formulation.

[0075] The framework and CDR regions of a humanized antibody do not need to strictly correspond to the parent sequence. For example, a donor antibody CDR or acceptor framework may be mutagenic by substitution, insertion, and / or deletion of at least one amino acid residue so that the CDR residue or framework residue at that site does not correspond to either the donor antibody or the consensus framework. In some exemplary embodiments, however, such mutations are not extensive. Typically, at least 80%, e.g., at least 85%, at least 90%, or at least 95% of the humanized antibody residues correspond to those of the parent FR and CDR sequences. Reverse mutations at specific framework sites to restore the same amino acid found at that position in the donor antibody are often used to maintain a specific loop structure or to properly orient the CDR sequence for contact with the target antigen.

[0076] The term "CDR" refers to the complementarity-determining region within the antibody variable domain sequence. Each of the heavy and light chain variable regions contains three CDRs, designated as CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. As used herein, the term "CDR set" refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The precise boundaries of these CDRs are defined differently for each system. (Kabat et al.) Sequences of Proteins of Immunological InterestThe system described by National Institutes of Health, Bethesda, Maryland (1987) and (1991) not only provides a clear residue numbering system applicable to any variable region of the antibody, but also provides precise residue boundaries that define the three CDRs.

[0077] The term "Kabat numbering," as recognized in the art, refers to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding moiety. (Kabat et al., Ann. NY Acad. Sci., 190: 382-391 (1971); and Kabat et al.,) Sequences of Protein s of Immunological Interest See, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

[0078] The growth and analysis of large-scale public databases of amino acid sequences of variable heavy and light chain regions over the past 20 years has led to an understanding of typical boundaries between framework regions (FRs) and CDR sequences within variable region sequences, enabling those skilled in the art to accurately determine CDRs according to Kabat numbering, Chothia numbering, or other systems. For example, Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains," In Kontermann and Duebel, eds. Antibody Engineering (Springer-Verlag, Berlin, 2001), Chapter 31, pp. 432-433.

[0079] The term "multivalent binding protein" refers to a binding protein comprising two or more antigen-binding sites. Multivalent binding proteins are preferably engineered to have three or more antigen-binding sites and are generally not naturally occurring antibodies. The term "bispecific binding protein" refers to a binding protein capable of binding to two targets with different specificities. The "Fabs-in-Tandem immunoglobulin" (FIT-Ig) binding protein of the present invention comprises two or more antigen-binding sites and is generally a tetravalent binding protein. FIT-Ig can be monospecific, i.e., capable of binding to one antigen, or multispecific, i.e., capable of binding to two or more antigens. The exemplary FIT-Ig according to the present invention binds to both CD3 and BCMA and is therefore bispecific. The FIT-Ig binding protein comprising two long (heavy) VCVC-Fc chain polypeptides and four short (light) VC chain polypeptides forms a hexamer exhibiting four Fab antigen-binding sites (VH-CH1 paired with VL-CL, sometimes denoted as VH-CH1::VL-CL). Each half of FIT-Ig comprises one heavy chain polypeptide and two light chain polypeptides, and the complementary immunoglobulin pairing of the VH-CH1 and VL-CL elements of these three chains gives rise to two tandem-configured Fab structured antigen-binding sites. In the present invention, it is preferable that the immunoglobulin domains comprising the Fab elements are directly fused within the heavy chain polypeptide without the use of interdomain linkers. That is, the N-terminal VC element of a long (heavy) polypeptide chain is directly fused at its C-terminus to the N-terminus of another VC element, which is then linked to the C-terminal Fc region. In a bispecific FIT-Ig binding protein, the tandem Fab elements are reactive with different antigens. Each Fab antigen-binding site comprises a heavy chain variable domain and a light chain variable domain, with a total of six CDRs per antigen-binding site. In some embodiments, the polyvalent binding protein of the present invention is a FIT-Ig Fab fragment (i.e., FIT-Fab), which is substantially FIT-Ig without a C-terminal Fc region.Such FIT-Fabs can be obtained by removing the C-terminal Fc region from existing FIT-Ig, or they can be produced by any of several techniques known in the art, such as expression from host cells comprising an expression vector encoding the corresponding peptide chain.

[0080] A description of the design, expression, and characterization of FIT-Ig molecules is presented in PCT Publication WO2015 / 103072. An example of such a FIT-Ig molecule comprises a heavy chain and two different light chains. The heavy chain is structurally defined as VL. A -CL-VH B -CH1-Fc(where CL is VH B (Directly fused) or VH B -CH1-VL A -CL-Fc(where CH1 is VL) A (Contains directly fused) and VL A This is a light chain variable domain derived from the parent antibody that binds to antigen A, and VH B is the heavy chain variable domain derived from the parent antibody that binds to antigen B, CL is the light chain constant domain, CH1 is the heavy chain constant domain, and Fc is the immunoglobulin Fc region (e.g., the C-terminal hinge-CH2-CH3 portion of the heavy chain of the IgG1 antibody). The two light polypeptide chains of FIT-Ig are each of formula VH A -CH1 and VL B It has -CL. In the embodiment of bispecific FIT-Ig, antigen A and antigen B are different antigens or different epitopes of the same antigen. In the present invention, one of A and B is CD3 and the other is BCMA.

[0081] As used herein, the term “directly fused” means that, when referring to a linear connection of two domains within a polypeptide structure, the domains are directly linked by a peptide bond without the use of an artificial polypeptide linker or connector.

[0082] The term "activity" includes properties such as the ability to bind to a target antigen with specificity, the affinity of the antibody to the antigen, the ability to neutralize the biological activity of the target antigen, and the ability to inhibit the interaction between the target antigen and its innate receptor. The exemplary antibodies and binding proteins of the present invention have the ability to inhibit the binding of CD3 to its ligand, the ability to inhibit the binding of BCMA to its ligand, or, in the case of bispecific binding proteins described herein, both.

[0083] "k on The term (also "Kon" or "kon"), as used herein, is intended to refer to the binding rate constant relating to the association of a binding protein (e.g., an antibody) and an antigen that forms an association complex, such as an antibody / antigen complex, as is known in the art. on This term is also known as the “association rate constant,” or “ka,” as interchangeably used herein. This value represents the binding rate of an antibody to its target antigen or the rate of complex formation between the antibody and the antigen, as shown in the following formula. Antibody (“Ab”) + Antigen (“Ag”) → Ab-Ag

[0084] "k off The term (also known as "Koff" or "koff"), as used herein, refers to the off-rate constant relating to the dissociation of a binding protein (e.g., an antibody) from an association complex (e.g., an antibody / antigen complex), as is known in the art, or This term refers to the "dissociation rate constant." This value indicates the rate at which an antibody dissociates from its target antigen, or the time-dependent separation of the Ab-Ag complex into free antibody and antigen, as shown in the following formula. Ab+Ag ← Ab-Ag

[0085] "K D The term (also known as "Kd"), as used herein, is intended to refer to the "equilibrium dissociation constant," which is measured in titration at equilibrium or the dissociation rate constant (k). off ) and the association rate constant (kon This refers to the value obtained by dividing by (k). Association rate constant (k on ), dissociation rate constant (k off ), and the equilibrium dissociation constant (K D ) is used to express the binding affinity between an antibody and an antigen. Methods for determining the association and dissociation rate constants are well known in the art. Fluorescence-based techniques offer high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as the BIAcore® (Biomolecular Interaction Analysis) assay are also available (e.g., instruments available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). For example, biolayer interferometry (BLI) using the Octet® RED96 system (Pall ForteBio LLC) is another affinity assay technique. Furthermore, the KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Idaho), is also available.

[0086] The term “isolated nucleic acid” means a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, by human intervention, is not associated with all or some of the polynucleotides that would normally be found together; is functionally linked with polynucleotides that are not normally linked; or is not naturally present as part of a longer sequence.

[0087] The term "vector," as used herein, refers to the other nucleic acid to which it is linked. The term "vector" is intended to refer to nucleic acid molecules capable of transporting additional DNA segments. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, to which additional DNA segments can be ligated to a viral genome. Certain types of vectors have the ability to autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell and thus replicate with the host genome. Furthermore, certain types of vectors can command the expression of genes to which they are operably ligated. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. Hereinafter, "plasmid" and "vector" can be used interchangeably as plasmids and are the most commonly used forms of vectors. However, the present invention is intended to include other forms of expression vectors, such as viral vectors that perform equivalent functions (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0088] The term “functionally linked” refers to the juxtaposition of components described in a way that enables them to function in their intended manner. “Functionally linked” regulatory sequences to a coding sequence are linked in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences. “Functionally linked” sequences include both expression regulatory sequences adjacent to the gene of interest and expression regulatory sequences acting in trans or at a distance to control the gene of interest. As used herein, the term “expression regulatory sequence” refers to the polynucleotide sequence necessary to perform the expression and processing of the coding sequence to which they are linked. Expression regulatory sequences include appropriate transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that promote protein secretion. The nature of such regulatory sequences varies depending on the host organism; in prokaryotes, such regulatory sequences generally include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such regulatory sequences generally include promoters and transcription termination sequences. The term “regulatory sequence” is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader or signal sequences and fusion partner sequences.

[0089] As defined herein, "transformation" refers to any process by which exogenous DNA enters a host cell. Transformation can occur naturally or artificially using various methods well known in the art. Transformation may be by any method known for the insertion of exogenous nucleic acid sequences into prokaryotic or eukaryotic host cells. This method may include, but is not limited to, transfection, viral infection, electroporation, lipofection, and particle impact, depending on the host cell to be transformed. Such "transformed" cells include stable transformed cells in which the inserted DNA can replicate autonomously as a plasmid or as part of a host chromosome. They also include cells that transiently express the inserted DNA or RNA for a limited time.

