Method for treating multiple myeloma with a bispecific anti-BCMA × anti-CD3 antibody

Abstract

B-cell maturation antigen (BCMA) is expressed on malignant plasma cells. The present invention provides a method for treating multiple myeloma using a bispecific antibody (bsAb) that binds to both BCMA and CD3 and activates T cells via the CD3 complex in the presence of BCMA-expressing tumor cells. In certain embodiments, the bispecific antigen-binding molecule of the present invention can inhibit the growth of BCMA-expressing tumors.

Description

Technical Field

[0001] Reference to Sequence Listing This application incorporates by reference a Sequence Listing submitted in computer-readable form as file 10702WO01-Sequence, created on December 4, 2020, and containing 81,956 bytes.

[0002] The present invention relates to bispecific antigen-binding molecules (e.g., bispecific antibodies) that bind to BCMA and CD3, and methods of using them.

Background Art

[0003] B cell maturation antigen (BCMA), also known as TNFRSF17 or CD269, is a type III transmembrane protein lacking a signal peptide and containing an extracellular domain rich in cysteine. BCMA, together with closely related proteins, promotes the survival of B cells at different stages of development. BCMA is expressed only in B cell lineage cells, particularly in the interfollicular regions of germinal centers, as well as in plasmablasts and differentiated plasma cells. BCMA is selectively induced during the differentiation of plasma cells and is required for the optimal survival of long-lived plasma cells in the bone marrow. In multiple myeloma, BCMA is widely expressed at high levels on malignant plasma cells, and the expression of BCMA increases with the progression from normal cells to active multiple myeloma. BCMA is also expressed in other B cell malignancies, including Waldenström macroglobulinemia, Burkitt lymphoma, and diffuse large B cell lymphoma. Non-Patent Document 1.

[0004] CD3 is a homodimeric or heterodimeric antigen that is expressed on T cells together with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed from the dimerization of two out of four different chains: epsilon, zeta, delta, and gamma. The dimer arrangements of CD3 include gamma / epsilon, delta / epsilon, and zeta / zeta. Antibodies against CD3 have been shown to cluster CD3 on T cells and thereby cause T cell activation in a manner similar to the engagement of the TCR by peptide-loaded MHC molecules. Thus, anti-CD3 antibodies have been proposed for therapeutic purposes including the activation of T cells. Furthermore, bispecific antibodies that can bind to both CD3 and a target antigen have been proposed for therapeutic use including targeting the T cell immune response to tissues and cells that express the target antigen.

[0005] Multiple myeloma patients who are resistant to multiple classes of therapy have a reduced overall survival (triple and quadruple refractory: 9.2 months, quintuple refractory: 5.6 months). Non-Patent Document 2. Antigen-binding molecules that target BCMA and include bispecific antigen-binding molecules that bind to both BCMA and CD3 may be useful in therapeutic settings where it is desired to specifically target cells that express BCMA and kill them in a T cell-mediated manner.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0007] In one aspect, the present invention provides an EC of less than about 100 nM as measured by an in vitro FACS binding assay 50a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA) on target tumor cells, and (b) about 10 -6 EC less than M 50 a second antigen-binding domain (D2) that specifically binds to human CD3, and provides an isolated bispecific antigen-binding molecule.

[0008] Optionally, the bispecific antigen-binding molecule is about 10 -9 EC less than M 50 activates T cells in vitro. Optionally, the bispecific antigen-binding molecule is about 10 -9 EC less than M 50 mediates in vitro T cell killing of tumor cell lines expressing BCMA with. Optionally, the bispecific antigen-binding molecule is about 10 -8 EC less than M 50 mediates in vitro autologous T cell killing of primary myeloma cells expressing BCMA with. In some embodiments, the bispecific antigen-binding molecule interacts with amino acid residues 1-43 of BCMA set forth in SEQ ID NO: 115.

[0009] In some cases, the target tumor cells are plasma cells. In some cases, the target tumor cells are from a patient suffering from multiple myeloma or from another B-cell disorder that is partially characterized by having B cells that express BCMA. In some cases, the bispecific antigen-binding molecule inhibits the proliferation of BCMA-expressing tumor cells at a dose of about 0.04 mg / kg to about 4.0 mg / kg. In some cases, the dose is 0.04 mg / kg, 0.4 mg / kg, or 4 mg / kg. In some cases, the dose is about 0.001 mg / kg, 0.002 mg / kg, 0.003 mg / kg, 0.004 mg / kg, 0.005 mg / kg, 0.006 mg / kg, 0.007 mg / kg, 0.008 mg / kg, 0.009 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg. In some embodiments, the dose is administered to a patient in need thereof at least twice a week for at least 7 doses. In some embodiments, the dose is administered to a patient in need thereof at least once a week. In some embodiments, the dose is administered to the patient at least every two weeks. In some embodiments, the dose is administered to the patient at least every four weeks. In some cases, the bispecific antigen-binding molecule inhibits the proliferation of BCMA+ tumor cells selected from the group consisting of myeloma cells, lymphoma cells, and leukemia cells.In some cases, the bispecific antigen-binding molecule inhibits the proliferation of BCMA+ tumor cells selected from the group consisting of H929 cells, MOLP-8 cells, and OPM cells.

[0010] In some cases, the bispecific antigen-binding molecule cross-reacts with cynomolgus BCMA. In some cases, the bispecific antigen-binding molecule does not cross-react with cynomolgus BCMA.

[0011] In some embodiments, the isolated bispecific antigen-binding molecule comprises a first antigen-binding domain comprising: (a) three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 66, and (b) three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 82. In some cases, the first binding domain of the bispecific antigen-binding molecule comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 68, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 70, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 72. In some cases, the first binding domain of the bispecific antigen-binding molecule comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 84, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 88. In some cases, the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 66 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82.

[0012] In some embodiments, the isolated bispecific antigen-binding molecule comprises a second antigen-binding domain comprising (a) three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 98, and (b) three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 82. Optionally, the second antigen-binding domain comprises (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 100, (b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 102, and (c) HCDR3 comprising the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 104. Optionally, the second antigen-binding domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 84, LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 88. Optionally, the second antigen-binding domain comprises (a) the HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequences of SEQ ID NO: 92, 94, and 96, respectively, and the LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequences of SEQ ID NO: 84, 86, and 88, respectively, or (b) the HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequences of SEQ ID NO: 100, 102, and 104, respectively, and the LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequences of SEQ ID NO: 84, 86, and 88, respectively. Optionally, the second antigen-binding domain comprises (a) an HCVR comprising the amino acid sequence of SEQ ID NO: 90 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82, or (b) an HCVR comprising the amino acid sequence of SEQ ID NO: 98 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82.

[0013] In another aspect, the present invention provides an isolated bispecific antigen-binding molecule comprising a first antigen-binding domain comprising: (a) HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequence of SEQ ID NO: 68, 70, 72, respectively, and LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequence of SEQ ID NO: 84, 86, 88, respectively.

[0014] In another aspect, the present invention provides an isolated bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequence of SEQ ID NO: 68, 70, 72, respectively, and LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequence of SEQ ID NO: 84, 86, 88, respectively; and (b) a second antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequence of SEQ ID NO: 92, 94, 96, respectively, and LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequence of SEQ ID NO: 84, 86, 88, respectively. Optionally, the isolated bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising HCVR comprising the amino acid sequence of SEQ ID NO: 66 and LCVR comprising the amino acid sequence of SEQ ID NO: 82; and (b) a second antigen-binding domain comprising HCVR comprising the amino acid sequence of SEQ ID NO: 90 and LCVR comprising the amino acid sequence of SEQ ID NO: 82.

[0015] In another aspect, the present invention provides an isolated bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequence of SEQ ID NO: 68, 70, 72, respectively, and LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequence of SEQ ID NO: 84, 86, 88, respectively; and (b) a second antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequence of SEQ ID NO: 100, 102, 104, respectively, and LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequence of SEQ ID NO: 84, 86, 88, respectively. Optionally, the isolated bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising HCVR comprising the amino acid sequence of SEQ ID NO: 66 and LCVR comprising the amino acid sequence of SEQ ID NO: 82; and (b) a second antigen-binding domain comprising HCVR comprising the amino acid sequence of SEQ ID NO: 98 and LCVR comprising the amino acid sequence of SEQ ID NO: 82.

[0016] In another aspect, the present invention provides an isolated bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain comprising a CDR of an HCVR that specifically binds to human BCMA and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 122, and 124, and a CDR of an LCVR that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 82, 123, and 125; and (b) a second antigen-binding domain that specifically binds to human CD3. Optionally, the first antigen-binding domain comprises CDRs from an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 122 / 123, 124 / 125, 2 / 82, 18 / 82, 34 / 82, 50 / 82, 66 / 82, 122 / 82, and 124 / 82. Optionally, the first antigen-binding domain comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-76-78-80, 4-6-8-84-86-88, 20-22-24-84-86-88, 36-38-40-84-86-88, 52-54-56-84-86-88, and 68-70-72-84-86-88. Optionally, the first antigen-binding domain comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 122 / 123, 124 / 125, 2 / 82, 18 / 82, 34 / 82, 50 / 82, 66 / 82, 122 / 82, and 124 / 82. Optionally, the second antigen-binding domain comprises CDRs from an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 90 / 82 and 98 / 82.

[0017] In another aspect, the present invention provides an isolated bispecific antigen-binding molecule that competes with binding to BCMA or binds to the same epitope on BCMA as a reference antibody, wherein the reference antibody comprises a first antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 66 / 82, and a second antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of either SEQ ID NO: 90 / 82 or SEQ ID NO: 98 / 82.

[0018] In another aspect, the present invention provides an isolated bispecific antigen-binding molecule that competes with binding to human CD3 or binds to the same epitope on human CD3 as a reference antibody, wherein the reference antibody comprises a first antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 66 / 82, and a second antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of either SEQ ID NO: 90 / 82 or SEQ ID NO: 98 / 82.

[0019] Any of the bispecific antigen-binding molecules described above or herein can be a bispecific antibody. Optionally, the bispecific antibody comprises a human IgG heavy chain constant region. Optionally, the human IgG heavy chain constant region is isotype IgG1. Optionally, the human IgG heavy chain constant region is isotype IgG4. In various embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to the wild-type hinge of the same isotype. Optionally, the bispecific antibody comprises a first heavy chain comprising a constant region comprising the amino acid sequence of SEQ ID NO: 130. Optionally, the bispecific antibody comprises a second heavy chain comprising a constant region comprising the amino acid sequence of SEQ ID NO: 131. In some embodiments, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 126, a second heavy chain comprising the amino acid sequence of either SEQ ID NO: 127 or SEQ ID NO: 128, and a common light chain comprising the amino acid sequence of SEQ ID NO: 129.

[0020] In another aspect, the present invention provides a pharmaceutical composition comprising the bispecific antigen-binding molecule (e.g., bispecific antibody) described above or discussed herein, and a pharmaceutically acceptable carrier or diluent.

[0021] In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antigen-binding molecule (e.g., bispecific antibody) described above or discussed herein.

[0022] In another aspect, the present invention provides an expression vector comprising the nucleic acid molecule discussed above.

[0023] In another aspect, the present invention provides a host cell comprising the nucleic acid molecule or expression vector discussed above.

[0024] In another aspect, the invention provides a method of inhibiting the growth of a plasmacytoma tumor in a subject, the method comprising administering to the subject an isolated bispecific antigen-binding molecule, or a pharmaceutical composition comprising a bispecific antigen-binding molecule, as described above or herein. Optionally, the plasmacytoma tumor is multiple myeloma. Optionally, the method further comprises administering a second therapeutic agent or regimen. In some embodiments, the second therapeutic agent comprises an anti-tumor agent (e.g., a chemotherapeutic agent including melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), oblimersen (Treanda), or any other known to be effective in treating a plasmacytoma tumor in a subject). In some embodiments, the second therapeutic agent comprises a steroid. In some embodiments, the second therapeutic agent comprises a targeted therapy including thalidomide, lenalidomide, and bortezomib, which are therapies approved for treating newly diagnosed patients. Lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab, and daratumumab are examples of second therapeutic agents effective for treating relapsed myeloma. In certain embodiments, the second therapeutic agent is a regimen including radiation therapy or stem cell transplantation. In certain embodiments, the second therapeutic agent can be an immunomodulatory agent. In certain embodiments, the second therapeutic agent can be a proteasome inhibitor including bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro). In certain embodiments, the second therapeutic agent can be a histone deacetylase inhibitor such as panobinostat (Farydak). In specific embodiments, the second therapeutic agent can be a monoclonal antibody, an antibody-drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a checkpoint inhibitor, or a combination thereof.

[0025] In another aspect, the invention provides a method of treating a patient afflicted with multiple myeloma or another BCMA-expressing B cell malignancy, the method comprising administering to the subject an isolated bispecific antigen-binding molecule, or a pharmaceutical composition comprising a bispecific antigen-binding molecule, as described above or herein. Optionally, the BCMA-expressing B cell malignancy is selected from the group consisting of Waldenström macroglobulinemia, Burkitt lymphoma, diffuse large B cell lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin lymphoma. Optionally, the method further comprises administering a second therapeutic agent. In some embodiments, the second therapeutic agent is an anti-tumor agent (chemotherapeutic agent), a DNA alkylating agent, an immunomodulatory agent, a proteasome inhibitor, a histone deacetylase inhibitor, radiation therapy, stem cell transplantation, an immunomodulatory agent, a monoclonal antibody that interacts with an antigen expressed on the surface of a tumor cell, a monoclonal antibody other than those described herein that can interact with a different antigen on the surface of a plasma cell, a bispecific antibody having one arm that binds to an antigen on the surface of a tumor cell and another arm that binds to an antigen on a T cell, an antibody-drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a checkpoint inhibitor, e.g., one that targets PD-1 or CTLA-4, or a combination thereof. In certain embodiments, the checkpoint inhibitor can be selected from PD-1 inhibitors such as pembrolizumab (Keytruda), nivolumab (Opdivo), or semiprimumab (REGN2810). In certain embodiments, the checkpoint inhibitor can be selected from PD-L1 inhibitors such as atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi). In certain embodiments, the checkpoint inhibitor can be selected from CTLA-4 inhibitors such as ipilimumab (Yervoy). Other combinations that can be used in combination with the antibodies of the invention are described above.

[0026] In another aspect, the present invention provides a method of treating a patient suffering from a BCMA-expressing tumor, the method comprising administering to the subject an isolated bispecific antigen-binding molecule, or a pharmaceutical composition comprising the same, as described above or discussed herein, in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof. Optionally, the anti-PD-1 antibody or antigen-binding fragment is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is cemiplimab (REGN2810). In various embodiments, the combination of an anti-BCMA×anti-CD3 bispecific antigen-binding molecule (e.g., a bispecific antibody) and an anti-PD-1 antibody or antigen-binding fragment (e.g., an anti-PD-1 antibody) produces a synergistic therapeutic effect in the treatment of BCMA-expressing tumors.

[0027] In another aspect, the present invention provides the use of a bispecific antigen-binding molecule as described above or discussed herein, or a pharmaceutical composition as described above or discussed herein, in the treatment of a disease or disorder associated with the expression of BCMA. Optionally, the disease or disorder is cancer. In some embodiments, the cancer is multiple myeloma. Optionally, the disease or disorder is Castleman disease. Optionally, the antigen-binding molecule is for use in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof, and optionally, the anti-PD-1 antibody is cemiplimab (REGN2810).

[0028] The present invention further comprises the use of a bispecific antigen-binding molecule as described above or discussed herein in the manufacture of a medicament for treating a disease or disorder associated with the expression of BCMA. Optionally, the disease or disorder is cancer. In some embodiments, the cancer is multiple myeloma. The present invention further comprises a bispecific antigen-binding molecule (e.g., a bispecific antibody) for use in the treatment of BCMA+ cancer (e.g., multiple myeloma) in a subject.

[0029] In another aspect, the present invention provides a method of treating multiple myeloma in a subject in need thereof, the method comprising administering to the subject a bispecific antibody comprising a pair of a first heavy chain and a common light chain comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA), and a pair of a second heavy chain and a common light chain comprising a second antigen-binding domain that specifically binds to human CD3, wherein the first antigen-binding domain comprises three heavy chain complementarity determining regions (CDRs) and three light chain CDRs each comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, 84, 86, and 88, and the second antigen-binding domain comprises three heavy chain CDRs and three light chain CDRs each comprising the amino acid sequences of SEQ ID NOs: 92, 94, 96, 84, 86, and 88, and the bispecific antibody is administered to the subject at a dose of 1 mg per week or every two weeks. Optionally, the bispecific antibody is 1 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg per week or every two weeks.Administered to a subject at a dose of 75 mg every at least one or two weeks, 80 mg every at least one or two weeks, 85 mg every at least one or two weeks, 90 mg every at least one or two weeks, 95 mg every at least one or two weeks, 100 mg every at least one or two weeks, 150 mg every at least one or two weeks, 200 mg every at least one or two weeks, 250 mg every at least one or two weeks, 300 mg every at least one or two weeks, 350 mg every at least one or two weeks, 400 mg every at least one or two weeks, 450 mg every at least one or two weeks, 500 mg every at least one or two weeks, 550 mg every at least one or two weeks, 600 mg every at least one or two weeks, 650 mg every at least one or two weeks, 700 mg every at least one or two weeks, 750 mg every at least one or two weeks, 800 mg every at least one or two weeks, 850 mg every at least one or two weeks, or 900 mg every at least one or two weeks.

[0030] Optionally, the first heavy chain comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 66, the second heavy chain comprises a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 90, and the common light chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82. Optionally, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 126, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 127, and the common light chain comprises the amino acid sequence of SEQ ID NO: 129.

[0031] In another aspect, the present invention provides a method of treating multiple myeloma in a subject in need thereof, the method comprising administering to the subject a bispecific antibody comprising a pair of a first heavy chain and a common light chain comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA), and a pair of a second heavy chain and a common light chain comprising a second antigen-binding domain that specifically binds to human CD3, wherein the first antigen-binding domain comprises three heavy chain complementarity-determining regions (CDRs) and three light chain CDRs, each comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, 84, 86, and 88, and the second antigen-binding domain comprises three heavy chain CDRs and three light chain CDRs, each comprising the amino acid sequences of SEQ ID NOs: 100, 102, 104, 84, 86, and 88, and the bispecific antibody is administered to the subject at a dose of 1 mg per week. Optionally, the bispecific antibody is administered at a dose of 1 mg every at least 1 or 2 weeks, 1.5 mg every at least 1 or 2 weeks, 2.0 mg every at least 1 or 2 weeks, 2.5 mg every at least 1 or 2 weeks, 3.0 mg every at least 1 or 2 weeks, 3.5 mg every at least 1 or 2 weeks, 4 mg every at least 1 or 2 weeks, 5 mg every at least 1 or 2 weeks, 6 mg every at least 1 or 2 weeks, 7 mg every at least 1 or 2 weeks, 8 mg every at least 1 or 2 weeks, 9 mg every at least 1 or 2 weeks, 10 mg every at least 1 or 2 weeks, 15 mg every at least 1 or 2 weeks, 20 mg every at least 1 or 2 weeks, 25 mg every at least 1 or 2 weeks, 30 mg every at least 1 or 2 weeks, 35 mg every at least 1 or 2 weeks, 40 mg every at least 1 or 2 weeks, 45 mg every at least 1 or 2 weeks, 50 mg every at least 1 or 2 weeks, 55 mg every at least 1 or 2 weeks, 60 mg every at least 1 or 2 weeks, 65 mg every at least 1 or 2 weeks, 70 mg every at least 1 or 2 weeks,Administered to a subject at a dose of 75 mg at least once or twice a week, 80 mg at least once or twice a week, 85 mg at least once or twice a week, 90 mg at least once or twice a week, 95 mg at least once or twice a week, 100 mg at least once or twice a week, 150 mg at least once or twice a week, 200 mg at least once or twice a week, 250 mg at least once or twice a week, 300 mg at least once or twice a week, 350 mg at least once or twice a week, 400 mg at least once or twice a week, 450 mg at least once or twice a week, 500 mg at least once or twice a week, 550 mg at least once or twice a week, 600 mg at least once or twice a week, 650 mg at least once or twice a week, 700 mg at least once or twice a week, 750 mg at least once or twice a week, 800 mg at least once or twice a week, 850 mg at least once or twice a week, or 900 mg at least once or twice a week.,

[0032] In some cases, the first heavy chain comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 66, the second heavy chain comprises a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 98, and the common light chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82. In some cases, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 126, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 128, and the common light chain comprises the amino acid sequence of SEQ ID NO: 129.

[0033] In any of the methods described above or discussed in this specification, the bispecific anti-BCMA × anti-CD3 antibody can be administered in a dosing regimen that includes divided primary doses. In some embodiments, the bispecific anti-BCMA × anti-CD3 antibody is administered to the subject at a dose of 1 mg per week or more. In some embodiments, the dose of the bispecific anti-BCMA × anti-CD3 antibody is administered to the subject at a dose of 3 mg to 900 mg per week or more. Optionally, the bispecific antibody is 1 mg every at least one week or two weeks, 1.5 mg every at least one week or two weeks, 2.0 mg every at least one week or two weeks, 2.5 mg every at least one week or two weeks, 3.0 mg every at least one week or two weeks, 3.5 mg every at least one week or two weeks, 4 mg every at least one week or two weeks, 5 mg every at least one week or two weeks, 6 mg every at least one week or two weeks, 7 mg every at least one week or two weeks, 8 mg every at least one week or two weeks, 9 mg every at least one week or two weeks, 10 mg every at least one week or two weeks, 15 mg every at least one week or two weeks, 20 mg every at least one week or two weeks, 25 mg every at least one week or two weeks, 30 mg every at least one week or two weeks, 35 mg every at least one week or two weeks, 40 mg every at least one week or two weeks, 45 mg every at least one week or two weeks, 50 mg every at least one week or two weeks, 55 mg every at least one week or two weeks, 60 mg every at least one week or two weeks, 65 mg every at least one week or two weeks, 70 mg every at least one week or two weeks, 75 mg every at least one week or two weeks, 80 mg every at least one week or two weeks, 85 mg every at least one week or two weeks, 90 mg every at least one week or two weeks, 95 mg every at least one week or two weeks, 100 mg every at least one week or two weeks, 150 mg every at least one week or two weeks,Administered to a subject at a dose of 200 mg every at least one or two weeks, 250 mg every at least one or two weeks, 300 mg every at least one or two weeks, 350 mg every at least one or two weeks, 400 mg every at least one or two weeks, 450 mg every at least one or two weeks, 500 mg every at least one or two weeks, 550 mg every at least one or two weeks, 600 mg every at least one or two weeks, 650 mg every at least one or two weeks, 700 mg every at least one or two weeks, 750 mg every at least one or two weeks, 800 mg every at least one or two weeks, 850 mg every at least one or two weeks, or 900 mg every at least one or two weeks.

[0034] In any of the methods described above or discussed herein, the BCMA+ cancer can be multiple myeloma, and the subject being administered the anti-BCMA × anti-CD3 bispecific antibody has been previously treated.

[0035] In any of the methods described above or discussed herein, the BCMA+ cancer can be multiple myeloma, and the subject being administered the anti-BCMA × anti-CD3 bispecific antibody has been previously treated with anti-CD38 antibody therapy. Optionally, the anti-CD38 antibody is daratumumab or isatuximab.

[0036] In any of the methods described above or discussed herein, the BCMA+ cancer can be multiple myeloma, and the subject being administered the anti-BCMA × anti-CD3 bispecific antibody has been previously treated with a proteasome inhibitor or an immunomodulatory agent. Optionally, the proteasome inhibitor is bortezomib, carfilzomib, or ixazomib. Optionally, the immunomodulatory agent is lenalidomide or pomalidomide.

[0037] In any of the methods described above or discussed herein, the subject may have relapsed or refractory multiple myeloma. In some cases, the subject has relapsed or refractory multiple myeloma after one or more (e.g., two or more, three or more, four or more, or five or more) systemic treatments, including any one or more of the treatments described above or discussed herein.

[0038] In any of the methods described above or discussed herein, the subject can be a patient having a multiple myeloma immunotype selected from immunoglobulin G, immunoglobulin A, lambda light chain, or kappa light chain.

[0039] In any of the methods described above or discussed herein, the subject may have extramedullary plasmacytoma.

[0040] In any of the methods described above or discussed herein, the subject is at least triple refractory to previous treatment (i.e., has progressed after at least three previous treatment lines). In some cases, the subject is quadruple refractory to previous treatment. In some cases, the subject is quintuple refractory to previous treatment.

[0041] In another aspect, the present invention provides a dosing regimen for use in a method of treating multiple myeloma in a subject in need thereof, the dosing regimen comprising administration of a bispecific antibody to the subject at a primary dose during the first week of the dosing regimen, a secondary dose during the second week of the dosing regimen, and a tertiary dose during the third week of the dosing regimen, wherein the tertiary dose is equal to or greater than the secondary dose, the secondary dose is equal to or greater than the primary dose, and the bispecific antibody comprises: (a) a pair of a first heavy chain and a common light chain comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA), and a pair of a second heavy chain and a common light chain comprising a second antigen-binding domain that specifically binds to human CD3, wherein the first antigen-binding domain comprises three heavy chain complementarity-determining regions (CDRs) and three light chain CDRs, each comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, 84, 86, and 88, and the second antigen-binding domain comprises three heavy chain CDRs and three light chain CDRs, each comprising the amino acid sequences of SEQ ID NOs: 92, 94, 96, 84, 86, and 88, or (b) a pair of a first heavy chain and a common light chain comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA), and a pair of a second heavy chain and a common light chain comprising a second antigen-binding domain that specifically binds to human CD3, wherein the first antigen-binding domain comprises three heavy chain CDRs and three light chain CDRs, each comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, 84, 86, and 88, and the second antigen-binding domain comprises three heavy chain CDRs and three light chain CDRs, each comprising the amino acid sequences of SEQ ID NOs: 100, 102, 104, 84, 86, and 88.

[0042] In some embodiments of the dosing regimen, the bispecific antibody comprises a pair of a first heavy chain and a common light chain comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA), and a pair of a second heavy chain and a common light chain comprising a second antigen-binding domain that specifically binds to human CD3, wherein the first antigen-binding domain comprises three heavy chain complementarity-determining regions (CDRs) and three light chain CDRs, each comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, 84, 86, and 88, and the second antigen-binding domain comprises three heavy chain CDRs and three light chain CDRs, each comprising the amino acid sequences of SEQ ID NOs: 92, 94, 96, 84, 86, and 88. Optionally, the first heavy chain comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 66, the second heavy chain comprises a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 90, and the common light chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82. Optionally, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 126, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 127, and the common light chain comprises the amino acid sequence of SEQ ID NO: 129.

[0043] In some embodiments of the dosing regimen, the bispecific antibody comprises a pair of a first heavy chain and a common light chain comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA), and a pair of a second heavy chain and a common light chain comprising a second antigen-binding domain that specifically binds to human CD3, wherein the first antigen-binding domain comprises three heavy chain complementarity determining regions (CDRs) and three light chain CDRs, each comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, 84, 86, and 88, and the second antigen-binding domain comprises three heavy chain CDRs and three light chain CDRs, each comprising the amino acid sequences of SEQ ID NOs: 100, 102, 104, 84, 86, and 88. Optionally, the first heavy chain comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 66, the second heavy chain comprises a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 98, and the common light chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82. Optionally, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 126, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 128, and the common light chain comprises the amino acid sequence of SEQ ID NO: 129.