[0090] The term “recombinant host cell” (or simply “host cell”) is intended to refer to a cell into which exogenous DNA has been introduced. In some embodiments, the host cell comprises two or more nucleic acids encoding antibodies, such as the host cell described in U.S. Patent No. 7,262,028. Such terminology is intended to refer not only to a specific target cell but also to the offspring of such a cell. Due to either mutation or environmental influence, specific Because modifications may occur in the next generation, such progeny may not actually be identical to the parent cell, but are still included within the scope of “host cell” as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any kingdom of life. In other embodiments, eukaryotic cells include protists, fungi, plant and animal cells. In other embodiments, host cells include, but are not limited to, prokaryotic Escherichia coli strains; mammalian cell lines CHO, HEK293, COS, NS0, SP2 and PER.C6; insect cell line Sf9; and fungal cells Saccharomyces cerevisiae.

[0091] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be carried out as described herein, according to the manufacturer's specifications or as commonly achieved in the art. The above techniques and procedures can generally be carried out according to the conventional methods well known in the art, as described in the various general and more specific references cited and discussed herein. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual See the 2nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0092] The term "agonist," as used herein, refers to a modifier that, upon contact with the molecule of interest, causes an increase in the magnitude of a particular activity or function of that molecule compared to the magnitude of that activity or function in the absence of the agonist. The terms "antagonist" and "inhibitor," as used herein, refer to a modifier that, upon contact with the molecule of interest, causes a decrease in the magnitude of a particular activity of that molecule compared to the magnitude of that activity or function in the absence of the antagonist. Specific antagonists of interest include those that block or reduce the biological or immunological activity of human CD3 and human BCMA.

[0093] As used herein, the term “effective dose” means a dose sufficient to reduce or improve the severity and / or duration of a disorder or one or more of its symptoms, prevent the progression of the disorder, cause regression of the disorder, prevent the recurrence, onset or progression of one or more symptoms associated with the disorder, detect the disorder, or enhance or improve the preventive or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic drug).

[0094] Production of anti-CD3 and anti-BCMA antibodies The anti-CD3 and anti-BCMA antibodies of the present invention may be produced by any of the many techniques known in the art. For example, in expression from host cells, an expression vector encoding the heavy and light chains is transfected into host cells by standard techniques. Various forms of the term “transfection” are intended to include various techniques commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. While it is possible to express the antibodies of the present invention in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, e.g., mammalian host cells, is preferred because such cells (particularly mammalian cells) are more likely to assemble and secrete immunologically active antibodies that are properly folded than those in prokaryotic cells.

[0095] Exemplary mammalian host cells for expressing the recombinant antibody of the present invention include Chinese hamster ovary (CHO cells) (dhfr cells as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980)). - Examples include CHO cells (which are used in combination with a DHFR selection marker, as described, for example, in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982)), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells... Antibodies are produced by culturing host cells for a sufficient time to express the antibodies, or by the secretion of antibodies into the culture medium in which the host cells are growing. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0096] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. Variations of the above procedure are understood to be within the scope of the present invention. For example, it may be desirable to transfect host cells with a functional fragment encoding either the light chain and / or heavy chain DNA of the antibody of the present invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not necessary to bind to the target antigen. Molecules expressed from such truncated DNA molecules are also included in the antibodies of the present invention. Furthermore, bifunctional antibodies in which one heavy chain and one light chain are the antibody of the present invention, and the other heavy and light chains are specific to an antigen other than the target antigen, can be produced by crosslinking the antibody of the present invention by standard chemical crosslinking methods to obtain a second antibody.

[0097] In the exemplary system for recombinant expression of an antibody or its antigen-binding moiety according to the present invention, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is transfected by calcium phosphate-mediated transfection (DHFR). - The recombinant expression vector is introduced into CHO cells. In this recombinant expression vector, the heavy and light chain genes of the antibody are functionally linked to CMV enhancer / AdMLP promoter regulators to drive high levels of gene transcription. The recombinant expression vector also carries the DHFR gene, which enables selection of vector-transfected CHO cells using methotrexate selection / amplification. The selected transfected host cells are cultured to enable expression of the antibody's heavy and light chains, and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to construct the recombinant expression vector, transfect host cells, select the transfectant, culture the host cells, and recover the antibody from the culture medium. The present invention also provides a method for producing the recombinant anti-CD3 antibody or anti-BCMA antibody of the present invention by culturing the transfected host cells of the present invention in a suitable culture medium until the recombinant antibody of the present invention is produced. This method further includes isolating the recombinant antibody from the culture medium.

[0098] Production of bispecific FIT-Ig that binds to CD3 and BCMA The present invention provides a Fabs-in-Tandem immunoglobulin-binding protein (FIT-Ig) that binds to both CD3 and BCMA. An exemplary embodiment of such a FIT-Ig molecule is (1) structural formula (i) VL A -CL-VH B -CH1-Fc(where CL is VH B (Directly fused), or structural formula (ii)VH B -CH1-VL A -CL-Fc(where CH1 is VL A A heavy polypeptide chain comprising one of the following (directly fused): (2) Formula VH A -CH1 light polypeptide chain; and (3) formula VL B -CL comprises another light polypeptide chain, where VL is the light chain variable domain, CL is the light chain constant domain, VH is the heavy chain variable domain, CH1 is the heavy chain constant domain, Fc is the immunoglobulin Fc region, A is an epitope of CD3 or BCMA, and B is an epitope of CD3 or BCMA, provided that A and B are different. In this invention, such a FIT-Ig binding protein binds to both CD3 and BCMA.

[0099] When recombinantly expressed in suitable host cells, the three chains of FIT-Ig typically associate as a 6-chain, polyvalent, monomeric protein, similar to native immunoglobulins, where two such heavy chains (1), two such light chains (2), and two such light chains (3) associate to form a 6-chain binding protein monomer exhibiting four functional Fab antigen-binding sites. Such a FIT-Ig binding protein comprises two identical subunits, each subunit consisting of one heavy chain (1), one light chain (2), and one light chain ( 3) comprises these, which together form a pair of tandem-arranged Fab binding sites. The pairing of the Fc regions of these two heavy chain subunits yields the 6-chain, bispecific, FIT-Ig binding protein of the present invention, having a total of four functional Fab binding units.

[0100] While it is possible to separate tandem-connected Fab portions using peptide linkers on the heavy chain, the omission of such linker sequences is preferable in the case of the bispecific FIT-Igs according to the present invention. In multivalent immunoglobulin forms having tandem binding sites, it has been understood in the art that adjacent binding sites interfere with each other unless flexible linkers are used to spatially separate these binding sites. However, in the BCMA / CD3 FIT-Ig of the present invention, the arrangement of immunoglobulin domains in the above chain form results in a polypeptide chain that is well expressed in transfected mammalian cells, is appropriately assembled, and is secreted as a bispecific multivalent immunoglobulin-like binding protein that binds to the target antigens CD3 and BCMA. Despite the absence of linker sequences between Fab binding sites, the BCMA / CD3 FIT-Ig of the present invention retains binding affinity to the target antigen and exhibits binding affinity equivalent to that of the parent mA. Furthermore, removing the synthetic linker sequence from the binding protein avoids the creation of an antigenic site recognized by the mammalian immune system. In this way, linker removal reduces the potential immunogenicity of FIT-Ig, resulting in a circulating half-life similar to that of native antibodies. That is, FIT-Ig is not rapidly removed by immune opsonization and is not captured in the liver.

[0101] Each variable domain (VH or VL) in FIT-Ig can be obtained from one or more "parent" monoclonal antibodies that bind to a target antigen, i.e., either CD3 or BCMA. The FIT-Ig binding protein is advantageously produced using variable domain sequences of anti-CD3 and anti-BCMA monoclonal antibodies, as disclosed herein. For example, the parent antibody is a humanized antibody. The variable domains can also be prepared or improved using affinity maturation techniques.

[0102] One aspect of the present invention relates to the selection of a parent antibody having at least one desired property in the FIT-Ig molecule. In some embodiments, these antibody properties are selected from the group consisting of antigen specificity, affinity for antigen, potency, biological function, epitope recognition, stability, solubility, production efficiency, lack of immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and orthologous antigen binding. Both CD3 and BCMA are cell surface proteins, and their interactions with their respective ligands impose intracellular signaling pathways; therefore, the optimal bispecific BCMA / CD3 FIT-Ig and FIT-Fab of the present invention may be able to inhibit or block CD3-mediated and / or BCMA-mediated signaling.

[0103] The antibodies, their functional fragments, and binding proteins according to the present invention can be purified (intended for use) using a variety of methods and materials available in the art for purifying antibodies and binding proteins. Such methods and materials include, but are not limited to, affinity chromatography (e.g., using a resin, particles, or membrane conjugated with specific ligands of protein A, protein G, protein L, or antibodies, their functional fragments, or binding proteins), ion exchange chromatography (e.g., using ion exchange particles or membranes), hydrophobic interaction chromatography ("HIC"; e.g., using hydrophobic particles or membranes), ultrafiltration, nanofiltration, diafiltration, size exclusion chromatography ("SEC"), low pH treatment (for inactivating contaminating viruses), and combinations thereof, for obtaining purity acceptable for intended use. An example of a low pH treatment for inactivating contaminating viruses is the antibody, its functional fragment, or binding protein of the present invention. The method comprises lowering the pH of a solution or suspension containing a substance to pH 3.5 with 0.5 M phosphoric acid at 18°C ​​to 25°C for 60 to 70 minutes.