[0044] In any of the various embodiments of the dosing regimen, the primary dose is 1 mg to 5 mg. In any of the various embodiments of the dosing regimen, the secondary dose is 3 mg to 400 mg. In any of the various embodiments of the dosing regimen, the tertiary dose is 3 mg to 800 mg. In some embodiments, the primary dose is 5 mg, the secondary dose is 25 mg, and the tertiary dose is 50 mg to 800 mg. In some cases, the dosing regimen includes administration of the tertiary dose once a week for at least 12 weeks (e.g., 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or more) during a once-a-week dosing period of the dosing regimen. In some cases, the dosing regimen further includes administration of the tertiary dose once every two weeks during an every-other-week dosing period of the dosing regimen following a once-a-week period of the dosing regimen. In some cases, the dosing regimen further includes administration of the tertiary dose once every three weeks or once every four weeks. In various embodiments, the dose is 1 mg at least once a week or every two weeks, 1.5 mg at least once a week or every two weeks, 2.0 mg at least once a week or every two weeks, 2.5 mg at least once a week or every two weeks, 3.0 mg at least once a week or every two weeks, 3.5 mg at least once a week or every two weeks, 4 mg at least once a week or every two weeks, 5 mg at least once a week or every two weeks, 6 mg at least once a week or every two weeks, 7 mg at least once a week or every two weeks, 8 mg at least once a week or every two weeks, 9 mg at least once a week or every two weeks, 10 mg at least once a week or every two weeks, 15 mg at least once a week or every two weeks, 20 mg at least once a week or every two weeks, 25 mg at least once a week or every two weeks, 30 mg at least once a week or every two weeks, 35 mg at least once a week or every two weeks, 40 mg at least once a week or every two weeks, 45 mg at least once a week or every two weeks, 50 mg at least once a week or every two weeks, 55 mg at least once a week or every two weeks, 60 mg at least once a week or every two weeks,65 mg at least once or twice a week, 70 mg at least once or twice a week, 75 mg at least once or twice a week, 80 mg at least once or twice a week, 85 mg at least once or twice a week, 90 mg at least once or twice a week, 95 mg at least once or twice a week, 100 mg at least once or twice a week, 150 mg at least once or twice a week, 200 mg at least once or twice a week, 250 mg at least once or twice a week, 300 mg at least once or twice a week, 350 mg at least once or twice a week, 400 mg at least once or twice a week, 450 mg at least once or twice a week, 500 mg at least once or twice a week, 550 mg at least once or twice a week, 600 mg at least once or twice a week, 650 mg at least once or twice a week, 700 mg at least once or twice a week, 750 mg at least once or twice a week, 800 mg at least once or twice a week, 850 mg at least once or twice a week, or 900 mg at least once or twice a week.

[0045] In any of the various embodiments of the dosing regimen, the subject has been previously treated with an anti-CD38 antibody therapy, a proteasome inhibitor, or an immunomodulatory agent. Optionally, the anti-CD38 antibody is daratumumab or isatuximab. Optionally, the proteasome inhibitor is bortezomib, carfilzomib, or ixazomib. Optionally, the immunomodulatory agent is lenalidomide or pomalidomide.

[0046] In any of the various embodiments of the dosing regimen, the multiple myeloma is relapsed or refractory multiple myeloma.

[0047] In any of the various embodiments of the dosing regimen, the subject is at least triple refractory to previous treatments. In some cases, the subject is quadruple or quintuple refractory to previous treatments.

[0048] In various embodiments, any of the features or components of the above or the embodiments discussed herein can be combined, and such combinations are included within the scope of the present disclosure. Any of the specific values discussed above or herein can be combined with another related value discussed above or herein to enumerate ranges that represent the upper and lower limits of the ranges, and such ranges are included within the scope of the present disclosure.

[0049] Other embodiments will become apparent from a review of the forms for carrying out the invention described hereinafter.

Brief Description of the Drawings

[0050]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0051] Before the present invention is described, it should be understood that because the specific methods and experimental conditions described may vary, the present invention is not limited to such methods and conditions. It should also be understood that the terms used herein are for the purpose of describing only particular embodiments and are not intended to be limiting since the scope of the present invention is limited only by the appended claims.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a particular recited numerical value means that the value can vary from the recited value by up to 1% therefrom. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0053] Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials will now be described. All patents, applications, and non-patent publications mentioned herein are incorporated herein by reference in their entirety.

[0054] Definitions As used herein, the expression "CD3" refers to an antigen that is expressed on T cells as part of the multi-molecular T cell receptor (TCR) and consists of a homodimer or heterodimer formed from the association of two of the four receptor chains, namely CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma. Human CD3-epsilon contains the amino acid sequence set forth in SEQ ID NO: 116, human CD3-delta contains the amino acid sequence set forth in SEQ ID NO: 117, human CD3-zeta contains the amino acid sequence set forth in SEQ ID NO: 118, and CD3-gamma contains the amino acid sequence set forth in SEQ ID NO: 119. All references to proteins, polypeptides, and protein fragments herein are intended to refer to the human form of each protein, polypeptide, or protein fragment unless specifically identified as being from a non-human species. Accordingly, the expression "CD3" means human CD3 unless specifically identified as being from a non-human species, e.g., "mouse CD3", "monkey CD3", etc.

[0055] As used herein, the term "antibody that binds to CD3" or "anti-CD3 antibody" includes antibodies that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta) and antigen-binding fragments thereof, as well as antibodies that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers) and antigen-binding fragments thereof. The antibodies and antigen-binding fragments of the invention are capable of binding to soluble CD3 and / or cell surface-expressed CD3. Soluble CD3 includes native CD3 protein, as well as recombinant CD3 protein variants that lack a transmembrane domain or are not associated with the cell membrane, such as monomeric and dimeric CD3 constructs.

[0056] As used herein, the expression "cell surface-expressed CD3" means one or more CD3 proteins expressed on the cell surface in vitro or in vivo, with at least a portion of the CD3 protein being exposed on the extracellular side of the cell membrane and accessible to the antigen-binding portion of an antibody. Examples of "cell surface-expressed CD3" include CD3 proteins contained within a functional T cell receptor in the cell membrane. The expression "cell surface-expressed CD3" includes CD3 proteins expressed as part of a homodimer or heterodimer on the surface of a cell (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The expression "cell surface-expressed CD3" also includes CD3 chains (e.g., CD3-epsilon, CD3-delta, or CD3-gamma) that are expressed on the surface of a cell by themselves without other CD3 chain types. Alternatively, "cell surface-expressed CD3" can include or consist of CD3 proteins expressed on the surface of a cell that normally expresses CD3 protein. Alternatively, "cell surface-expressed CD3" can include or consist of CD3 proteins expressed on the surface of a cell that is artificially engineered to express CD3 on its surface although it normally does not express human CD3 on its surface.

[0057] As used herein, the expression "BCMA" refers to B cell maturation antigen. BCMA (also known as TNFRSF17 and CD269) is a cell surface protein expressed on malignant plasma cells and plays a central role in the regulation of B cell maturation and differentiation into immunoglobulin-producing plasma cells. The amino acid sequence of human BCMA is shown in SEQ ID NO: 115 and can also be found in GenBank accession number NP_001183.2.

[0058] As used herein, "antibody that binds to BCMA" or "anti-BCMA antibody" includes antibodies that specifically recognize BCMA and antigen-binding fragments thereof.

[0059] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0060] As used in the present invention, the term "antibody" means any antigen-binding molecule or molecular complex that includes at least one complementarity-determining region (CDR) that specifically binds to or interacts with a specific antigen (e.g., BCMA or CD3). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains, as well as multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules consisting of four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains. Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or V H and a heavy chain constant region. The heavy chain constant region includes three domains, C H 1, C H 2, and C H 3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or V L and a light chain constant region. The light chain constant region includes one domain (C L 1). The V H region and the V LThe region can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) in which more conserved regions called framework regions (FRs) are interspersed. Each V H and V L consists of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. In different embodiments of the invention, the FRs of the anti-BCMA antibody or anti-CD3 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. Amino acid consensus sequences can be defined based on the parallel analysis of two or more CDRs.

[0061] As used herein, the term "antibody" also includes antigen-binding fragments of complete antibody molecules. The "antigen-binding portion" of an antibody, the "antigen-binding fragment" of an antibody, and similar terms, as used herein, include polypeptides or glycoproteins that specifically bind an antigen and form a complex, which are natural, enzymatically obtainable, synthetic, or genetically engineered. Antibody-binding fragments of an antibody can be derived from a complete antibody molecule using any suitable standard techniques, such as protein digestion techniques or recombinant genetic engineering techniques related to the manipulation and expression of DNA encoding the antibody variable domain and optionally the constant domain. Such DNA is known and / or can be readily obtained, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques to, for example, arrange one or more variable domains and / or constant domains in a suitable conformation, or to introduce codons, create cysteine residues, modify, add, or delete amino acids, and the like.

[0062] Non-limiting examples of antibody-binding fragments include: (i) Fab fragments, (ii) F(ab’)2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides), or minimal recognition units consisting of constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, mini-bodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0063] An antigen-binding fragment of an antibody typically includes at least one variable domain. The variable domain can be of any size or amino acid composition and generally includes at least one CDR that is adjacent to or in-frame with one or more framework sequences. V H domain is V L domain associated antigen-binding fragment, V H domain and V L domains may be positioned relative to each other in any suitable arrangement. For example, the variable regions can be dimers and include V H -V H 、V H -V L 、or V L -V L dimers. Alternatively, an antigen-binding fragment of an antibody can include a monomeric V H domain or V L domain.

[0064] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently attached to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within the antigen-binding fragments of the antibodies of the present invention include (i) V H -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2, (v) V H -C H 1-C H 2-C H 3, (vi) V H -C H 2-C H 3, (vii) V H -C L , (viii) V L -C H 1, (ix) V L -C H 2, (x) V L -C H 3, (xi) V L -C H 1-C H 2, (xii) V L -C H 1-C H 2-C H 3, (xiii) V L -C H 2-C H 3, and (xiv) V L -C Linclude. Any three-dimensional arrangement of the variable and constant domains, including any of the exemplary three-dimensional arrangements listed above, can be such that the variable and constant domains are either directly linked to each other or linked by a complete or partial hinge region or linker region. The hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a mobile or semi-mobile linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibodies of the present invention can include homodimers or heterodimers (or other multimers) of any of the variable domain three-dimensional arrangements and constant domain three-dimensional arrangements listed above in non-covalent linkages with each other and / or with one or more monomeric V H or V L domains (e.g., by disulfide bonds).

[0065] Similar to full antibody molecules, antibody-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically include at least two different variable domains, each of which can specifically bind to a distinct antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of the antibodies of the present invention using conventional techniques available in the art.

[0066] The antibodies of the present invention may function via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of the present invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcR) recognize the antibody bound on the target cell, thereby resulting in the lysis of the target cell. CDC and ADCC are well-known in the art and can be measured using available assays. (See, for example, U.S. Patent Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of the antibody is important for the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0067] In certain embodiments, the anti-BCMA monospecific antibody or anti-BCMA × anti-CD3 bispecific antibody of the present invention is a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by somatic mutations in vivo), for example, in the CDRs, particularly CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted into human framework sequences.

[0068] In some embodiments, the antibodies of the present invention can be recombinant human antibodies. As used herein, the term "recombinant human antibody" refers to any human antibody (described below) prepared, expressed, created, or isolated by recombinant means, such as an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant combinatorial human antibody library (described below), an antibody isolated from an animal that is transgenic for human immunoglobulin genes (e.g., a mouse) (e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or an antibody prepared, expressed, created, or isolated by any other means including splicing to other DNA sequences of the human immunoglobulin gene sequence. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when transgenic animals for human Ig sequences are used), and thus, the amino acid sequences of the V H and V L regions of the recombinant antibody are related to and derived from the human germline V H and V L sequences but are sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0069] Human antibodies can exist in two forms related to hinge heterogeneity. In the first form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by interchain heavy-chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds and a molecule of approximately 75-80 kDa consisting of covalently linked light and heavy chains is formed (half-antibody). These forms are extremely difficult to separate even after affinity purification.

[0070] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences related to the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form to levels typically observed using the human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention encompasses antibodies having one or more mutations in the hinge region, C H 2 region, or C H 3 region and may be desirable, for example, in improving the yield of the desired antibody form in production.

[0071] The antibodies of the present invention may be isolated antibodies. "Isolated antibody" as used herein means an antibody that has been identified, separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism or from a tissue or cell in which the antibody may naturally occur or be naturally produced is an "isolated antibody" for the purposes of the present invention. Isolated antibodies also include antibodies in situ within recombinant cells. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0072] The present invention also includes one-armed antibodies that bind to BCMA. As used herein, "one-armed antibody" means an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain. The one-armed antibodies of the present invention may comprise any of the HCVR / LCVR or CDR amino acid sequences set forth in Table 1.

[0073] The anti-BCMA or anti-BCMA × anti-CD3 antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework regions and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to, for example, germline sequences available from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate many antibodies and antibody-binding fragments containing one or more individual germline mutations or combinations thereof starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within the domain mutate back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., the mutated residues are found within the first 8 amino acids of FR1, or the mutated residues are found within the last 8 amino acids of FR4, or the mutated residues are found only within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues mutate to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Further, the antibodies of the invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues mutate to the corresponding residues of a particular germline sequence while certain other residues different from the original germline sequence are maintained or mutate to the corresponding residues of a different germline sequence. Once obtained, antibodies and antibody binding fragments containing one or more germline mutations can be readily tested for one or more desired properties such as improved binding specificity, increased binding affinity, improved or enhanced (where appropriate) biological properties of an antagonist or agonist, reduced immunogenicity, etc. Antibodies and antibody binding fragments obtained in this general manner are encompassed within the scope of the invention.

[0074] The present invention also includes anti-BCMA or anti-BCMA × anti-CD3 antibodies comprising variants of any of the HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention relates to HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences described in Tables 1 and 3 herein, or anti-CD3 antibodies disclosed in WO2014 / 047231 or WO2017 / 053856 (each of which is incorporated herein by reference).

[0075] The term "epitope" refers to an antigenic determinant that interacts with the specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have two or more epitopes. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are produced by amino acids that are spatially juxtaposed from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues within a polypeptide chain. In certain situations, an epitope can include a saccharide, phosphoryl group, or sulfonyl group moiety on an antigen.

[0076] The terms "substantially identical" or "substantially the same," when referring to a nucleic acid or fragment thereof, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, as discussed below, when measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or Gap, indicate that there is at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% nucleotide sequence identity of nucleotide bases. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0077] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity when optimally aligned by programs such as GAP or BESTFIT using a default gap weight. Preferably, the residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. If the conservative substitutions of two or more amino acid sequences differ from each other, the percent sequence identity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0078] Sequence similarity to a polypeptide, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measurements of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software can be used with default parameters for determining sequence homology or sequence identity between closely related polypeptides such as homologous polypeptides from organisms of different species or between a wild-type protein and its mutant protein, including programs such as Gap and Bestfit. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters of the GCG version 6.1 program. FASTA (e.g., FASTA2 and FASTA3) provides an alignment of the best overlapping regions between a query sequence and a search sequence and a percent sequence identity (Pearson (2000) (cited supra)). Another preferred algorithm for comparing the sequences of the present invention to a database containing numerous sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0079] Germline variation The anti-CD3 antibodies disclosed herein contain one or more amino acid substitutions, insertions, and / or deletions in the framework region and / or CDR regions of the heavy chain variable domain compared to the corresponding germline sequences.

[0080] The invention also includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"), and the detectable binding to the CD3 antigen is weak or absent.

[0081] Furthermore, the antibodies of the invention may include any combination of two or more germline mutations within the framework and / or CDR regions. For example, while a particular individual residue is mutated to the corresponding residue of a particular germline sequence, a particular other residue that is different from the original germline sequence is either maintained or mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antibody-binding fragments containing one or more germline mutations can be tested for one or more desired properties such as improved binding specificity, reduced or decreased binding affinity, improved or enhanced pharmacokinetic properties, and reduced immunogenicity. Antibodies and antigen-binding fragments obtained in this general manner, taking into account the guidance of the present disclosure, are encompassed within the scope of the invention.

[0082] The present invention also includes an antigen-binding molecule comprising an antigen-binding domain having an HCVR and / or CDR amino acid sequence that is substantially identical to any of the HCVR and / or CDR amino acid sequences disclosed herein, while maintaining or improving the desired weak affinity for the CD3 antigen. When referring to amino acid sequences, the terms "substantial identity" or "substantially identical" mean that two amino acid sequences share at least 95% sequence identity, more preferably at least 98%, or 99% sequence identity when optimally aligned by programs such as GAP or BESTFIT using a defined gap weight. Preferably, the residue positions that are not identical differ by conservative amino acid substitutions. When the conservative substitutions of two or more amino acid sequences differ from each other, the percent sequence identity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331.

[0083] Sequence similarity to a polypeptide, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measurements assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software can be used with default parameters for determining sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant protein, including programs such as Gap and Bestfit. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters of the GCG version 6.1 program. FASTA (e.g., FASTA2 and FASTA3) provides an alignment of the best overlapping regions and the percent sequence identity between a query sequence and a search sequence (Pearson (2000) (supra)). Another preferred algorithm when comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0084] Binding characteristics of the antibody As used herein, the term "binding" in the context of an antibody, immunoglobulin, antibody binding fragment, or Fc-containing protein binding to a given antigen, such as a cell surface protein or a fragment thereof, typically refers to an interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0085] For example, when the binding affinity is determined by surface plasmon resonance (SPR) technology in, for example, a BIAcore 3000 instrument using an antigen as a ligand and an antibody, Ig, antibody-binding fragment, or Fc-containing protein as an analyte (or anti-ligand), it is typically about 10 -7 M or less, for example about 10 -8 M or less, for example about 10 -9 M or less of K D value. Cell-based binding strategies such as fluorescence-activated cell sorting (FACS) binding assays are also routinely used, and FACS data correlates well with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31, Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0086] Thus, the antibody or antigen-binding protein of the present invention binds to a predetermined antigen or cell surface molecule (receptor) having an affinity corresponding to a K D value that is at least 10-fold lower than its affinity for binding to non-specific antigens (e.g., BSA, casein). According to the present invention, an antibody affinity corresponding to a K D value that is 10-fold or less lower than that for a non-specific antigen can be regarded as undetectable binding, but such an antibody can pair with a second antigen-binding arm for producing the bispecific antibody of the present invention.

[0087] The term "K D " (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment that binds to an antigen. There is an inverse relationship between K D and the binding affinity, and thus, the smaller the K D value, the higher the affinity, i.e., the stronger. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, i.e., a smaller K DConversely, with respect to values, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction, i.e., a larger K D value. Depending on the situation, a higher binding affinity (or K D ) of a particular molecule (e.g., an antibody) for an interaction partner molecule (e.g., antigen X), compared to the binding affinity of the molecule (e.g., the antibody) for another interaction partner molecule (e.g., antigen Y), is represented as a binding ratio determined by dividing the larger K D value (lower, or weaker, affinity) by the smaller K D (higher, or stronger, affinity), and is represented, for example, as 5-fold or 10-fold higher binding affinity in some cases.

[0088] The term "k d " (seconds-1 or 1 / second) refers to the dissociation rate constant of a specific antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. Its value is also called the k off value.

[0089] The term "k a " (M-1 × seconds-1 or 1 / M) refers to the association rate constant of a specific antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.

[0090] The term "K A " (M-1 or 1 / M) refers to the association equilibrium constant of a specific antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is obtained by dividing k a by k d .

[0091] The term "EC50" or "EC 50 " refers to the half maximal effective concentration and includes the concentration of an antibody that induces a response midway between the baseline and the maximum value after a specific exposure time. EC 50Essentially represents the concentration of the antibody at which 50% of its maximum effect is observed. In certain embodiments, the EC 50 value is equal to the concentration of the antibody of the present invention that gives half-maximal binding to cells expressing CD3 or a tumor-associated antigen (e.g., BCMA) when determined, for example, by a FACS binding assay. Thus, reduced or weak binding is observed at an increase in EC 50 or at the half-maximal effective concentration.

[0092] In one embodiment, the decrease in binding can be defined as an increase in the EC 50 antibody concentration that enables half-maximal binding to target cells.

[0093] In another embodiment, the EC 50 value represents the concentration of the antibody of the present invention that induces half-maximal depletion of target cells by the cytotoxic activity of T cells. Thus, an increase in cytotoxic activity (e.g., T cell-mediated tumor cell killing) is observed at a decrease in EC 50 or at the half-maximal effective concentration value.

[0094] Bispecific antigen-binding molecules The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies can be specific for different epitopes of one target polypeptide or can contain antigen-binding domains specific for two or more target polypeptides. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69, Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-BCMA monospecific antibody or anti-BCMA × anti-CD3 bispecific antibody of the present invention can be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, the antibody or a fragment thereof can be functionally linked (e.g., by chemical bonding, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second or additional binding specificity.

[0095] The use of the expressions "anti-CD3 antibody" or "anti-BCMA antibody" in this specification is intended to include both monospecific anti-CD3 antibodies or anti-BCMA antibodies, as well as bispecific antibodies containing a CD3-binding arm and a BCMA-binding arm. Thus, the present invention includes bispecific antibodies in which one arm of the immunoglobulin binds to human CD3 and the other arm of the immunoglobulin is specific for human BCMA. The CD3-binding arm can include any of the HCVR / LCVR or CDR amino acid sequences described in Table 3 of this specification, or anti-CD3 antibodies disclosed in WO2014 / 047231 or WO2017 / 053856.

[0096] In certain embodiments, the CD3-binding arm binds to human CD3 and induces human T cell activation. In certain embodiments, the CD3-binding arm binds weakly to human CD3 and induces human T cell activation. In other embodiments, the CD3-binding arm binds weakly to human CD3 and induces tumor-associated antigen-expressing cell death in the context of a bispecific or multispecific antibody. In other embodiments, the CD3-binding arm binds weakly or associates with human and cynomolgus monkey (monkey) CD3, but the binding interaction is still undetectable by in vitro assays known in the art. The BCMA-binding arm can include any of the HCVR / LCVR or CDR amino acid sequences described in Table 1 of this specification.

[0097] According to certain exemplary embodiments, the present invention includes a bispecific antigen-binding molecule that specifically binds to CD3 and BCMA. Such molecules can be referred to herein, for example, as "anti-BCMA×anti-CD3" or "anti-CD3 / anti-BCMA", or "anti-CD3×BCMA" or "CD3×BCMA" bispecific molecules, or other similar terms (e.g., anti-BCMA / anti-CD3).

[0098] As used herein, the term "BCMA" refers to the human BCMA protein, unless otherwise specified as being derived from a non-human species (e.g., "mouse BCMA", "monkey BCMA", etc.). The human BCMA protein has the amino acid sequence set forth in SEQ ID NO: 115.

[0099] The foregoing bispecific antigen-binding molecule that specifically binds to CD3 and BCMA has a weak binding affinity for CD3, as measured in an in vitro affinity binding assay, with a K greater than about 40 nM. D It can include an anti-CD3 antigen-binding molecule that binds to CD3 having a weak binding affinity as shown.

[0100] As used herein, the expression "antigen-binding molecule" means a protein, polypeptide, or molecular complex that alone or in combination with one or more additional complementarity-determining regions (CDRs) and / or framework regions (FRs) that specifically bind to a particular antigen includes or consists of at least one complementarity-determining region (CDR). In certain embodiments, the antigen-binding molecule is an antibody or a fragment of an antibody as those terms are defined elsewhere herein.

[0101] As used herein, the expression "bispecific antigen-binding molecule" means a protein, polypeptide, or molecular complex that includes at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within the bispecific antigen-binding molecule includes at least one CDR that specifically binds to a particular antigen, alone or in combination with one or more additional CDRs and / or FRs. In the context of the present invention, the first antigen-binding domain specifically binds to a first antigen (e.g., BCMA) and the second antigen-binding domain specifically binds to a second different antigen (e.g., CD3).

[0102] In certain exemplary embodiments of the present invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of the bispecific antibody comprises a heavy-chain variable domain (HCVR) and a light-chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising first and second antigen-binding domains, the CDRs of the first antigen-binding domain are designated with the prefix "D1", and the CDRs of the second antigen-binding domain may be designated with the prefix "D2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as D1-HCDR1, D1-HCDR2, and D1-HCDR3, and the CDRs of the second antigen-binding domain may be referred to herein as D2-HCDR1, D2-HCDR2, and D2-HCDR3.

[0103] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises a first antigen-binding domain comprising: (a) three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 66, and (b) three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 82. Optionally, the isolated bispecific antigen-binding molecule comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 68, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 70, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 72. Optionally, the isolated bispecific antigen-binding molecule comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 84, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 88. Optionally, the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 66 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82.

[0104] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises a second antigen-binding domain comprising: (a) three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 98; and (b) three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 82. Optionally, the second antigen-binding domain comprises: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 100; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 102; and (c) HCDR3 comprising the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 104. Optionally, the second antigen-binding domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 84, LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 88. Optionally, the second antigen-binding domain comprises: (a) HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 92, 94, and 96, respectively, and LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively; or (b) HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 100, 102, and 104, respectively, and LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively. Optionally, the second antigen-binding domain comprises: (a) an HCVR comprising the amino acid sequence of SEQ ID NO: 90 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82; or (b) an HCVR comprising the amino acid sequence of SEQ ID NO: 98 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82.

[0105] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 68, 70, and 72, respectively, and LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively, and (b) a second antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 92, 94, and 96, respectively, and LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively. Optionally, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 66 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82, and (b) a second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 90 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82.

[0106] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 68, 70, and 72, respectively, and LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively, and (b) a second antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 100, 102, and 104, respectively, and LCDR1, LCDR2, and LCDR3 domains each comprising the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively. Optionally, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 66 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82, and (b) a second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 98 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82.

[0107] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain that specifically binds to human BCMA and comprises CDRs of an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 122, and 124, and CDRs of an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 82, 123, and 125, and (b) a second antigen-binding domain that specifically binds to human CD3. Optionally, the first antigen-binding domain comprises CDRs from an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 122 / 123, 124 / 125, 2 / 82, 18 / 82, 34 / 82, 50 / 82, 66 / 82, 122 / 82, and 124 / 82. Optionally, the first antigen-binding domain comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-76-78-80, 4-6-8-84-86-88, 20-22-24-84-86-88, 36-38-40-84-86-88, 52-54-56-84-86-88, and 68-70-72-84-86-88. Optionally, the first antigen-binding domain comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 122 / 123, 124 / 125, 2 / 82, 18 / 82, 34 / 82, 50 / 82, 66 / 82, 122 / 82, and 124 / 82. Optionally, the second antigen-binding domain comprises CDRs from an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 90 / 82 and 98 / 82.

[0108] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule competes for binding to BCMA or binds to the same epitope on BCMA as a reference antibody, the reference antibody comprising a first antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 66 / 82, and a second antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of either SEQ ID NO: 90 / 82 or SEQ ID NO: 98 / 82.

[0109] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule competes for binding to human CD3 or binds to the same epitope on human CD3 as a reference antibody, the reference antibody comprising a first antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 66 / 82, and a second antigen-binding domain comprising an HCVR / LCVR pair comprising the amino acid sequence of either SEQ ID NO: 90 / 82 or SEQ ID NO: 98 / 82.

[0110] The bispecific antigen-binding molecules described above or discussed herein can be bispecific antibodies. Optionally, the bispecific antibody comprises a human IgG heavy chain constant region. Optionally, the human IgG heavy chain constant region is isotype IgG1. Optionally, the human IgG heavy chain constant region is isotype IgG4. In various embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding as compared to the wild-type hinge of the same isotype.

[0111] The first antigen-binding domain and the second antigen-binding domain may bind directly or indirectly to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be linked to a separate multimerization domain. The association of one multimerization domain with another multimerization domain promotes the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerization domain" is any polymer, protein, polypeptide, peptide, or amino acid having the ability to associate with a second multimerization domain of the same or similar structure or composition. For example, the multimerization domain may be a polypeptide containing an immunoglobulin C H polypeptide containing 3 domains. Non-limiting examples of multimerization components include the Fc portion of an immunoglobulin (C H 2-C H including 3 domains), for example, the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4, and any allotype within each isotype group.