[0104] Use of antibodies and binding proteins of the present invention Given their ability to bind to human CD3 and / or BCMA, the antibodies, functional fragments thereof, and bispecific polyvalent binding proteins described herein can be used to detect CD3 or BCMA, or both, in a biological sample containing, for example, cells expressing one or both of these target antigens. The antibodies, functional fragments, and binding proteins of the present invention can be used in conventional immunoassays such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), or immunohistochemistry. The present invention provides a method for detecting CD3 or BCMA in a biological sample, comprising contacting the biological sample with the antibody, its antigen-binding moiety, or binding protein of the present invention, detecting whether it binds to a target antigen, and thereby determining the presence or absence of the target in the biological sample. The antibody, functional fragment, or binding protein may be directly or indirectly labeled with a detectable substance to facilitate the detection of bound or unbound antibody / fragment / binding protein. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholine esterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent substances include luminol; and examples of suitable radioactive substances include, 3 H 、 14 C 、 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm is one example.

[0105] The antibodies, their functional fragments, and binding proteins of the present invention can preferably neutralize human CD3 and / or human BCMA activity both in vitro and in vivo. Therefore, the antibodies, their functional fragments, and binding proteins of the present invention can be used, for example, to inhibit cell signaling mediated by CD3 / T cell interactions and / or BCMA / B cell interactions in cell cultures containing CD3-expressing and / or BCMA-expressing cells in human subjects or other mammalian subjects having CD3 or BCMA with which the antibodies, their functional fragments, or binding proteins of the present invention cross-react.

[0106] In another embodiment, the present invention provides a method for treating a subject suffering from a disease or disorder in which CD3 activity and / or BCMA activity are adverse, the method comprising administering to the subject an antibody or binding protein of the present invention in an effective amount such that the CD3 binding and / or BCMA binding-mediated activity in the subject is reduced.

[0107] As used herein, the term “disorders in which CD3 activity and / or BCMA activity are adverse” includes diseases and other disorders in which the interaction between CD3 and its ligand or between BCMA and its ligand is a contributing factor to the pathophysiology of the disorder or to its exacerbation in the affected subject. Thus, a disorder in which CD3 activity and / or BCMA activity are adverse is a disorder in which inhibition of CD3 activity and / or BCMA activity is expected to alleviate the symptoms and / or progression of the disorder.

[0108] In another embodiment, the present invention provides a method for treating an autoimmune disease or cancer in a subject of interest, comprising administering to the subject an antibody, a functional fragment thereof, or a binding protein described herein that can bind to CD3, BCMA, or both CD3 and BCMA, the autoimmune disease or cancer being a disease responsive to immunotherapy. In another embodiment, the method of the present invention is used to treat an autoimmune disease or cancer not associated with immunotherapy. In another embodiment, the method of the present invention is used to treat cancer that is refractory or recurrent. In another embodiment, the CD3 antibody or BCMA antibody, a functional fragment thereof, or a BCMA / CD3 bispecific binding protein of the present invention is used in a method for inhibiting the proliferation or survival of tumor cells.

[0109] In another embodiment, the present invention provides a method for treating cancer in a subject, comprising the step of administering to the subject an antibody against CD3 or BCMA as described herein, a functional fragment thereof, or a BCMA / CD3 bispecific binding protein as described herein, such as a Fabs-in-tandem immunoglobulin (FIT-Ig) binding protein, or a MAT-Fab binding protein, wherein the cancer is selected from any of the group consisting of melanoma (e.g., metastatic melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, primary bone cancer (e.g., osteosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma, and chondrosarcoma), metastatic cancer, and other neoplastic malignancies.

[0110] The present invention also provides pharmaceutical compositions comprising the antibody of the present invention, or its antigen-binding moiety, or a bispecific polyvalent binding protein (i.e., a major active ingredient), or a bispecific monovalent binding protein of the present invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising the proteins of the present invention are for use in, but not limited to, the diagnosis, detection, or monitoring of disorders; the treatment, management, or improvement of a disorder or one or more symptoms thereof; and / or research. In certain embodiments, the composition comprises one or more antibodies or binding proteins of the present invention. In other embodiments, the pharmaceutical composition comprises one or more antibodies or binding proteins of the present invention, as well as one or more prophylactic or therapeutic agents other than the antibodies or binding proteins of the present invention for the treatment of disorders in which CD3 activity and / or BCMA activity is harmful. In some embodiments, these prophylactic or therapeutic agents are known, have been used, or are currently used for the prevention, treatment, management, or improvement of a disorder or one or more symptoms thereof. According to these embodiments, the composition may further comprise a carrier, diluent, or excipient. Excipients are generally any compound or combination of compounds that provide the composition with desired features other than the main active ingredient (i.e., other than the antibody, its functional portion, or the binding protein of the present invention).

[0111] The antibodies (including their functional fragments) and binding proteins of the present invention can be incorporated into pharmaceutical compositions suitable for administration to a target. Generally, a pharmaceutical composition comprises the antibody or binding protein of the present invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any physiologically compatible solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, isotonic agent and absorption retarder, etc. Examples of pharmaceutically acceptable carriers include one or more of water, physiological saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Often, it is preferable to include an isotonic agent in the composition, such as sugars, polyalcohols (e.g., mannitol or sorbitol), or sodium chloride. The pharmaceutically acceptable carrier further does not include trace amounts of auxiliary substances such as wetting agents or emulsifiers, preservatives, or buffers that enhance the shelf life or efficacy of the antibody or binding protein present in the composition. ru.

[0112] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration, but not limited to, include parenteral (e.g., intravenous, intradermal, subcutaneous, intramuscular), oral, intranasal (e.g., inhalation), transdermal (e.g., topical), intratumoral, transmucosal, and rectal administration. In specific embodiments, the compositions are formulated according to conventional methods as pharmaceutical compositions adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans. Generally, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizer and a local anesthetic such as lidocaine (xylocaine, lignocaine) to reduce pain at the injection site.

[0113] The method of the present invention may involve administering a composition formulated for parenteral administration by injection (e.g., bolus injection or continuous infusion). The injectable formulation may be provided in unit dose form (e.g., ampoule or multi-dose container) with the addition of a preservative. The composition may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and may contain formulations such as suspending agents, stabilizers and / or dispersants. Alternatively, the main active ingredient may be in powder form for preparation with a suitable vehicle (e.g., sterile pyrogenic substance-free water) before use.

[0114] The method of the present invention may further comprise the administration of a composition formulated as a depot formulation. Such long-acting formulations may be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the composition may be formulated using a suitable polymer or hydrophobic substance (e.g., an emulsion in an acceptable oil), an ion exchange resin, or a poorly soluble derivative (e.g., a poorly soluble salt).

[0115] The antibodies, functional fragments, or binding proteins of the present invention can also be administered together with one or more additional therapeutic agents useful for treating various diseases. The antibodies, functional fragments, and binding proteins described herein can be used alone or in combination with additional agents, such as therapeutic agents, which are selected by those skilled in the art for their intended purpose. For example, an additional agent may be a therapeutic agent recognized in the art as useful for treating a disease or condition treated by the antibodies or binding proteins of the present invention. Additional agents may also be agents that confer beneficial attributes, such as agents that affect the viscosity of the composition.

[0116] The present invention has been described in detail above, but this will be understood more clearly by referring to the following examples, which are included for illustrative purposes only and are not intended to limit the present invention. [Examples]

[0117] Example 1: Preparation of anti-human CD3 monoclonal antibody Anti-human CD3 monoclonal antibodies were prepared as follows. Example 1.1: Immunoinduction by human CD3 antigen Anti-CD3 antibodies were used in 10 Balb / c and SJL / J mice (Shanghai The immunogens were obtained by inducing immunization in a group of animals (Laboratory Animal Center) using an alternating immunization strategy. Four different CD3 immunogens were used, including two peptides (CD3ε fragments: LSLKEFSELEQSGYYVC (SEQ ID NO: 2) and QDGNEEMGGITQTPYK (SEQ ID NO: 3) and a recombinant huCD3εγ / Fc fusion protein (fusion protein heterodimer: first chain). [ka] and the second chain [ka] This included CHO cell lines (CHOK1 / CD3 / TCR) transfected with full-length human CD3γ, CD3ε, CD3δ, CD3ζ (zeta), TCRα, and TCRβ chains to express the human T cell receptor complex.

[0118] Example 1.2: Preparation of hybridomas Mice were immunized with one of the immunogens at two-week intervals (with some groups receiving a booster immunization with a different immunogen than the one used in the initial immunization), and serum titers were monitored weekly after the second injection. After 4-6 immunizations, splenocytes were collected and fused with mouse myeloma cells to create hybridoma cell lines. Next, the supernatant of the hybridoma cells was screened for huCD3 / Fc dimer targets, and cell lines producing CD3-specific mouse antibodies were identified by counterstaining with unrelated protein / Fc dimers. Positive hybridomas were tested with cell binding assays against Jurkat cell targets and TCR complex transfected CHO cell targets to confirm antibody cell surface binding. Finally, the identified cell binding agents were tested for cynomolgus monkey cross-reactivity by ELISA using cynoCD3εγ / Fc fusion protein as a target, and anti-CD3 agonist activity was characterized using the Jurkat-NFAT-luciferase reporter cell line. Of the hybridomas tested in this manner, only two produced monoclonal antibodies that were positive in all assays. These were designated mAbCD3-001 and mAbCD3-002.