[0112] The bispecific antigen-binding molecule of the present invention typically includes two multimerization domains, for example, two Fc domains that are each part of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains may be of different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0113] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence 1 to about 200 amino acids in length that includes at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides that contain or consist of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0114] Using any bispecific antibody format or technology, the bispecific antigen-binding molecules of the present invention can be produced. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical bonding, genetic fusion, non-covalent bonding, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to generate a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, for example, scFv-based formats or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knob-into-hole, common light chain (e.g., common light chain with knob-into-hole, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, duo-body, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2 Bispecific formats are included but not limited to these (for reviews of the above formats, see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

[0115] In the context of the bispecific antigen-binding molecules of the present invention, multimerization domains, such as the Fc domain, may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to the wild-type, naturally occurring Fc domain. For example, the present invention includes bispecific antigen-binding molecules that include one or more modifications in the Fc domain that result in a modified Fc domain having a modified binding interaction (e.g., enhanced or decreased) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule is C H 2 or C HIt contains modifications in three regions, and these modifications increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., within endosomes with a pH range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or position 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F) and position 434. In one embodiment, the modifications include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), modifications of 307 and / or 308 (e.g., 308F or 308P).

[0116] The present invention also includes a bispecific antigen-binding molecule comprising a first Ig C H 3 domain and a second Ig C H 3 domain, wherein the first and second Ig C H 3 domains have at least one amino acid different from each other, and due to at least one amino acid difference, the binding of the bispecific antibody to protein A is reduced compared to a bispecific antibody lacking that amino acid difference. In one embodiment, the first Ig C H 3 domain binds to protein A, and the second Ig C H 3 domain contains a mutation that reduces or abolishes protein A binding, such as the H95R modification (according to IMGT exon numbering, H435R according to EU numbering). The second C H3 may further include a Y96F modification (by IMGT, Y436F by EU). See, for example, U.S. Patent No. 8,586,713. A second C H Additional modifications that may be found within C3 include, for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT, D356E, L358M, N384S, K392N, V397M, and V422I by EU), for IgG2 antibodies, N44S, K52N, and V82I (by IMGT, N384S, K392N, and V422I by EU), and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

[0117] In certain embodiments, the Fc domain may be a chimeric that combines Fc sequences from two or more immunoglobulin isotypes. For example, the chimeric Fc domain may be from human IgG1, human IgG2, or human IgG4 C H 2 region-derived C H 2 sequences, in part or in whole, and C H 3 sequences, in part or in whole. The chimeric Fc domain may also include a chimeric hinge region. For example, the chimeric hinge may include an "upper hinge" sequence derived from the human IgG1 hinge region, the human IgG2 hinge region, or the human IgG4 hinge region combined with a "lower hinge" sequence derived from the human IgG1 hinge region, the human IgG2 hinge region, or the human IgG4 hinge region. Specific examples of chimeric Fc domains that may be included in any of the antigen-binding molecules described herein include, from N-terminus to C-terminus, [IgG4 C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein includes, from N-terminus to C-terminus, [IgG1 C HIt comprises [upper hinge of IgG1]-[lower hinge of IgG2]-[CH2 of IgG4]-[CH3 of IgG1]. These and other examples of chimeric Fc domains that can be included in any of the antigen-binding molecules of the present invention are described in US Publication No. 2014 / 0243504, published on August 28, 2014, which is hereby incorporated by reference in its entirety. Chimeric Fc domains having these general structural arrangements and their variants can have altered Fc receptor binding, which in turn affects Fc effector functions.

[0118] Sequence variant The antibodies and bispecific antigen-binding molecules herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains are derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein, for example, to germline sequences available from public antibody sequence databases. The antigen-binding molecules of the present invention may comprise antigen-binding domains derived from any of the exemplary amino acid sequences disclosed herein, and one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate many antibodies and antibody-binding fragments that contain one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V LAll framework and / or CDR residues within the domain mutate back to the residues found in the original germline sequence from which the antigen-binding domain originally derived. In other embodiments, only certain residues mutate back to the original germline sequence, e.g., only the mutated residues are found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues are found within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues mutate to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antigen-binding domain originally derived). Further, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues mutate to the corresponding residues of a particular germline sequence while certain other residues different from the original germline sequence are maintained or mutate to the corresponding residues of a different germline sequence. Once obtained, an antigen-binding domain containing one or more germline mutations can be readily tested for one or more desired properties such as improved binding specificity, increased binding affinity, improved or enhanced (where appropriate) biological properties of an antagonist or agonist, reduced immunogenicity, etc. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are encompassed within the scope of the present invention.

[0119] pH-dependent binding The present invention includes anti-BCMA antibodies and anti-BCMA×anti-CD3 bispecific antigen-binding molecules having pH-dependent binding characteristics. For example, the anti-BCMA antibodies of the present invention may exhibit a decrease in binding to BCMA at acidic pH as compared to neutral pH. Alternatively, the anti-BCMA antibodies of the present invention may exhibit enhanced binding to BCMA at acidic pH as compared to neutral pH. The expression "acidic pH" includes pH values of less than about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. As used herein, the expression "neutral pH" means a pH of from about 7.0 to about 7.4. The expression "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0120] In some cases, "decreased binding at acidic pH as compared to neutral pH" is represented with respect to the ratio of the K D value of the antibody that binds to the antigen at acidic pH to the K D value of the antibody that binds to the antigen at neutral pH. (Or vice versa). For example, when an antibody or its antigen-binding fragment exhibits an acidic / neutral K D ratio of about 3.0 or more, for the purposes of the present invention, the antibody or its antigen-binding fragment can be considered to exhibit "decreased binding to BCMA at acidic pH as compared to neutral pH". In certain exemplary embodiments, the acidic / neutral K D ratio of the antibody or antigen-binding fragment of the present invention is about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

[0121] Antibodies having pH-dependent binding characteristics can be obtained, for example, by screening a population of antibodies for a reduction (or enhancement) in binding to a particular antigen at acidic pH compared to neutral pH. Further, modification of the antigen-binding domain at the amino acid level can produce antibodies having pH-dependent characteristics. For example, by substituting one or more amino acids in the antigen-binding domain (e.g., within a CDR) with histidine residues, an antibody can be obtained in which antigen binding is reduced at acidic pH relative to neutral pH.

[0122] Antibodies comprising Fc variants According to certain embodiments of the invention, there are provided, for example, anti-BCMA antibodies and anti-BCMA × anti-CD3 bispecific antigen-binding molecules comprising an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the invention provides for C H 2 or C HAn antibody comprising a mutation in the 3 domain, the mutation increasing the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), at positions 250 and 428 (e.g., L or F), at position 252 (e.g., L / Y / F / W or T), at position 254 (e.g., S or T), and at position 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or at position 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and at position 434. In one embodiment, the modifications include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), modifications of 307 and / or 308 (e.g., 308F or 308P). All positions are indicated by EU numbering.

[0123] For example, the present invention includes an anti-BCMA antibody and an anti-BCMA × anti-CD3 bispecific antigen-binding molecule comprising an Fc domain comprising one or more mutation pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T and 256E (e.g., M252Y, S254T and T256E), 428L and 434S (e.g., M428L and N434S), and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are contemplated within the scope of the present invention.

[0124] Biological properties of antibodies and bispecific antigen-binding molecules The present invention includes antibodies that bind to human BCMA with high affinity (e.g., a K D value) below nanomolar) and antigen-binding fragments thereof.

[0125] According to certain embodiments, the present invention has a K D of less than about 5 nM when measured using surface plasmon resonance, e.g., the assay format defined in Example 4 herein, for human BCMA (e.g., at 25°C). Antibodies that bind to BCMA having a K D of less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, or less than about 25 pM, as measured using surface plasmon resonance, e.g., the assay format defined in Example 4 herein, or in a substantially similar assay. The present invention also includes bispecific antigen-binding molecules (e.g., bispecific antibodies that bind to human BCMA having a K D of less than about 25 pM and bind to cynomolgus BCMA having a K D of less than about 170 pM).

[0126] The present invention also includes antibodies and antigen-binding fragments thereof that bind to BCMA and have a dissociation half-life (t1 / 2) of greater than about 10 minutes or greater than about 125 minutes when measured using surface plasmon resonance at 25°C, for example, using the assay format defined in Example 4 herein or in a substantially similar assay. In certain embodiments, when measured using surface plasmon resonance at 25°C, for example, using the assay format defined in Example 4 herein or in a substantially similar assay, the antibodies or antigen-binding fragments of the present invention bind to BCMA and have a t1 / 2 of greater than about 3 minutes, greater than about 4 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, or greater than about 120 minutes. The present invention also includes bispecific antigen-binding molecules (e.g., bispecific antibodies that bind to BCMA with a t1 / 2 of greater than about 10 minutes when measured using surface plasmon resonance at 25°C, for example, using the assay format defined in Example 4 herein or in a substantially similar assay).

[0127] The present invention also includes antibodies and antigen-binding fragments thereof that specifically bind to human cell lines expressing endogenous BCMA (e.g., NCI-H929, MOLP-8, or OMP-2) as determined by the FACS binding assay described in Example 6 or a substantially similar assay.

[0128] The present invention also includes anti-BCMA×anti-CD3 bispecific antigen-binding molecules that exhibit one or more characteristics selected from the group consisting of: (a) inhibiting tumor growth in immunodeficient mice bearing human multiple myeloma xenografts; (b) suppressing the tumor growth of established tumors in immunodeficient mice bearing human multiple myeloma xenografts (see, e.g., Examples 10-15); and (c) suppressing the tumor growth of syngenic melanoma and colon cancer cells engineered to express human BCMA in immune-responsive mice expressing human CD3.

[0129] The present invention includes antibodies and antigen-binding fragments thereof that bind to human CD3 with high affinity. The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD3 with moderate or low affinity, depending on the treatment situation and the specific targeting properties desired. In some cases, low affinity refers to a K D or EC 50 (e.g., when measured by surface plasmon resonance assay) greater than 300 nM, greater than 500 nM, or greater than 1 μM for antibodies that bind to CD3. The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD3 with undetectable affinity. For example, in the context of a bispecific antigen-binding molecule where one arm binds to CD3 and the other arm binds to a target antigen (e.g., BCMA), it is desirable for the target antigen-binding arm to bind to the target antigen with high affinity, while the anti-CD3 arm binds to CD3 with moderate or low affinity or no affinity. In this manner, preferential targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved while avoiding general / non-targeted CD3 binding and the associated harmful side effects.

[0130] The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies) that can bind simultaneously to human CD3 and human BCMA. The binding arm that interacts with cells expressing CD3 can have weak to undetectable binding when measured in an appropriate in vitro binding assay. The extent to which the bispecific antigen-binding molecule binds to cells expressing CD3 and / or BCMA can be evaluated by fluorescence-activated cell sorting (FACS) as shown in Examples 5 and 6 herein.

[0131] For example, the present invention includes antibodies, antibody-binding fragments, and bispecific antibodies thereof that specifically bind to human cell lines expressing CD3 but do not express BCMA (e.g., Jurkat) and / or cells expressing BCMA.

[0132] The present invention includes antibodies that bind to human CD3 with weak (i.e., low) or even undetectable affinity, antibody binding fragments thereof, and bispecific antibodies thereof.

[0133] The present invention includes antibodies that bind to cynomolgus monkey CD3 with weak (i.e., low) or even undetectable affinity, antibody binding fragments thereof, and bispecific antibodies thereof.

[0134] The present invention includes antibodies that bind to human CD3 and induce T cell activation, antibody binding fragments thereof, and bispecific antibodies thereof.

[0135] The present invention includes an anti-BCMA×anti-CD3 bispecific antigen-binding molecule that can deplete or reduce tumor antigen-expressing cells in a subject (see, e.g., Examples 8-16, or assays substantially similar thereto). For example, according to certain embodiments, an anti-BCMA×anti-CD3 bispecific antigen-binding molecule is provided, and a single or multiple administrations of 0.04 mg / kg, 0.4 mg / kg, or 4 mg / kg of the bispecific antigen-binding molecule to a subject causes a decrease in the number of BCMA-expressing cells in the subject (e.g., tumor growth in the subject is suppressed or inhibited).

[0136] Epitope mapping and related techniques The epitopes on CD3 and / or BCMA to which the antigen-binding molecules of the present invention bind may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acids of the CD3 or BCMA protein. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of CD3 or BCMA. The antibodies of the present invention can interact with amino acids contained within a single CD3 chain (e.g., CD3-epsilon, CD3-delta, or CD3-gamma) or with amino acids on two or more different CD3 chains. As used in the present invention, the term "epitope" refers to an antigenic determinant that interacts with the specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have two or more epitopes. Thus, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are produced by amino acids that are spatially juxtaposed from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include a portion of a saccharide, phosphoryl group, or sulfonyl group on the antigen.

[0137] Using various techniques known to those skilled in the art, it is possible to determine whether the antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutagenesis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be employed (Tomer (2000) Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which the antigen-binding domain of an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves labeling the protein of interest with deuterium and then binding the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259, Engen and Smith (2001) Anal.Chem.73:256A-265A. X-ray crystallography of the antigen / antibody complex may also be used for epitope mapping purposes.

[0138] The present invention further includes an anti-BCMA antibody that binds to the same epitope as any of the specific exemplary antibodies described herein (e.g., an antibody comprising any of the amino acid sequences described in Table 1 herein). Similarly, the present invention also includes an anti-BCMA antibody that competes with any of the specific exemplary antibodies described herein for binding to BCMA (e.g., an antibody comprising any of the amino acid sequences described in Table 1 herein).

[0139] The present invention also includes a bispecific antigen-binding molecule comprising a second antigen-binding domain that specifically binds to human CD3 and / or cynomolgus CD3 with low or undetectable binding affinity, and a second antigen-binding domain that specifically binds to human BCMA, wherein the second antigen-binding domain binds to the same epitope on CD3 as any of the specific exemplary CD3-specific antigen-binding domains described herein, and / or the second antigen-binding domain binds to the same epitope on BCMA as any of the specific exemplary BCMA-specific antigen-binding domains described herein.

[0140] Similarly, the present invention also includes a bispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds to human BCMA and a second antigen-binding domain that specifically binds to human CD3, wherein the first antigen-binding domain competes with any of the specific exemplary BCMA-specific antigen-binding domains described herein for binding to BCMA, and / or the second antigen-binding domain competes with any of the specific exemplary CD3-specific antigen-binding domains described herein for binding to CD3.

[0141] Whether a particular antigen-binding molecule (e.g., an antibody) or its antigen-binding domain binds to the same epitope as the reference antigen-binding molecule of the present invention or competes with the reference antigen-binding molecule of the present invention for binding can be readily determined using conventional methods known in the art. For example, to determine whether a test antibody binds to the same epitope on BCMA (or CD3) as the reference bispecific antigen-binding molecule of the present invention, the reference bispecific molecule is first bound to the BCMA protein (or CD3 protein). Next, the ability of the test antibody to bind to the BCMA (or CD3) molecule is evaluated. If the test antibody can bind to BCMA (or CD3) after saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to an epitope of BCMA (or CD3) that is different from the reference bispecific antigen. On the other hand, if the test antibody cannot bind to the BCMA (or CD3) molecule after saturation binding with the reference bispecific antigen-binding molecule, the test antibody may bind to the same epitope of BCMA (or CD3) as the epitope bound by the reference bispecific antigen-binding molecule of the present invention. Next, to confirm whether the observed loss of binding of the test antibody is actually due to binding to the same epitope as the reference bispecific antigen-binding molecule or whether steric hindrance (or another phenomenon) is the cause of the observed loss of binding, additional routine experiments (e.g., peptide mutagenesis and binding analysis) can be performed. This type of experiment can be carried out using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody-binding assay available in the art. According to a particular embodiment of the present invention, for example, if one antigen-binding protein in a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess inhibits at least 50%, but preferably 75%, 90%, or even 99% of the binding of the other in a competitive binding assay, the two antigen-binding proteins bind to the same (or overlapping) epitope (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, if essentially all amino acid mutations in an antigen that reduce or eliminate the binding of one antigen-binding protein also reduce or eliminate the binding of the other, the two antigen-binding proteins are considered to bind to the same epitope. If only a subset of the amino acid mutations that reduce or eliminate the binding of one antigen-binding protein reduce or eliminate the binding of the other, the two antigen-binding proteins are considered to have "overlapping epitopes".

[0142] To determine whether an antibody or its antigen-binding domain competes with a reference antigen-binding molecule for binding, the above-described binding method is performed in two directions. In the first direction, after binding the reference antigen-binding molecule to the BCMA protein (or CD3 protein) under saturation conditions, the binding of the test antibody to the BCMA (or CD3) molecule is evaluated. In the second direction, after binding the test antibody to the BCMA (or CD3) molecule under saturation conditions, the binding of the reference antigen-binding molecule to the BCMA (or CD3) molecule is evaluated. In both directions, if only the first (saturated) antigen-binding molecule can bind to the BCMA (or CD3) molecule, it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to BCMA (or CD3). As will be recognized by those skilled in the art, antibodies that compete for binding with a reference antigen-binding molecule do not necessarily bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0143] Preparation of Antigen-Binding Domains and Construction of Bispecific Molecules Antigen-binding domains specific for a particular antigen can be prepared by any antibody production technique known in the art. Once obtained, two different antigen-binding domains specific for two different antigens (e.g., CD3 and BCMA) can be appropriately arranged relative to each other and the bispecific antigen-binding molecules of the invention can be produced using conventional methods. (Consideration of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of the invention is provided elsewhere herein). In certain embodiments, one or more individual components of the multispecific antigen-binding molecules of the invention (e.g., heavy and light chains) are derived from chimeric, humanized, or fully human antibodies. Methods for making such antibodies are well known in the art. For example, one or more heavy and / or light chains of the bispecific antigen-binding molecules of the invention can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generation technology), high-affinity chimeric antibodies against a particular antigen (e.g., CD3 or BCMA) having human variable regions and murine constant regions are first isolated. The antibodies are characterized and selected for desirable features including affinity, selectivity, epitope, etc. The murine constant regions are replaced with the desired human constant regions to generate fully humanized heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules of the invention.

[0144] The genetically engineered animals can be used to produce human bispecific antigen-binding molecules. For example, genetically modified mice that are unable to rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, where the mice express only one or two human light chain variable domains encoded by a human immunoglobulin sequence operably linked to the mouse kappa constant gene of the endogenous mouse kappa locus. Such genetically modified mice can be used to produce a fully human bispecific antigen-binding molecule comprising two different heavy chains that associate with the same light chain containing a variable domain derived from one of two different human light chain variable region gene segments. (See, e.g., US2011 / 0195454). By "fully human" is meant an antibody, or an antigen-binding fragment or immunoglobulin domain thereof, comprising an amino acid sequence encoded by DNA derived from a human sequence over the full length of each polypeptide of the antibody or antigen-binding fragment or immunoglobulin domain. In some instances, the fully human sequence is derived from a human endogenous protein. In other instances, the fully human protein or protein sequence comprises a chimeric sequence where each component sequence is derived from a human sequence. Without being bound by any theory, chimeric proteins or chimeric sequences are generally designed to minimize the creation of immunogenic epitopes at the junctions of the component sequences as compared to, for example, any wild-type human immunoglobulin region or domain.

[0145] Biological equivalents The present invention encompasses antigen-binding molecules having an amino acid sequence that, although different from those of the exemplary molecules disclosed herein, retains the ability to bind to CD3 and / or BCMA. Such variant antibodies contain one or more additions, deletions, or substitutions of amino acids as compared to the parental sequence, but exhibit a biological activity that is essentially equivalent to the biological activity of the bispecific antigen-binding molecules described.

[0146] The present invention includes antigen-binding molecules that are biologically equivalent to any of the exemplary antigen-binding molecules described herein. Two antigen-binding proteins or antibodies are considered to be biological equivalents, for example, if they are pharmaceutical equivalents or pharmaceutical alternatives that show no significant difference in absorption rate and extent of absorption when administered at the same molar dose, either as a single dose or multiple doses, under similar experimental conditions. When these extents of absorption are equivalent but the absorption rates are not, such differences in absorption rate are intentional and reflected in the labeling, and are considered not to be medically significant for the particular pharmaceutical product being tested, for example, because they are not essential for achieving an effective body drug concentration for long-term use, some antigen-binding proteins may be considered equivalents or pharmaceutical alternatives.

[0147] In one embodiment, two antigen-binding proteins are biologically equivalent if there are no clinically significant differences in their safety, purity, or efficacy.

[0148] In one embodiment, two antigen-binding proteins are biologically equivalent if a patient can be switched one or more times compared to a therapy that is sustained without switching between a reference product and a biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or a decrease in efficacy.

[0149] In one embodiment, two antigen-binding proteins are biologically equivalent if they both act by a common mechanism or mode of action with respect to the conditions or conditions of use, to the extent that such a mechanism is known.

[0150] Biological equivalence can be demonstrated by in vivo and in vitro methods. Biological equivalence measurements include, for example, (a) in vivo tests in humans or other mammals where the concentration of an antibody or its metabolite is measured as a function of time in blood, plasma, serum or other biological fluids, (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo tests in humans or other mammals where the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or biological equivalence of the antigen-binding protein.

[0151] Biologically equivalent variants of the exemplary bispecific antigen-binding molecules shown herein can be constructed, for example, by introducing various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unwanted or inaccurate intramolecular disulfide bridges during regeneration. In other contexts, biologically equivalent antigen-binding proteins can include variants of the exemplary bispecific antigen-binding molecules described herein that contain amino acid changes that modify the glycosylation properties of the molecule, such as mutations that eliminate or remove glycosylation.

[0152] Species selectivity and species cross-reactivity According to certain embodiments of the invention, antigen-binding molecules are provided that bind to human CD3 but not to CD3 from other species. Antigen-binding molecules that bind to human BCMA but not to BCMA from other species are also provided. The invention also includes antigen-binding molecules that bind to human CD3 and CD3 from one or more non-human species, and / or antigen-binding molecules that bind to human BCMA and BCMA from one or more non-human species.

[0153] According to certain exemplary embodiments of the present invention, provided are antigen-binding molecules that bind to human CD3 and / or human BCMA and, optionally, may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey or chimpanzee CD3 and / or BCMA. For example, in certain exemplary embodiments of the present invention, provided is a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds to human BCMA and cynomolgus monkey BCMA and a second antigen-binding domain that specifically binds to human CD3, or a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds to human BCMA and cynomolgus monkey BCMA and a second antigen-binding domain that specifically binds to human CD3.

[0154] Therapeutic Formulations and Administration The present invention provides a pharmaceutical composition comprising an antigen-binding molecule of the present invention. The pharmaceutical composition of the present invention is formulated with a suitable carrier, excipient, and other agents that provide improved movement, delivery, tolerance, etc. Many suitable formulations can be found in the formulary known to all pharmacists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) including vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA complexes, anhydrous absorbent pastes, oil-in-water emulsions and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0155] The dosage of the antigen-binding molecule administered to a patient can vary depending on the patient's age and size, target disease, condition, route of administration, etc. Preferred dosages are typically calculated according to body weight or body surface area. When the bispecific antigen-binding molecule of the present invention is used for the treatment of adult patients, the bispecific antigen-binding molecule of the present invention is usually administered by a single intravenous administration of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dosage and schedule for administering the bispecific antigen-binding molecule are determined empirically, for example, the patient's course can be monitored by regular evaluation and the dosage adjusted accordingly. Furthermore, interspecies scaling of the dosage can be carried out using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0156] Various delivery systems are known and can be used to administer the pharmaceutical composition of the present invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). The introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, such as by injection or bolus injection, by absorption through the inner lining of the epithelium or mucous membrane (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0157] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices facilitate the application in delivering the pharmaceutical composition of the present invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, this empty cartridge can be easily discarded and replaced easily with a new cartridge containing the pharmaceutical composition. Next, the pen-type delivery device can be reused. In disposable pen-type delivery devices, there are no replaceable cartridges. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. When the pharmaceutical composition runs out from the reservoir, the entire device is discarded.

[0158] Numerous reusable pen-type delivery devices and auto-injector delivery devices have use in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), and only a few of these are listed. Examples of disposable pen delivery devices having use in the subcutaneous delivery of the pharmaceutical compositions of the present invention include SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.) and HUMIRA™ pen (Abbott Labs, Abbott Park IL), but are not limited thereto.

[0159] In certain circumstances, the pharmaceutical composition can be delivered in a sustained release system. In one embodiment, a pump can be used (see Langer, supra, Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the sustained release system can be placed in the vicinity of the target of the composition, whereby only a fraction of the systemic dose is required (see, for example, Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other sustained release systems are discussed in a review by Langer, 1990, Science 249:1527-1533.

[0160] Injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, infusion, etc. These injectable preparations can be prepared by publicly known methods. For example, an injectable preparation may be prepared by dissolving, suspending or emulsifying the above-described antibody or its salt in a sterile aqueous medium or an oily medium conventionally used for injection. Examples of the injectable aqueous medium include physiological saline, glucose-containing isotonic solutions, and other adjuvants, which may be used in combination with appropriate solubilizing agents such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As the oily medium, for example, sesame oil, soybean oil, etc. are used, and they may be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol. The injection thus prepared is preferably filled into an appropriate ampoule.

[0161] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms of suitable unit dosages to conform to the dosages of the active ingredients. Such dosage forms of unit dosages include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. The amount of the antibody contained therein is generally about 5 to about 500 mg per dosage form of unit dosage, and particularly in the form of injection, the aforementioned antibody is preferably contained at about 5 to about 100 mg, and for other dosage forms, at about 10 to about 250 mg.

[0162] Therapeutic Use of Antigen-Binding Molecules The present invention includes a method comprising administering to a subject in need thereof a therapeutic composition comprising an anti-BCMA antibody or an antigen-binding fragment thereof, or a bispecific antigen-binding molecule that specifically binds to CD3 and BCMA. The therapeutic composition may comprise any of the antibodies or bispecific antigen-binding molecules disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression "a subject in need thereof" means a human or non-human animal (e.g., a subject expressing a tumor or a subject suffering from any of the cancers described hereinafter) showing one or more symptoms or signs of cancer, or alternatively, a person who would benefit from inhibition or reduction of BCMA activity or depletion of BCMA+ cells (e.g., multiple myeloma cells).

[0163] The antibodies and bispecific antigen-binding molecules of the present invention (and therapeutic compositions containing the same) are useful, inter alia, for the treatment of any disease or disorder in which stimulation, activation, and / or targeting of the immune response is beneficial. In particular, the anti-BCMA antibodies or anti-BCMA × anti-CD3 bispecific antigen-binding molecules of the present invention can be used for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by BCMA expression or activity or the proliferation of BCMA+ cells. The mechanism of action by which the therapeutic methods of the present invention are achieved involves the killing of cells expressing BCMA in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. Cells expressing BCMA that can be inhibited or killed using the bispecific antigen-binding molecules of the present invention include, for example, multiple myeloma cells.

[0164] The antigen-binding molecules of the present invention can be used to treat diseases or disorders associated with BCMA expression, including, for example, cancers or other B-cell or plasma cell cancers, such as Waldenström macroglobulinemia, Burkitt lymphoma, and diffuse large B-cell lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin lymphoma, including multiple myeloma. According to certain embodiments of the present invention, the anti-BCMA antibody or anti-BCMA × anti-CD3 bispecific antibody is useful for treating patients suffering from multiple myeloma. According to other related embodiments of the present invention, provided are methods comprising administering to a patient suffering from multiple myeloma an anti-BCMA antibody or an anti-BCMA × anti-CD3 bispecific antigen-binding molecule disclosed herein. Analytical / diagnostic methods known in the art, such as tumor scanning, can be used to determine whether a patient has multiple myeloma or another B-cell lineage cancer.