[0119] Example 1.3: Heavy and light chain variable region arrangement To amplify the heavy and light chain variable regions, the total RNA of each hybridoma clone was divided into 5 × 10⁻¹⁶ units. 6 From cells exceeding [number], RNA was isolated using TRIzol® RNA extraction reagent (Invitrogen, catalog number 15596018). cDNA was synthesized using the Invitrogen® SuperScript® III First-Strand Synthesis SuperMix kit (ThermoFisher Scientific, catalog number 18080) according to the manufacturer's instructions, producing light and heavy macules. cDNA encoding the variable region of a mouse immunoglobulin chain was amplified using MilliporeSigma® Novagen® mouse Ig primer set (Fisher Scientific catalog no. 698313). PCR products were analyzed by electrophoresis using SYB® Safe DNA gelstain (ThermoFisher catalog no. S33102) on a 1.2% agarose gel. DNA fragments of appropriate size were purified using NucleoSpin® gel and PCR Clean-up kit (Macherey-Nagel catalog no. 740609) according to the manufacturer's instructions and individually subcloned into pMD18-T vectors. Fifteen colonies were selected from each transformation, and the sequences of the inserted fragments were analyzed by DNA sequencing. At least eight colony fragments were sequence-verified to match the VH and VL consensus sequences. The protein sequences of the variable regions of mouse monoclonal antibodies were analyzed by sequence homology alignment and are listed in Table 1. The complementarity determination region (CDR) is underlined based on Kabat numbering.

[0120] [Table 5]

[0121] Example 1.4: Anti-CD3 antibody that binds to both human CD3 and cynomolgus monkey CD3 The binding properties of isolated mouse anti-CD3 antibody were measured using ELISA as follows: Heterodimeric CD3εγ / Fc fusion protein was coated onto a 96-well plate at 1 μg / mL overnight at 4°C. The plate was washed once with washing buffer (PBS containing 0.05% Tween 20) and blocked at room temperature for 2 hours with ELISA blocking buffer (1% BSA in PBS containing 0.05% Tween 20). Next, the anti-CD3 antibody was added and incubated at 37°C for 1 hour. The plate was washed three times with washing buffer. HRP-labeled anti-mouse IgG secondary antibody (Sigma, catalog no. A0168) was added, the plate was incubated at 37°C for 30 minutes, and washed five times with washing buffer. 100 μl of tetramethylbenzidine (TMB) chromogenic solution was added to each well. After color development, the reaction was stopped with 1 N HCl, and the absorbance at 450 nm was measured using a Varioskan® LUX microplate reader (ThermoFisher Scientific). The binding signal was plotted against antibody concentration using GraphPad Prism 5.0 software, and EC50 was calculated accordingly. As shown in Figures 1 and 2, mAbCD3-002 showed the best binding activity, while mAbCD3-001 exhibited the variable domain sequence reported in U.S. Patent No. 8,236,308. It had a binding EC50 equivalent to that of the reference anti-CD3ε antibody expressed using this method.

[0122] Example 1.5: Anti-CD3 antibody activates human T cells in vitro. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors using Lymphoprep® mononuclear cell isolation medium (STEMCELL Technologies, catalog no. 07851), and T cells were isolated from the PBMCs using a CD3-negative T cell selection kit (EasySep® STEMCELL Technologies, catalog no. 17951). Proliferation (CTG) and cytokine (IFN-γ) production data were obtained using the following protocol: 100 μl of test antibody (mAbCD3-001, OKT3, or negative control IgG) was coated onto a high-binding 96-well plate (Nunc®, catalog no. 3361) overnight at 4°C, and then washed with DPBS. A commercially available OKT3 antibody was used as a positive anti-CD3 control, and unrelated mouse IgG was used as a negative control. 1 × 10⁶ cells were collected per well. 5 T cells were seeded in 200 μl of culture medium (RPMI1640 + 10% FBS + 1% penicillin-streptomycin solution + 1% GlutaMAX® additive) and incubated at 37°C for 96 hours. Growth was measured using ATP-catalyzed quantification kits (CellTiter-Glo®, Promega). IFN-γ was measured using the LANCE® (Lanthanide Chelate Excite) TR-FRET assay kit (PerkinElmer, catalog number TRF1217M). Data were analyzed using GraphPad Prism 5.0 software. Results are shown in Figures 3 and 4. Cell proliferation and IFN-γ production data demonstrated that mAbCD3-001 activates human T cells in vitro.

[0123] Example 2: Humanization of anti-CD3 antibody Example 2.1: Humanization of mAbCD3-001 Humanized mAbs were created using the anti-CD3 mAbCD3-001 variable region gene. In the first step of this process, the VH and VK amino acid sequences of mAbCD3-001 (see Table 1 above) were compared with available human IgV gene sequence databases to find the best overall matching human germline IgV gene sequence. Furthermore, the VH or VL framework 4 was compared with the J region database to find the human framework with the greatest homology to the mouse VH and VL regions, respectively. For the light chain, the closest human V gene match was the B3 gene (V-base database), and for the heavy chain, the closest human match was the VH1-2 gene. Next, we designed humanized variable domain sequences in which the CDR-L1, CDR-L2, and CDR-L3 of the mAbCD3-001 light chain were grafted onto the framework sequence of the B3 gene along with the JK4 framework 4 sequence following CDR-L3; and the CDR-H1, CDR-H2, and CDR-H3 sequences of mAbCD3-001 VH were grafted onto the framework sequence of VH1-2 along with the JH6 framework 4 sequence following CDR-H3. Next, we constructed a 3D Fv model of mAbCD3-001 and analyzed it to determine whether there were framework residues that were most likely important for supporting loop structures or VH / VL interference, located less than 4 Å away from the CDR residues. These residues in the humanized sequence should be reverse-mutated to mouse residues at the same position to maintain affinity / activity. Q1E mutations were always included, where applicable, to eliminate the formation of the N-terminal pyroglutamate. For the heavy chain, potential mutations Y27F, T28S, V37M, M48I, V67A, M69L, and R71A (Kabat numbering) were identified. For the light chain, T5S and N22T were identified as reverse mutations. Based on the hierarchy of importance of each reverse mutation as determined by their interaction with the CDR, the most important reverse mutations were introduced into the humanized VH sequence in order of priority, followed by the introduction of other reverse mutations as a sequential design. In addition, the VK CDR-L1 sequence had an NS pattern, which is a potential deamidation site. To eliminate this deamidation of asparagine, NS was replaced with QS and NT in the humanized κ chain. or NA were mutated. The humanized VH and VL constructs are shown in Table 2 (below). (The framework amino acid residues that underwent the reverse mutation are double-underlined, and the mouse CDR from the original parental antibody is underlined.)

[0124] [Table 6-1] [Table 6-2]

[0125] Humanized VH and VK genes were de novo synthesized by backtranslating from corresponding amino acid sequence designs, and then cloned into vectors containing human IgG1 and human κ constant domains (SEQ ID NO: 26 and SEQ ID NO: 27, respectively).

[0126] [Table 7]

[0127] Pairing human VH and human VK resulted in the creation of 27 humanized antibodies, designated HuEM0006-01-1 to HuEM0006-01-27 (Table 3). Chimeric antibodies (HuEM0006-01c) possessing both parental mouse VH / VL and human constant sequences were also created and used as positive controls for ranking the humanized antibodies. All recombinant humanized mAbs were transiently expressed in HEK293 cells and purified by protein A chromatography.

[0128] [Table 8-1] [Table 8-2]

[0129] Example 2.2: Humanized anti-CD3 antibodies exhibited various CD3 binding activities. Humanized anti-CD3 mutants with various CD3 binding affinities were obtained from various humanized VH and VK combinations. The binding activity of the humanized mutant mAbCD3-001 was tested by flow cytometry in a human CD3-expressing Jurkat T cell line. 5 × 10⁶ in FACS buffer 5 Jurkat cells were seeded into each well of a 96-well plate. The cells were centrifuged at 400g for 5 minutes, and the supernatant was discarded. Next, 100 μl of serially diluted antibody was added to each well and mixed with the cells. After incubation at 4°C for 40 minutes, the plate was washed several times to remove excess antibody. Next, a secondary fluorescent dye conjugate antibody (Alexa Fluor® 647 goat anti-human IgG1 H&L; Jackson) was added. ImmunoResearch (catalog no. 109-606-170) was added and incubated with the cells at room temperature for 20 minutes. After another centrifugation and washing cycle, the cells were resuspended in FACS buffer for reading using a CytoFLEX flow cytometer (Beckman Coulter). Median fluorescence intensity (MFI) readings were plotted against antibody concentration and analyzed using GraphPad Prism 5.0 software.

[0130] As shown in Figures 5A–5H, humanized anti-CD3 antibodies exhibited broad affinity for cell surface CD3 targets on Jurkat cells. Since differences in the κ chain (i.e., between EM0006-01VK.1 and EM0006-01VK.1A) did not appear to have a significant effect on binding, the VH mutant was clearly a key component in this CD3 binding regulation. Several humanized antibodies, particularly HuEM0006-01-08 and HuEM0006-01-17 (see Table 4 and SEQ ID NO: 18) possessing the VH mutant EM006-01VH.1H, showed similarly high CD3 binding compared to the chimeric control antibody HuEM0006-01c possessing the parental mouse VH region EM0006-01VH (see Table 4).