[0165] The present invention also includes a method for treating residual cancer in a subject. As used herein, the term "residual cancer" means the presence or persistence of one or more cancerous cells in a subject after treatment with anti-cancer therapy.

[0166] According to certain embodiments, the present invention provides a method for treating a disease or disorder associated with BCMA expression (e.g., multiple myeloma), comprising administering to a subject one or more of the anti-BCMA or bispecific antigen-binding molecules described elsewhere herein after the subject has been determined to have multiple myeloma. For example, the present invention includes a method for treating multiple myeloma, comprising administering an anti-BCMA antibody or an anti-BCMA x anti-CD3 bispecific antigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received other immunotherapy or chemotherapy.

[0167] Combination Therapies and Formulations The present invention provides a method comprising administering a pharmaceutical composition comprising any of the exemplary antibodies and bispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that can be combined with or administered in combination with the antigen-binding molecules of the present invention include, for example, anti-tumor agents (e.g., melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), oblimustine (Treanda), and any other agent known to be effective in the treatment of plasma cell tumors in a subject). In some embodiments, the second therapeutic agent comprises a steroid. In some embodiments, the second therapeutic agent comprises targeted therapies including thalidomide, lenalidomide, and bortezomib, which are therapies approved for treating newly diagnosed patients. Lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab, and daratumumab are examples of second therapeutic agents effective for treating relapsed multiple myeloma. In certain embodiments, the second therapeutic agent is a regimen comprising radiation therapy or stem cell transplantation. In certain embodiments, the second therapeutic agent can be an immunomodulatory agent. In certain embodiments, the second therapeutic agent can be a proteasome inhibitor including bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro). In certain embodiments, the second therapeutic agent can be a histone deacetylase inhibitor such as panobinostat (Farydak). In certain embodiments, the second therapeutic agent can be a monoclonal antibody, an antibody-drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a checkpoint inhibitor, or a combination thereof.Other agents that can be beneficially administered in combination with the antigen-binding molecules of the present invention include small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or cytokine inhibitors that include their respective receptors. The pharmaceutical compositions of the present invention (e.g., pharmaceutical compositions comprising an anti-BCMA × anti-CD3 bispecific antigen-binding molecule as disclosed herein) can also be administered as part of a treatment regimen comprising one or more therapeutic combinations selected from monoclonal antibodies other than those described herein that can interact with different antigens on the surface of plasma cells, bispecific antibodies having one arm that binds to an antigen on the surface of a tumor cell and another arm that binds to an antigen on a T cell, antibody-drug conjugates, bispecific antibodies conjugated to anti-tumor agents, checkpoint inhibitors, e.g., those that target PD-1 or CTLA-4, or combinations thereof. In certain embodiments, the checkpoint inhibitor can be selected from PD-1 inhibitors such as pembrolizumab (Keytruda), nivolumab (Opdivo), or semiprimumab (REGN2810). In certain embodiments, the checkpoint inhibitor can be selected from PD-L1 inhibitors such as atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi). In certain embodiments, the checkpoint inhibitor can be selected from CTLA-4 inhibitors such as ipilimumab (Yervoy). Other combinations that can be used in combination with the antibodies of the present invention are described above.

[0168] The present invention also includes a therapeutic combination comprising any of the antigen-binding molecules described herein, and one or more inhibitors of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementioned cytokines, wherein the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody or antibody fragment (e.g., Fab fragment, F(ab’)2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or diabody, triabody, tetrabody, minibody, and minimal recognition unit). The antigen-binding molecules of the present invention can also be administered and / or co-formulated in combination with antiviral agents, antibiotics, analgesics, corticosteroids and / or NSAIDs. The antigen-binding molecules of the present invention may also be administered as part of a treatment regimen that includes radiotherapy and / or conventional chemotherapy.

[0169] The additional therapeutic active ingredient may be administered immediately before, simultaneously with, or immediately after administration of the antigen-binding molecule of the present invention. (For the purposes of this disclosure, such an administration regimen is considered to administer the antigen-binding molecule "in combination with" the additional therapeutic active ingredient.)

[0170] The present invention includes a pharmaceutical composition in which the antigen-binding molecule of the present invention is co-formulated with one or more of the additional therapeutic active ingredients described elsewhere herein.

[0171] Administration regimen According to certain embodiments of the present invention, multiple doses of an antigen-binding molecule (e.g., an anti-BCMA antibody or a bispecific antigen-binding molecule that specifically binds to BCMA and CD3) can be administered to a subject over a predetermined period of time. The methods according to this aspect of the present invention include continuously administering multiple doses of the antigen-binding molecule of the present invention to a subject. As used herein, "administering continuously" means that each dose of the antigen-binding molecule is administered to the subject on different days separated by different time points, e.g., a predetermined interval (e.g., several hours, several days, several weeks or several months). The present invention includes methods that include continuously administering to a patient a single primary dose of an antigen-binding molecule, followed by one or more secondary doses of the antigen-binding molecule, and then optionally one or more tertiary doses of the antigen-binding molecule.

[0172] The terms "primary dose", "secondary dose", and "tertiary dose" refer to the time sequence of administration of the antigen-binding molecules of the present invention. Thus, the "primary dose" is the dose administered at the start of the treatment regimen (also referred to as the "baseline dose"), the "secondary dose" is the dose administered after the primary dose, and the "tertiary dose" is the dose administered after the secondary dose. The primary dose, secondary dose, and tertiary dose may all contain the same amount of antigen-binding molecule, but generally may differ from each other with respect to the frequency of administration. However, in certain embodiments, the amounts of antigen-binding molecule contained in the primary dose, secondary dose, and / or tertiary dose differ from each other (e.g., are adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered as a "loading dose" at the start of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered at a lower frequency. In any of the embodiments, the primary dose (e.g., a once-weekly dose) may be divided into two doses administered on separate days (e.g., consecutive days) at intervals within three days. In any of the embodiments, the first nominal dose (i.e., the secondary dose) may be divided into two doses administered on separate days (e.g., consecutive days) at intervals within three days. For example, if the primary dose or secondary dose is 6 mg, the dose may be divided into two 3 mg doses administered, for example, on consecutive days or on separate days at intervals within three days. In various embodiments, the dose (e.g., a once-weekly, single dose or a dose administered as two divided fractions of a dose) is 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg,61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg, 445 mg, 450 mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg, 490 mg, 495 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1.5 g, 2 g, 2.5 g, 3 g3.5 g, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, 10 g, or more, or at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg, 445 mg, 450 mg, 455 mg,460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg, 490 mg, 495 mg, 500 mg, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg, 640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg, 750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg, 860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg, 970 mg, 980 mg, 990 mg, 1000 mg, 1.5 g, 2 g, 2.5 g, 3 g, 3.5 g, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, 10 g, or more. Any of these amounts can be used to define the primary, secondary, or tertiary dosage ranges discussed herein, which are included within the scope of the present disclosure. In some embodiments, all dosages are administered as a single dose (e.g., a single injection) including the dosages administered during the first and second weeks of the dosing regimen. For example, a primary dosage of 1 mg to 5 mg can be administered as a single dose in the first week, a secondary dosage of 3 mg to 400 mg can be administered as a single dose in the second week, a tertiary dosage of 50 mg to 800 mg can be administered as a single dose in the third week, and thereafter can be administered during the once-weekly dosing portion of the dosing regimen. In another example, a primary dosage of 5 mg can be administered as a single dose in the first week, a secondary dosage of 25 mg can be administered as a single dose in the second week, a tertiary dosage of 50 mg to 800 mg can be administered as a single dose in the third week, and thereafter can be administered during the once-weekly dosing portion of the dosing regimen. In some cases, the dosing schedule can include administrations every two weeks, every three weeks, once a month, etc. thereafter (e.g., 12 to 16 weeks later).

[0173] In certain exemplary embodiments of the present invention, each secondary dose and / or tertiary dose is administered 1 to 26 (e.g., 1, 1 and 1 / 2, 2, 2 and 1 / 2, 3, 3 and 1 / 2, 4, 4 and 1 / 2, 5, 5 and 1 / 2, 6, 6 and 1 / 2, 7, 7 and 1 / 2, 8, 8 and 1 / 2, 9, 9 and 1 / 2, 10, 10 and 1 / 2, 11, 11 and 1 / 2, 12, 12 and 1 / 2, 13, 13 and 1 / 2, 14, 14 and 1 / 2, 15, 15 and 1 / 2, 16, 16 and 1 / 2, 17, 17 and 1 / 2, 18, 18 and 1 / 2, 19, 19 and 1 / 2, 20, 20 and 1 / 2, 21, 21 and 1 / 2, 22, 22 and 1 / 2, 23, 23 and 1 / 2, 24, 24 and 1 / 2, 25, 25 and 1 / 2, 26, 26 and 1 / 2, or more) weeks after the immediately preceding dose. As used herein, the phrase "immediately preceding dose" means, in a series of multiple administrations, the dose of the antigen-binding molecule that is administered to the patient prior to the administration of the immediately succeeding dose in the absence of an intervening dose.

[0174] The method according to this aspect of the invention may comprise administering to the patient any number of secondary doses and / or tertiary doses of an antigen-binding molecule (e.g., an anti-BCMA antibody or a bispecific antigen-binding molecule that specifically binds to BCMA and CD3). For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

[0175] In embodiments involving multiple secondary doses, each secondary dose can be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, the frequency at which the secondary dose and / or tertiary dose is administered to the patient can vary over the course of the treatment regimen. The dosing frequency can also be adjusted by the physician during the course of treatment according to the needs of the individual patient after clinical examination.

[0176] Diagnostic uses of the antibody The anti-BCMA antibodies of the present invention may also be used, for example, for diagnostic purposes, to detect and / or measure BCMA, or BCMA-expressing cells, in a sample. For example, an anti-BCMA antibody or a fragment thereof may be used to diagnose a condition or disease characterized by abnormal expression of BCMA (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for BCMA may, for example, include contacting a sample obtained from a patient with an anti-BCMA antibody of the present invention, wherein the anti-BCMA antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-BCMA antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. Detectable labels or reporter molecules can be 3 H, 14 C, 32 P, 35 S or 125 radioisotopes such as I, fluorescent moieties or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine, or enzymes such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Another exemplary diagnostic use of the anti-BCMA antibodies of the present invention is 89 for non-invasive identification and tracking of tumor cells in a subject, such as Zr-desferrioxamine labeling 89Includes Zr-labeled antibodies (e.g., positron emission tomography (PET) imaging). (See, for example, Tavare, R. et al. Cancer Res. 2016 Jan 1;76(1):73-82, and Azad, B. B. et al. Oncotarget. 2016 Mar 15;7(11):12344-58.) Specific exemplary assays that can be used to detect or measure BCMA in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0177] Samples that can be used in the BCMA diagnostic assay according to the present invention include any tissue or body fluid sample that can be obtained from a patient and contains a detectable amount of BCMA protein or a fragment thereof, under normal or pathological conditions. Generally, the level of BCMA in a specific sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal BCMA levels or activity) will be measured first to establish a baseline or standard level of BCMA. This baseline level of BCMA can then be compared to the level of BCMA measured in a sample obtained from an individual suspected of having a disease or condition associated with BCMA (e.g., a tumor containing BCMA-expressing cells).

Examples

[0178] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope that the inventors regard as the present invention. Efforts have been made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0179] Example 1: Generation of anti-BCMA antibodies Anti-BCMA antibodies were obtained by immunizing genetically modified mice with human BCMA antigen (e.g., hBCMA, SEQ ID NO: 115), or by immunizing transgenic mice containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions with human BCMA antigen.

[0180] After immunization, splenocytes were harvested from each mouse and (1) fused with mouse myeloma cells to maintain their viability, form hybridoma cells, and screen for BCMA specificity, or (2) B cells were sorted using a human BCMA fragment as a sorting reagent that binds to reactive antibodies (antigen-positive B cells) for identification (as described in US2007 / 0280945A1).

[0181] Chimeric antibodies against BCMA having human variable regions and mouse constant regions were first isolated. Antibodies were characterized and selected for desirable features including affinity, selectivity, etc. If necessary, the mouse constant regions were replaced with desired human constant regions, such as wild-type or modified IgG1 or IgG4 constant regions, to generate fully human anti-BCMA antibodies. The constant regions selected may vary depending on the specific application, but the high-affinity antigen-binding features and target specificity features reside in the variable regions.

[0182] Amino acid and nucleic acid sequences of the heavy and light chain variable regions of anti-BCMA antibodies: Table 1 shows the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of the selected anti-BCMA antibodies of the present invention. The corresponding nucleic acid sequence identifiers are listed in Table 2. [Table 1] [Table 2]

[0183] Example 2: Generation of anti-CD3 antibodies The anti-CD3 antibodies were generated as described in WO2017 / 053856, which is incorporated herein by reference. Two such anti-CD3 antibodies were selected from the production of the bispecific anti-BCMA×anti-CD3 antibodies according to the present invention. Table 3 shows the amino acid sequence identifiers of the heavy chain variable region, light chain variable region and CDRs of the selected anti-CD3 antibodies. The corresponding nucleic acid sequence identifiers are shown in Table 4. Other anti-CD3 antibodies for use in the preparation of the bispecific antibodies according to the present invention can be found, for example, in WO2014 / 047231.

Table 3

Table 4

[0184] Example 3: Generation of Bispecific Antibodies that Bind to BCMA and CD3 The present invention provides a bispecific antigen-binding molecule that binds to CD3 and BCMA, and such bispecific antigen-binding molecules are also referred to herein as "anti-BCMA×anti-CD3 or anti-CD3×BCMA or anti-BCMA×anti-CD3 bispecific molecules". The anti-BCMA portion of the anti-BCMA×anti-CD3 bispecific molecule is useful for targeting tumor cells that express BCMA (also known as CD269), and the anti-CD3 portion of the bispecific molecule is useful for activating T cells. Simultaneous binding of BCMA on tumor cells and CD3 on T cells promotes the direct killing (cytolysis) of target tumor cells by activated T cells.