[0131] Example 3: Preparation of anti-BCMA monoclonal antibody Anti-BCMA antibodies are used to fused human Fc regions in Balb / c or SJL mice. It was obtained by immunoinduction with recombinant BCMA extracellular domain / Fc dimers formed by homodimerization of BCMA(ECD).

[0132] [ka]

[0133] Mice were immunized at two-week intervals, and serum titers were monitored weekly after the second injection.

[0134] Example 3.1: Preparation of a hybridoma After 4-6 immunotherapy cycles, splenocytes were collected and fused with mouse myeloma cells to form hybridoma cell lines. The fusion products were then plated in 96-well plates in a selective medium containing hypoxanthine-aminopterin-thymidine (HAT), with 1 × 10⁶ cells per well. 5 Splenocytes were seeded at a density. Visible hybridoma colonies were obtained 7-10 days after fusion. Next, the supernatant of the hybridoma cells was screened to select cell lines that produced BCMA-specific mouse antibodies. Five anti-BCMA antibodies were selected and sequenced.

[0135] Example 3.2: Heavy and light chain variable region arrangement To amplify the heavy and light chain variable regions, the total RNA of each hybridoma clone was divided into 5 × 10⁻¹⁶ units. 6Cells exceeding a certain size were isolated using TRIzol® RNA extraction reagent (Invitrogen, catalog no. 15596018). cDNA was synthesized using the Invitrogen® SuperScript® III First-Strand Synthesis SuperMix kit (ThermoFisher Scientific, catalog no. 18080) according to the manufacturer's instructions, and cDNA encoding the variable regions of light and heavy mouse immunoglobulin chains was amplified using the MilliporeSigma® Novagen® Mouse Ig Primer Set (Fisher Scientific, catalog no. 698313). PCR products were analyzed by electrophoresis on a 1.2% agarose gel using SYBR® Safe DNA gelstain (ThermoFisher, catalog no. S33102). DNA fragments of appropriate size were purified using NucleoSpin® gel and PCR Clean-up kit (Macherey-Nagel, catalog number 740609) according to the manufacturer's instructions, and individually subcloned into pMD18-T vectors. Fifteen colonies were selected from each transformation, and the sequences of the inserted fragments were analyzed by DNA sequencing. The sequences of at least eight colonies were confirmed to match the VH and VL consensus sequences. The protein sequences of the mouse mAb variable region were analyzed by sequence homology alignment.

[0136] Example 3.3: Binding rate of anti-BCMA antibody by surface plasmon resonance (SPR) The binding affinity and rate constant of the anti-BCMA antibody were determined using a standard procedure at Biacore. The determination was made by surface plasmon resonance at 25°C using a T200 instrument (GE Healthcare). Briefly, goat anti-mouse IgG Fc antibody was immobilized directly onto a biosensor chip, and the antibody sample was injected into the reaction matrix at a flow rate of 5 μl / min. It was injected onto the surface immobilized with the mouse anti-BCMA IgG test antibody and captured by the immobilized anti-Fc antibody. Next, human and cynomolgus monkey BCMA (ECD) / Fc target polypeptides were injected onto the captured mouse anti-BCMA IgG surface. The association and dissociation rate constants, k on (M -1 s -1 ) and k off (s -1 ) were determined at a continuous flow rate of 30 μl / min, respectively. The rate constants were derived by performing kinetic binding measurements at five different concentrations of the target BCMA (ECD) polypeptide.Next, the equilibrium dissociation constant K D (M) of the reaction between the antibody and the target protein was calculated from the reaction rate constants using the equation K D =k off / k on . The rate constants were determined by processing the data using Biacore analysis software and fitting it to a 1:1 binding model. The results are shown in Table 7.

[0137]

Table 9

[0138] Two anti-BCMA antibodies that show high affinity for both human and cynomolgus monkey BCMA targets were further developed and analyzed. The variable domain sequences of these selected anti-BCMA monoclonal mAb BCMA-002 and mAb BCMA-003 are shown in Table 8 below. The complementarity-determining regions (CDRs) were underlined based on Kabat numbering.

[0139]

Table 10

[0140] Example 3.4: Anti-BCMA antibodies exhibited various BCMA blocking activities. The ability of a monoclonal anti-BMCA antibody to block NFκB phosphorylation stimulated by BCMA ligand BAFF in the human myeloma cell line NCI-H929 was demonstrated using the Phospho-NFκB(Ser536) cell assay kit (Cisbio; catalog number 64). Evaluation was performed using NFBPEG. NCI-H929 human myeloma cells were starved overnight in assay medium (RPMI1640, 0.1% BSA) at 37°C. The cells were washed, resuspended, and placed in a 384-well microplate (PerkinElmer, catalog no. 6008280) at a rate of 2 × 10⁶ cells per well. 5 Cells were seeded. Next, antibodies were added to the wells and incubated with the cells at 37°C for approximately 10 minutes. Anti-BAFF antibody (R&D Systems, catalog no. BAF124) was used as a positive control, and an unrelated anti-RAC1 monoclonal antibody was used as a negative control. Reference anti-BCMA antibodies TAb1 and TAb2 (clone CA8 from international publication WO2012 / 163805 and clone 83A10 from international publication WO2014 / 122143, respectively) were tested for comparison.

[0141] Next, recombinant BAFF was added to each well at a concentration of 5 μg / ml and incubated for 30 minutes. The cells were lysed with kit lysis buffer and incubated at room temperature for at least 30 minutes with shaking. The cell lysates were then transferred to a 384-well microplate (PerkinElmer, catalog no. 6008280). The assay kit reagents were prepared according to the manufacturer's instructions and added to the wells. After a final incubation of 4 hours at room temperature, fluorescence was read from the plate at wavelengths of 665 nm and 620 nm. The inhibition percentage was calculated and plotted against antibody concentration using GraphPad Prism 5.0 software. As shown in Figure 6, the selective anti-BCMA antibodies mAbBCMA-002 and mAbBCMA-003 isolated as described above showed superior inhibitory activity against BAFF-induced NF-κB phosphorylation compared to the negative control (anti-RAC).

[0142] Furthermore, a luminescence assay system based on another reporter gene was used to characterize antibody blockade of ligand-binding activity to BCMA. The cells were transfected to express BCMA. We established a stable HEK293F cell line (HEK293F-BCMA-NF-kB-luc clone 1H2) in-house that emits a luciferase signal when NF-κB phosphorylation is induced, and used it in this luminescence assay. Cells were harvested, washed, and resuspended in assay medium (RPMI1640 containing 10% FBS). Next, the cells were placed in a 96-well microplate (Costar, catalog number 3903) at a rate of 5 × 10⁴ cells per well. 4 Cells were seeded and incubated with test antibodies: mAbBCMA-002, mAbBCMA-003, TAb1 (anti-BCMA), TAb2 (anti-BCMA), or unrelated mouse IgG. BAFF or APRIL (TNFSF13, CD286) were added as BCMA ligands and incubated with antibody solution for 10 minutes. One-Glo® luciferase assay system (Promega, catalog number E6130) reagents were prepared according to the manufacturer's instructions and added to the wells. The luminescence signal from the plates was read using a Varioskan® LUX microplate reader (Thermo Scientific). Inhibition percentages were calculated and plotted against antibody concentration using GraphPad Prism 5.0 software. As shown in Figure 7 (BAFF blockade) and Figure 8 (APRIL blockade), mAbBCMA-002 showed no activity or weak blocking activity, while mAbBCMA-003 showed strong NF-κB signaling pathway blocking activity, similar to the positive reference antibodies TAb1 and TAb2.

[0143] Next, we used the binding domains of mAbBCMA-002 and mAbBCMA-003 to construct bispecific BCMA / CD3 FIT-Ig binding proteins.

[0144] Example 4: Preparation of BCMA / CD3 Fabs-in-Tandem Immunoglobulin (FIT-Ig) Example 4.1: Preparation of BCMA / CD3 FIT-Ig binding protein (FIT-Ig) We constructed a bispecific Fabs-in-Tandem immunoglobulin (FIT-Ig) binding protein that recognizes both human CD3 and human BCMA. The FIT-Ig construct was manipulated to eliminate the use of synthetic linker sequences between immunoglobulin domains, following the general procedure described in International Publication No. WO2015 / 103072.

[0145] The DNA constructs used to generate FIT-Ig antibodies capable of binding to CD3 and BCMA encoded the variable domains of the parental anti-CD3 and anti-BCMA monoclonal antibodies (mAbs). Each FIT-Ig binding protein consists of three polypeptide chains having the following structure: Chain 1 (long chain): VL CD3 -CL-VH BCMA -CH1-hinge-CH2-CH3; Chain 2 (first short chain): VH CD3 -CH1; Chain 3 (second short chain): VL BCMA -CL; Here, VL BCMA This is the light chain variable domain of a monoclonal antibody that recognizes BCMA, and VH CD3 This is the heavy chain variable domain of a monoclonal antibody that recognizes CD3, and VL CD3 This is the light chain variable domain of a monoclonal antibody that recognizes CD3, and VH BCMA is the heavy chain variable domain of a monoclonal antibody that recognizes BCMA, each CL is a light chain constant domain, each CH1 is the first heavy chain constant domain, and hinge-CH2-CH3 is the antibody C-terminal Fc region.

[0146] To construct a long-chain vector, VL CD3 -CL-VH BCMAThe cDNA encoding the segment was de novo synthesized and inserted into the multiple cloning site (MCS) of a vector containing the coding sequence for human CH1-hinge-CH2-CH3. In the resulting vector, the MCS sequence was removed during homologous recombination to ensure that all domain fragments were within the correct reading frame. Similarly, to construct the first and second short chains, VH CD3 and VL BCMA Structural genes are synthesized de novo, and The coding segments of the human CH1 and CL domains were inserted into the MCS of appropriate vectors.

[0147] Three plasmids were mixed in a 1:2:1.5 ratio and then co-transfected into HEK293 cells. After 7 days of expression, the cell culture supernatant was collected and purified by protein A chromatography. The concentration of the purified FIT-Ig protein was measured using A280, and homogeneity was analyzed by size exclusion chromatography (SEC).

[0148] To investigate the binding characteristics of mAbBCMA-002 and mAbBCMA-003, novel mouse anti-BCMA antibodies, in the bispecific FIT-Ig format, VH was used with chain 1 and chain 3 polypeptides (shown above). BCMA and VL BCMA The domains were the VH and VL domain sets shown in Table 8 (i.e., for VH, either SEQ ID NO: 29 or SEQ ID NO: 31; for VL, either SEQ ID NO: 30 or SEQ ID NO: 32). VH of Chain 1 and Chain 2 (shown above) CD3 and VL CD3In the domain, the parental anti-CD3 antibody was one of three selected humanized anti-CD3 antibodies: HuEM0006-01-24 antibody (VH=SEQ ID NO: 18; VL=SEQ ID NO: 25), HuEM0006-01-25 antibody (VH=SEQ ID NO: 15; VL=SEQ ID NO: 25), or HuEM0006-01-26 antibody (VH=SEQ ID NO: 12; VL=SEQ ID NO: 25). For the constant domains CH1-hinge-CH2-CH3 and CL of the FIT-Ig structure, human sequences, namely SEQ ID NOs. 26 and 27, were used. A FIT-I expression vector was constructed using cDNA encoding these polypeptide domains, and HEK293 cells were transfected with this vector. Cultures of FIT-Ig constructs without each linker were grown, and FIT-Ig was purified as described above. The six FIT-Ig binding proteins were given the names shown in Table 9 below.

[0149] [Table 11]

[0150] Example 4.2: Production of BCMA / CD3 FIT-Ig Fab fragment-binding protein (FIT-Fab) Full-length FIT-Ig protein was digested and purified using the Pierce® Fab Preparation Kit (ThermoFisher Scientific, catalog number 44985). During this process, the Fc domain of the FIT-Ig protein was removed by enzymatic cleavage using papain immobilized on agarose beads. Next, the FIT-Ig Fab fragment (FIT-Fab) was purified from the flow-through of protein A chromatography. The concentration of the purified FIT-Fab protein was measured using A280, and homogeneity was analyzed by size exclusion chromatography (SEC).

[0151] Example 4.3: The FIT-Ig bispecific antibody showed binding to both the CD3 target and the BCMA target. The binding activity of the chimeric bispecific BCMA / CD3 FIT-Ig antibody was examined by flow cytometry using a human CD3 / TCR complex-transfected CHO cell line (CHOK1 / CD3 / TCR cells) and BCMA-expressing NCI-H929 cells. Briefly, 5×10 5 cells were seeded in a 96-well plate. The cells were centrifuged at 400×g for 5 minutes and the supernatant was discarded. Next, for each well, 100 μl of serial diluted FIT-Ig antibody or FIT-Fab antibody was added and mixed with the cells. After incubation at 4°C for 40 minutes, the plate was washed several times to remove the excess antibody. Then, a secondary antibody (goat anti-huIgGκ chain specific) was added and incubated with the cells at room temperature for 20 minutes. After centrifugation and washing once more, the cells were resuspended in FACS buffer for reading on a CytoFLEX flow cytometer. The results were analyzed and plotted using GraphPad Prism 5.0 software. The results are shown in Figures 9 and 10.

[0152] As shown in Figure 9, the binding activity to BCMA with the Fab fragment of the bispecific chimeric BCMA / CD3 FIT-Fab antibody accurately showed the same binding curve when they consisted of the same BCMA binding domain. As shown in Figure 10, the chimeric BCMA / CD3 FIT-Ig binding protein maintained a binding activity curve similar to that of the parental monoclonal humanized CD3 antibody (see Figures 5B and 5C).

[0153] Example 4.4: Chimeric FIT-Fab and FIT-Ig demonstrated redirection of CD3 activation. A co-cultured reporter gene assay was used to measure the redirection of CD3 activation by BCMA / CD3 bispecific FIT-Ig and FIT-Fab antibodies. Jurkat-NFAT-luc cells elicit downstream luciferase signaling once cell surface CD3 is activated. NCI-H929 cells were used as BCMA-expressing target cells, as they can crosslink the CD3 / TCR complex on T cells via bispecific BCMA / CD3 antibodies upon BCMA binding. Jurkat-NFAT-luc and NCI-H929 cells were washed and resuspended in assay medium (RPMI1640 containing 10% FBS), respectively. Both cell types were placed in a 1:1 ratio in a 96-well plate (Costar #3903) at a rate of 1 × 10⁶ cells per well. 5 Cells were seeded. FIT-Ig antibody or FIT-Fab antibody was added and mixed with the cells, and incubated at 37°C for 4 hours. At the end of incubation, the ONE-Glo® luminescence assay kit (Promega, catalog no. E6130) reagent was prepared according to the manufacturer's instructions and added to the wells. The luminescence signal from the plate was read using a Varioskan® LUX microplate reader (ThermoFisher Scientific). The results are shown in Figures 11 and 12.

[0154] Referring to Figure 11, there are two antibodies with high affinity: an anti-BCMA antibody and an anti-CD3 antibody, that is, The FIT-Ig concentrations that induce T cell activation are plotted for FIT-Ig binding proteins prepared using mAbBCMA-003 and HuEM0006-01-24 as described in Example 4.1. In this figure, FIT1006-4a is a FIT-Ig having an external CD3-binding Fab site and an internal BCMA-binding Fab site (see Table 9 above); FIT1006-4b is a FIT-Ig construct that uses the same amino acid sequence but has a reversed binding domain, i.e., an external BCMA-binding Fab site and an internal CD3-binding Fab site; and FIT1006-4a-Fab was prepared from FIT1006-4a by papain digestion (see Example 4.2). The performance of these binding proteins was compared to two negative controls: (i) FIT-Ig ("FIT1002-5a") with binding sites reactive to two unrelated antigen targets, and (ii) a humanized IgG monoclonal antibody ("hIgG") reactive to an unrelated antigen. Two anti-CD3 binding proteins, namely a humanized anti-CD3 monoclonal antibody (HuEM0006-01-24) and a Fab fragment derived from it (HuEM0006-01-24-Fab), were also tested. All bispecific BCMA / CD3 binding proteins were observed to result in increased T cell activation compared to monospecific anti-CD3 binding proteins with BCMA binding activity in the presence of BCMA-expressing target cells.

[0155] Referring to Figure 12, the concentrations of various bispecific BCMA / CD3 FIT-Fab-binding proteins that cause T cell activation in the presence of BCMA-expressing target cells are plotted for FIT-Fab (referred to as FIT1006-3a-Fab, FIT1006-4a-Fab, FIT1006-5a-Fab, FIT1006-6a-Fab, FIT1006-7a-Fab, and FIT1006-8a-Fab) prepared from the FIT-Ig-binding proteins listed in Table 9 above. The performance of these FIT-Fabs was compared to a reference anti-CD3 Fab and mAbBCMA-002 combination, a reference FIT-Fab antibody designated FIT1006-1a-Fab using anti-CD3 and anti-BCMA binding regions disclosed in WO2016 / 020332, and a negative control FIT-Fab designated FIT1002-5a-Fab prepared using parental antibody binding sites directed to two unrelated antigen targets.

[0156] These results demonstrated that BCMA / CD3 bispecific antibodies can activate CD3 by crosslinking upon binding to BCMA on the surface of tumor cells. In this assay, not only FIT-Ig binding protein (Figure 11) but also FIT-Fab binding protein (Figure 12) showed activation redirection. Furthermore, FIT1006-4a-Fab showed remarkably rapid activation polarity even at low concentrations.

[0157] Example 4.5: Chimeric BCMA / CD3 FIT-Fab showed T cell cytotoxic redirection. The tumor cell killing efficacy of BCMA / CD3 bispecific binding protein was measured using a redirected T cell cytotoxicity assay with the human myeloma cell line NCI-H929 as the target cell and human T cells as the effector cells. Briefly, cells were collected, washed, and resuspended in assay medium (RPMI1640 containing 10% FBS). NCI-H929 cells were placed in a flat-bottomed 96-well plate (Corning, catalog no. 3599) at a rate of 5 × 10⁶ cells per well. 4Cells were seeded. T cells were purified from human PBMCs using a commercially available PBMC isolation kit (EasySep®, Stemcell Technologies, catalog number 17951), and 2 × 10⁶ cells were extracted per well. 5 The test antibody was added to the cells and incubated with the cell mixture at 37°C for 48 hours. Lactate dehydrogenase (LDH) release was measured using the CytoTox 96® Cytotoxic Assay Kit (Promega, catalog number G1780). OD490 readings were obtained according to the manufacturer's instructions. Target cells NCI-H929 maximal lysis (100%) - maximum Low lysis (0%) was used as the denominator for normalization. The percentage of LDH release was plotted against the concentration of the bispecific antibody. As shown in Figure 13, bispecific FIT-Fab with anti-BCMA and anti-CD3 specificity showed T-cell cytotoxic redirection against NCI-H929 tumor cells, while monospecific humanized anti-CD3 Fab and the combination of anti-CD3 Fab (Fab fragment of HuEM0006-01-24) and anti-BCMA mAb (TAB1) did not show cytotoxic activity.

[0158] Example 4.5: Chimeric FIT-Ig showed limited non-targeted CD3 activation redirection in vitro. Non-targeted CD3 activation redirection was tested using a reporter gene assay based on Jurkat-NFAT-luc in the absence of target cells. Jurkat-NFAT-luc cells were harvested, washed, resuspended in assay medium (RPMI1640 containing 10% FBS), and loaded into a 96-well plate (Costar #3903) at a rate of 1 × 10⁶ cells per well. 5 Cells were seeded. The test antibody was added and mixed with the cells, and incubated at 37°C for 4 hours. After incubation, the ONE-Glo® luminescence assay kit (Promega, catalog number E6130) reagent was prepared according to the manufacturer's instructions. The luminescence signal from the plate was read using a Varioskan® Lux plate reader. The results are shown in Figure 14.

[0159] This assay was similar to the one performed in Example 4.4 above, except for the absence of cells expressing a bispecific binding protein, in this case a co-target for BCMA. These results showed that bispecific BCMA / CD3 FIT-Ig antibodies (FIT1006-4a and FIT1006-4b) and BCMA / CD3 FIT-Fab designated as FIT1006-4a-Fab (all possessing the same CD3 binding domain as the humanized anti-CD3 monoclonal antibody HuEM0006-01-24) exhibited significantly lower non-target activation redirection than anti-CD3 antibodies alone in the absence of BCMA-expressing target cells (see Figure 11).

[0160] Example 5: Preparation of humanized bispecific BCMA / CD3 FIT-Ig binding protein The anti-BCMA monoclonal mAbBCMA-003 showed higher BCMA binding affinity and better cell death redirection when used in BCMA / CD3 FIT-Ig and FIT-Fab forms. Therefore, mAbBCMA-003 was selected for use in humanization and subsequent construction of humanized bispecific binding proteins.

[0161] Example 5.1: Humanization of anti-BCMA antibody mAbBCMA-003 Humanized mAbs were created using the mAbBCMA-003 variable region gene. In the first step of this method, the amino acid sequences of VH and VK of mAbBCMA-003 (see Table 8, previously mentioned) were compared with available databases of human IgV gene sequences to find the best overall matching human germline IgV gene sequence. Furthermore, the VH or VL framework 4 was compared with the J region database to find the human framework with the greatest homology to the mouse VH and VL regions, respectively. For the light chain, the closest human V gene match was the VK1-39(02) gene, and for the heavy chain, the closest human match was the VH1-03 gene. Next, we designed humanized variable domain sequences in which the CDR-L1, CDR-L2, and CDR-L3 of the mAbBCMA-003 light chain were grafted onto the framework sequence of the VK1-39(02) gene along with the JK2 framework 4 sequence following CDR-L3; and the CDR-H1, CDR-H2, and CDR-H3 sequences of the mAbBCMA-003 VH were grafted onto the framework sequence of the VH1-03 gene along with the JH6 framework 4 sequence following CDR-H3. Then, we constructed a 3D Fv model of mAbBCMA-003 and identified the framework residues that were most likely to be important for supporting loop structures or VH / VL interference, located less than 4 Å away from the CDR residues. These were analyzed to determine whether or not to perform this action. These residues in the humanized sequence should be reverse-mutated to mouse residues at the same position to maintain affinity / activity. Q1E mutations were always included, where applicable, to eliminate the formation of the N-terminal pyroglutamate. For the heavy chain, P30T, I48M, K66R, A67V, L69I, and A71R Potential mutations in the (Kabat numbering) were identified as desirable reverse mutations. For the light chain, V58I and R69T were identified as reverse mutations. Based on the hierarchy of importance of each reverse mutation as determined by their interaction with the CDR, the most important reverse mutations were introduced into the humanized VH sequence in order of priority, followed by the introduction of other reverse mutations as a sequential design. Furthermore, the DG dipeptide resulting at the C-terminus of CDR-L2 exhibits a potential aspartate isomerization site, which was eliminated in the light chain mutants by the following alternative substitutions: D56A, D56E, D56S, D56T, or G57A. The humanized VH and VL design constructs are shown in Table 10 (below). (Reverse-mutated framework amino acid residues are double-underlined, and the mouse CDR from the original parental antibody is underlined.)

[0162] [Table 12-1] [Table 12-2] [Table 12-3]

[0163] Humanized anti-BCMA VH and VL genes were synthesized and then individually cloned into FIT-Ig vectors, as described in Example 4.1, which also contained the VH and VL genes derived from anti-CD3 monoclonal HuEM0006-01-24. Pairing of humanized VH and humanized VL produced the humanized BCMA / CD3 FIT-Ig binding proteins listed in Table 11 below. Chimeric antibodies containing the parental mouse VH / VL and human constant sequences of mAbBCMA-003 were also produced as positive controls for ranking the humanized binding proteins. All recombinant FIT-Ig were expressed and purified as described in Example 4.1.

[0164] [Table 13]

[0165] Bispecific FIT-Ig and FIT-Fab binding proteins capable of binding to both BCMA and CD3 antigens were constructed using cDNA encoding the humanized variable domains listed in Table 11 above and human constant region sequences (SEQ ID NOs. 26 and 27) as shown in Table 3, in the same manner as described in Examples 4.1 and 4.2 above. Since no linkers were used between immunoglobulin domains, the complete sequences of the FIT-Ig binding proteins may be derived from the sequence information in Tables 11 and 3. For example, with respect to FIT1006-29b(DA), FIT1006-31b(DT), and FIT1006-35b(DT) as FIT-Ig, the amino acid sequences of the three polypeptide chains of the three exemplary FIT-Ig binding proteins disclosed in Table 11 are shown in Tables 12, 13, and 14 below. These FIT-Ig molecules have a BCMA binding site at the N-terminus of the assembled chain, and the CD3 binding site is adjacent to the Fc region (N-terminus) and located inside the C-terminus of the FIT-Ig structure of the BCMA binding site. In other words, the domain structure of the component polypeptide chain is as follows: Chain 1 (long chain): VL BCMA -CL-VH CD3 -CH1-Hinge-CH2-CH3; Chain 2 (first short chain): VH BCMA -CH1; Chain 3 (second short chain): VL CD3 -CL And here, VL BCMA This is a light chain of a humanized monoclonal antibody that recognizes BCMA. It is a variable domain, VH CD3 This is the heavy chain variable domain of a humanized monoclonal antibody that recognizes CD3, and VL CD3 This is the light chain variable domain of a humanized monoclonal antibody that recognizes CD3, and VH BCMAis the heavy chain variable domain of a humanized monoclonal antibody that recognizes BCMA, where each CL is a light chain constant domain (SEQ ID NO: 27), each CH1 is the first heavy chain constant domain, and CH1-hinge-CH2-CH3 is the C-terminal heavy chain constant region from CH1 to the end of the Fc region (see SEQ ID NO: 26).

[0166] [Table 14]

[0167] [Table 15]

[0168] [Table 16]

[0169] Example 5.3: Binding rate of humanized BCMA / CD3 to FIT-Ig The binding affinity and rate constant of the BCMA / CD3 bispecific FIT-Ig antibody were measured by surface plasmon resonance (SPR) at 25°C using a Biacore® T200 instrument (GE Healthcare) with a standard procedure. The results are shown in Table 15.

[0170] Simply put, heterodimer CD3 / Fc antibody according to a typical amine coupling method. The proto-antigen or BCMA / Fc antigen was immobilized directly onto a biosensor chip, and then the antibody was injected into the reaction matrix at a flow rate of 5 μl / min to record the binding response. The binding rate constant and dissociation rate constant were recorded, respectively. on (M -1 s -1 ) and k off (s -1 The rate constant was determined at a continuous flow rate of 30 μl / min. The rate constant was derived by performing kinetic binding measurements at five different concentrations of human CD3 / Fc protein or human BCMA / Fc protein. Next, the equilibrium dissociation constant K of the reaction between the antibody and the associated target protein was determined. D(M) is given by formula K D =k off / k on The reaction rate constant was calculated using [a specific method]. The affinity of the humanized anti-CD3 / humanized anti-BCMAFIT-Ig antibody was measured as shown in Table 15 below.

[0171] [Table 17]

[0172] Example 5.4: Humanized bispecific FIT-Ig exhibited CD3 activation and cytotoxic redirection. The efficacy of BCMA / CD3 humanized bispecific FIT-Ig antibody in tumor cell death was measured using a redirected T cell cytotoxicity assay with the human myeloma cell line NCI-H929 as the target cell and human T cells as the effector cells. In short, the cells The cells were collected, washed, and resuspended in assay medium (RPMI1640 containing 10% FBS). NCI-H929 cells were placed in a flat-bottom 96-well plate (Corning #3599) at a rate of 5 × 10⁶ cells per well. 4 Cells were seeded. T cells were purified from human PBMCs using a commercially available kit (Stemcell #17951) and placed in the same plate at a rate of 2 × 10⁶ per well. 5Cells were added. Next, the FIT-Ig binding protein was added and incubated with the cell mixture. After incubation at 37°C for 48 hours, LDH release was measured using the assay kit (Promega #G1780). OD490 readings were obtained according to the manufacturer's instructions. The denominator for normalization was shown as maximum (100%)-minimum (0%) lysis of target cells NCI-H929. The percentage of LDH release was plotted against the concentration of bispecific Ab. In this example, humanized FIT-Ig showed similar T cell cytotoxic redirection as parental chimeric FIT-Ig. The results are shown in Figure 15. These results demonstrate that the humanized BCMA / CD3 FIT-Ig according to the present invention was able to redirect T cell cytotoxicity against NCI-H929 tumor cells in co-culture. Referring to Figures 16 and 17, the binding of the two BCMA / CD3 bispecific FIT-Ig binding proteins according to the present invention to BCMA-expressing target cells and CD3-expressing target cells is confirmed.

[0173] Two alternative constructs of exemplary BCMA / CD3 FIT-Ig binding proteins were prepared, in which the external binding site is a tandem-configured CD3 Fab binding site in the Fab region and the internal binding site is a BCMA Fab binding site. These were designated FIT1006-31a(DT) and FIT1006-35a(DT). The polypeptide chain formulas for these two FIT-Ig proteins are: Chain 1 (long chain): VL CD3 -CL-VH BCMA -CH1-Hinge-CH2-CH3; Chain 2 (first short chain): VH CD3 -CH1; Chain 3 (second short chain): VL BCMA -CL The amino acid sequences of the polypeptide chains of FIT1006-31a(DT) and FIT1006-35a(DT) are shown below.

[0174] [Table 18]

[0175] [Table 19]

[0176] The binding activity of these two different configurations to BCMA-expressing cells and CD3-expressing target cells is shown in Figures 18 and 19, respectively. Comparing the two configurations for each target, the corresponding binding domain located distal to the Fc showed relatively higher binding activity than the same binding domain located proximal to the Fc, indicating a specific influence of the configuration on binding. Nevertheless, these results confirm that both configurations possess desirable target binding activity for both BCMA and CD3.

[0177] Example 6: Treatment with BCMA×CD3 FIT-Ig reduces the volume of NCI-H929 tumors in human PBMC-grafted NPSG mice. Antitumor efficacy was evaluated in NPSG mice, an immunodeficient line lacking T cells, B cells, and natural killer cells. NCI-H929 cells (5 × 10⁶) 6 The drug was subcutaneously injected into the right dorsal flank of NPSG mice. On the same day, the mice received a single intravenous dose of 5 × 10⁶. 6 Hi PBMC was performed. On the 11th day, the tumor size was 70-140 mm. 3 Animals were randomized based on the criteria and treatment was initiated on the same day. Tumor growth was monitored by caliper measurement. The study was completed on day 25, when the tumor size reached 3000 mm. 3 Mice were euthanized when the threshold was exceeded. Mice were treated intraperitoneally (ip) with 6 mg / kg of FIT1006-31b(DT) or FIT1006-35b(DT) or a vehicle once a week (QW x 3) for 3 weeks. As shown in Figure 20, mice treated with FIT-Ig showed significant inhibition of tumor growth compared to the vehicle group. In particular, tumors were completely eradicated in the FIT1006-35b(DT) treated group.

[0178] Example 7: BCMA×CD3 FIT-Ig depletes the B cell population in cynomolgus monkeys and exhibits limited cytokine release characteristics. Cynomolgus monkey B cells have been reported to show higher BCMA expression than human B cells (Seckinger, A. et al., (2017). Target Expression, Generation, Preclinical Activity, and Pharmacokinetics of the BCMA-T Cell Bispecific Antibody EM801 for Multiple Myeloma Treatment. Cancer Cell, 31(3), 396-410). To evaluate the ability of BCMA×CD3 FIT-Ig to deplete B cell populations in these animals, a pilot non-GLP toxicological and pharmacological study was conducted in cynomolgus monkeys. This study consisted of three groups of monkeys, each consisting of one male and one female with comparable body weight. Group 1 was provided with a vehicle, Group 2 received a single injection of 0.5 mg / kg FIT1006-31b(DT), and Group 3 received a single injection of 0.5 mg / kg FIT1006-35b(DT). All were administered intravenously on day 1. Blood samples were collected from subcutaneous veins of the forelimb or hindlimb two days prior to administration (-1 day, baseline), 2, 4, 6, and 24 hours after administration on day 1, and on days 8 and 15. Blood samples were analyzed by FACS for B cell markers and T cell markers, and the relative percentage changes in each population were determined by comparing them to baseline levels at -1 day. Serum samples were also analyzed for cytokine levels (INFγ, IL-2, IL-6, and TNFα) using a commercially available cytometry bead array (CBA) kit.

[0179] Figure 21 shows the depletion of over 50% of circulating B cells caused by BCMA×CD3 FIT-Ig administration from the first dose (2 hours post-administration) to the last dose (day 15). Temporary B cell depletion was also observed in the vehicle group by the second and third dose times (2 and 4 hours on day 1), which may be related to the blood collection schedule. However, the B cell population in the vehicle group showed rapid recovery, reaching a plateau by 6 hours post-administration.

[0180] As shown in Figure 22, circulating T cell levels in the FIT-Ig treatment group showed a transient loss, which recovered to the vehicle group level by day 8 and remained there until the end of the study. This transient T cell loss was thought to be due to T cell activation and redistribution during treatment.

[0181] The present invention can be embodied in other specific forms without departing from the essential features of the invention described above. Therefore, the above embodiments are considered illustrative rather than limiting of the invention described herein. The scope of the invention is indicated by the appended claims.

Claims

1. The following CD-R set: Table 1 An anti-BCMA antibody or its antigen-binding moiety comprising a set of six CDRs selected from the group CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.

2. The following VH / VL pair: Table 2 The anti-BCMA antibody or its antigen-binding moiety according to claim 1, comprising a VH domain and a VL domain having an amino acid sequence selected from the above.

3. (i) A VH domain having a sequence selected from SEQ ID NO: 29, SEQ ID NO: 34, or SEQ ID NO: 36 and a CH1-hinge-CH2-CH3 human stationary IgG1 having the sequence of SEQ ID NO: 26; and (ii) A VL domain having a sequence selected from SEQ ID NO: 30, SEQ ID NO: 43, or SEQ ID NO: 48 and a CLκ having a sequence of SEQ ID NO: 27 The anti-BCMA antibody or its antigen-binding moiety according to claim 1, comprising the above.

4. A pharmaceutical composition comprising at least one anti-BCMA antibody or its antigen-binding fragment as described in claim 1, and a pharmaceutically acceptable carrier.

5. Use of an anti-BCMA antibody or its antigen-binding moiety according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4 for the manufacture of a drug for treating a disease or disorder in which CD3-mediated activity and / or BCMA-mediated activity is harmful.

6. The use according to claim 5, wherein the disease is cancer, and optionally selected from multiple myeloma, melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and primary bone cancer (e.g., osteosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma, or chondrosarcoma).

7. The following CD-R set: Table 3 CDR-H1-1, CDR-H2-1, CDR-H3-1, CDR-L1-1, CDR-L2-1 and CDR-L3-1 are selected from the group, and Table 4 CDR-H1-2, CDR-H2-2, CDR-H3-2, CDR-L1-2, CDR-L2-2, and CDR-L3-2 are selected from the group. A set of 12 CDRs comprising an anti-BCMA and anti-CD3 bispecific antibody or its antigen-binding moiety.

8. The following VH / VL pair: Table 5 and Table 6 An anti-BCMA and anti-CD3 bispecific antibody or its antigen-binding moiety comprising a VH domain and a VL domain having an amino acid sequence selected from the group.

9. (i) A first VH domain having a sequence selected from SEQ ID NO: 29, SEQ ID NO: 34, or SEQ ID NO: 36, and a CH1-hinge-CH2-CH3 human constant IgG1 having the sequence of SEQ ID NO: 26; (ii) A first VL domain having a sequence selected from SEQ ID NO: 30, SEQ ID NO: 43, or SEQ ID NO: 48, and a CLκ having the sequence of SEQ ID NO: 27; (iii) Sequence ID: 8, Sequence ID: 11, Sequence ID: 12, Sequence ID: 15, Sequence ID: 17 or CH1-hinge-CH2-CH3 human constant IgG1 having sequences of sequence number 18 and sequence number 26; and (iv) A second VL domain having a sequence selected from sequence number 9 or sequence number 25 CLκ having sequence number: 27 The anti-BCMA and anti-CD3 bispecific antibody or its antigen-binding moiety according to claim 7 or 8, comprising the above.

10. The anti-BCMA and anti-CD3 bispecific antibody or its antigen-binding moiety according to claim 7 or 8, comprising three polypeptides, wherein the first polypeptide has the sequence of SEQ ID NO: 80, the second polypeptide has the sequence of SEQ ID NO: 81, and the third polypeptide has the sequence of SEQ ID NO:

82.

11. A pharmaceutical composition comprising at least one anti-BCMA and anti-CD3 bispecific antibody or its antigen-binding moiety as described in claim 7, and a pharmaceutically acceptable carrier.

12. Use of an anti-BCMA and anti-CD3 bispecific antibody or its antigen-binding fragment according to claim 7 or 8, or the pharmaceutical composition according to claim 11, for the preparation of a pharmacopoeia for treating a disease or disorder in which CD3-mediated activity and / or BCMA-mediated activity is harmful.

13. The use according to claim 12, wherein the disease is cancer, and optionally selected from multiple myeloma, melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and primary bone cancer (e.g., osteosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma, or chondrosarcoma).

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