[0185] A bispecific antibody comprising an anti-BCMA specific binding domain and an anti-CD3 specific binding domain was constructed using standard methods, wherein the anti-BCMA antigen binding domain and the anti-CD3 antigen binding domain each comprise distinct HCVRs paired with a common LCVR. In the exemplified bispecific antibody, these molecules were constructed using the heavy chain from an anti-CD3 antibody, the heavy chain from an anti-BCMA antibody, and a common light chain from an anti-CD3 antibody (e.g., SEQ ID NO: 82). In other examples, the bispecific antibody can be constructed using the heavy chain from an anti-CD3 antibody, the heavy chain from an anti-BCMA antibody, and an antibody light chain that is scrambled or known to effectively pair with various heavy chain arms. [Table 5]

[0186] Table 6 shows the amino acid sequence identifiers of the bispecific anti-BCMA × anti-CD3 antibodies exemplified herein. [Table 6]

[0187] The bsAb25441D bispecific antibody (REGN4548) identified in Table 6 comprises a first heavy chain (comprising a first antigen binding domain) having the amino acid sequence of SEQ ID NO: 126, a second heavy chain (comprising a second antigen binding domain) having the amino acid sequence of SEQ ID NO: 127, and a common light chain having the amino acid sequence of SEQ ID NO: 129. The first heavy chain of the bsAb25441D bispecific antibody (REGN5458) comprises a constant region having the amino acid sequence of SEQ ID NO: 130. The second heavy chain of the bsAb25441D bispecific antibody (REGN5458) comprises a constant region having the amino acid sequence of SEQ ID NO: 131. The common light chain of the bsAb25441D bispecific antibody (REGN5458) comprises a constant region having the amino acid sequence of SEQ ID NO: 132.

[0188] The bsAb25442D bispecific antibody (REGN5459) identified in Table 6 comprises a first heavy chain (including a first antigen-binding domain) comprising the amino acid sequence of SEQ ID NO: 126, a second heavy chain (including a second antigen-binding domain) comprising the amino acid sequence of SEQ ID NO: 128, and a common light chain comprising the amino acid sequence of SEQ ID NO: 129. The first heavy chain of the bsAb25442D bispecific antibody (REGN5459) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 130. The second heavy chain of the bsAb25442D bispecific antibody (REGN5459) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 131. The common light chain of the bsAb25442D bispecific antibody (REGN5459) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 132.

[0189] Example 4: Binding Affinity and Kinetic Constants from Surface Plasmon Resonance of Anti-BCMA Antibodies and Anti-BCMA × Anti-CD3 Bispecific Antibodies Equilibrium dissociation constant (K) of hBCMA.mmh (SEQ ID NO: 106) binding to purified anti-BCMA mAb and anti-BCMA × anti-CD3 bispecific mAb DThe value) was determined using a real-time surface plasmon resonance biosensor using a Biacore 4000 instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture purified anti-BCMA mAb and anti-BCMA × anti-CD3 bispecific mAb. All Biacore binding tests were performed in a buffer composed of 0.01M HEPES pH7.4, 0.15M NaCl, 3mM EDTA, 0.05 volume / volume% surfactant P20 (HBS-ET running buffer). For monomer affinity, different concentrations of the extracellular domain of human BCMA expressed with a C-terminal myc-myc-hexahistidine tag (human BCMA-MMH; SEQ ID NO: 106) or cynomolgus BCMA expressed with a C-terminal myc-myc-hexahistidine tag (cynomolgus BCMA-MMH; SEQ ID NO: 110) were prepared in HBS-ET running buffer (90 to 1.11 nM, 3-fold dilution). For dimer affinity, different concentrations of the extracellular domain of human BCMA expressed with a C-terminal mFc tag (human BCMA-MFC; SEQ ID NO: 108) or cynomolgus BCMA expressed with a C-terminal mFc tag (cynomolgus BCMA-MFC; SEQ ID NO: 112) were prepared in HBS-ET running buffer (30 to 0.37 nM, 3-fold dilution), or 30 nM BCMA expressed with a C-terminal mFc tag (mouse BCMA-MFC; SEQ ID NO: 114) was prepared. The antigen sample was then injected across the anti-BCMA and anti-BCMA × anti-CD3 bispecific mAb captured on the surface at a flow rate of 30 μL / min. The antibody-reagent conjugate was monitored for 5 minutes while dissociation in HBS-ET running buffer was monitored for 10 minutes. All binding kinetics experiments were performed at 25°C. By fitting the real-time sensorgram to a 1:1 binding model using Scrubber 2.0c curve fitting software, the kinetic binding (k a ) rate constant and the kinetic dissociation (k d ) rate constant were measured. The binding dissociation equilibrium constant (K D ) and the dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows:

Number

[0190] As shown in Table 7, at 25°C, all anti-BCMA antibodies of the present invention bound to human BCMA-MMH with K values in the range of 1.06 nM to 3.56 nM. D As shown in Table 8, at 25°C, all anti-BCMA antibodies of the present invention bound to human BCMA-MFC with K values in the range of 22.3 pM to 103 pM. D As shown in Table 9, at 25°C, two anti-BCMA antibodies of the present invention bound to cynomolgus BCMA-MMH with K values in the range of 38.8 nM to 49.92 nM. D As shown in Table 10, at 25°C, four anti-BCMA antibodies of the present invention bound to cynomolgus BCMA-MFC with K values in the range of 148 pM to 14.7 nM. D As shown in Table 11, at 25°C, four anti-BCMA antibodies of the present invention bound to mouse BCMA-MFC with K values in the range of 677 pM to 18.8 nM. D

Table 7

Table 8

Table 9

Table 10

Table 11

[0191] Example 5: FACS Binding of Anti-BCMA × Anti-CD3 Bispecific Antibodies to Human and Cynomolgus CD3-Expressing Cells Flow cytometry analysis was used to determine the binding of BCMA×CD3 bispecific antibodies to human and cynomolgus CD3 (Jurkat cells, mfCD3 engineered Jurkat cells, primary human CD8+ and cynomolgus CD8+ T cells). Briefly, 1e05 cells / well were incubated on ice for 30 minutes in the presence of FACS wash with block (PBS + 1% filtered FBS + 5% mouse serum) with serial dilutions of BCMA×CD3 and control antibodies. After incubation, cells were washed twice with cold FACS wash (PBS + 1% filtered FBS) and the bound antibodies were detected by incubating for an additional 30 minutes on ice with anti-human secondary antibody conjugated with Alexa647. Wells containing no antibody or secondary antibody only were used as controls. To detect monkey and human T cells, a cocktail of human and cynomolgus cross-reactive antibodies against CD4, CD8, and CD16 was added to the anti-human secondary antibody. After incubation, cells were washed and resuspended in 200 μL of cold PBS containing 1% filtered FBS and analyzed by flow cytometry on a BD FACS Canto II. Cells were gated by FSC-H versus FSC-A to select single events, followed by side scatter and forward scatter to select live events. For cynomolgus T cells, additional gating was performed on CD8+ / CD16− cells.

[0192] The EC50 values for FACS binding were calculated using 4-parameter non-linear regression analysis in Prism software.

[0193] Jurkat cells are human CD3 expressing T cell lymphoblastoid cell lines. REGN5458 bound to human CD3 on Jurkat cells and primary human CD8+ T cells with median EC50s of 1.50×10 -8 M and 3.20×10 -8 M, respectively. The binding of REGN5459 was weak to human CD3 with median EC50s of 5.58×10 -7 M in Jurkat cells and 4.71×10 -6It was. Using CRISPR / Cas9 technology, the Jurkat cell line was engineered to express cynomolgus CD3ε and CD3δ chains instead of the human version. The median EC50 of REGN5458 binding to the engineered Jurkat cell line expressing mfCD3 was 1.51x10 -8 M, and the median EC50 of its binding to primary cynomolgus CD8+ T cells was 4.66×10 -8 M. REGN5459 did not bind to mfCD3-expressing cells.

[0194] No binding was observed in any cell line with the negative isotype control antibody called mAb15260. [Table 12]

[0195] Example 6: FACS Binding Assay to Evaluate Cell Surface Antigen Binding Ability The ability of the anti-BCMA×CD3 antibody, mAb25442D, to bind to the surface of BCMA-positive multiple myelomas (NCI-H929, MM.1S, OPM-2, and RPMI-8226), BCMA-positive lymphomas (Raji and Daudi), and BCMA-negative (HEK293) cells was determined by flow cytometry. Cells were harvested from flasks using cell dissociation buffer (Millipore, catalog number S-004-C) and seeded at a density of 500,000 cells per well in 96-well V-bottom plates in staining buffer (PBS without calcium and magnesium (Irving 9240) + 2% FBS (ATCC 30-2020)). Cells were stained with Alexa647-conjugated anti-BCMA×CD3 antibody (mAb25442D-A647) or an Alexa647-conjugated isotype control with the same CD3-binding arm as the unrelated tumor target arm (Isotype-A647) at a 2-fold serial dilution for 30 minutes at 4°C. Cells were washed twice with staining buffer and labeled with the LIVE / DEAD™ Fixable Green Dead Cell Stain Kit (Invitrogen, L34970) according to the manufacturer's instructions to distinguish live and dead cells. Cells were then washed and fixed with a 50% solution of BD Cytofix (BD, catalog number 554655) diluted in PBS for 25 minutes at 4°C. Samples were run on an Accuri C6 flow cytometer (BD Biosciences) and analyzed with Flowjo 10.2 (Tree Star). After gating on live cells and single cells, the mean fluorescence intensity (MFI) was determined, and the MFI values were plotted in Graphpad Prism using a 4-parameter logistic equation on a 10-point response curve to calculate EC 50 50 ​The value was in the range of 27 - 83 nM. In HEK293 cells, no detectable binding was observed.

Table 13

[0196] Example 7: T cell activation via bispecific anti - BCMA × anti - CD3 antibody in the presence of BCMA - expressing cells The activity of the anti - BCMA × anti - CD3 bispecific antibody was evaluated by a Jurkat / NFATLuc reporter bioassay using several cell lines with different levels of BCMA surface expression. Jurkat cells were engineered to express an NFAT - luciferase reporter (Jurkat / NFATLuc.3C7), and 50,000 Jurkat reporter cells were mixed with 50,000 BCMA - positive (Daudi, MM1 - S, NCI - H929, OPM - 2, RPMI - 8226, MOLP - 8, or Raji) or BCMA - negative (HEK293) cells in 50 ul of assay medium (RPMI medium containing 10% FBS and 1% P / S / G) in a Thermo Nunclon delta 96 - well white microplate (Thermo Scientific, catalog number 136102). Serial three - fold dilutions of the BCMA×CD3 bispecific antibody (mAb25441D or mAb25442D) or the bivalent anti - BCMA antibody (mAb21581) were immediately added into 50 uL of assay buffer. The plate was gently agitated and incubated for 4 - 6 hours in a 37°C, 5% CO2 incubator. NFAT - luciferase activity was determined using Promega One - Glo (catalog number E6130) and a Perkin Elmer Envision plate reader. RLU was plotted in GraphPad Prism using a four - parameter logistic equation on a 12 - point response curve to calculate the EC 50 To calculate the value, the antibody - free treatment conditions of each dose - response curve were also included in the analysis as a continuation of the serial three - fold dilutions and represented as the lowest dose. The signal - to - noise ratio (S:N) was determined by taking the ratio of the highest RLU to the lowest RLU on the curve.

[0197] mAb25441D activated Jurkat / NFATLuc cells in the presence of BCMA-expressing cells with an EC50 in the range of 0.61 nM to 2.1 nM and an S:N in the range of 8 to 123. mAb25442D activated Jurkat / NFATLuc cells in the presence of BCMA-expressing cells with an EC50 in the range of 2.6 nM to 11 nM and an S:N in the range of 7 to 120. BCMA×CD3 bispec mAb25441D with a high-affinity CD3-binding arm was consistently more potent than mAb25442D with a low-affinity CD3-binding arm, while the S:N was similar for the two bispecifics. Neither antibody activated Jurkat / NFATLuc cells in the presence of HEK293 cells, and the control bispecific antibody did not significantly increase Jurkat reporter activity in any of the cell lines tested. The results are shown in Tables 14A and 14B below. [Table 14] [Table 15]

[0198] Example 8: A FACS-based cytotoxicity assay to evaluate T cell-mediated killing of BCMA-expressing multiple myeloma cells in the presence of anti-BCMA×anti-CD3 bispecific antibodies. The antibody binding capacity (ABC) of a commercially available anti-human BCMA antibody (clone 19F2) was determined on a panel of multiple myeloma cell lines using the Quantum Simply Cellular anti-human IgG kit according to the manufacturer's instructions (Bangs Laboratories).

[0199] Briefly, multiple myeloma (MM) cell lines (H929, MM1S, U266, MOLP8, and RPMI8226) and Quantum Simply Cellular beads were incubated at 4°C for 30 minutes by titration of APC-conjugated anti-hBCMA-19F2 antibody. After incubation, the cells and beads were washed three times and resuspended in 200 μL of cold PBS containing 1% filtered FBS and analyzed by flow cytometry. Using the QuickCal® template (Bangs Labs), the saturation level of ABC of anti-BCMA 19F2 of each cell line was interpolated from a standard curve generated by the channel intensity of the bead population at saturation.

[0200] Killing of BCMA-expressing target cells by arresting human or cynomolgus monkey T cells was determined by flow cytometry. Briefly, human or cynomolgus monkey peripheral blood mononuclear cells (PBMCs) were seeded in RPMI (human) or X-Vivo (cynomolgus monkey) medium supplemented with 1 × 10 6 cells / mL and incubated overnight at 37°C to enrich lymphocytes by depleting adherent macrophages, dendritic cells, and some monocytes. The next day, BCMA-expressing target cells were labeled with 1 μM Violet CellTrace and co-cultured at 37°C with adherent cell-depleted PBMCs (effector / target cell ratio of 4:1) and serial dilutions of BCMA×CD3 bispecific or control antibody. After 48 - 72 hours, the cells were removed from the cell culture plates, stained with cocktail phenotypic antibodies and live / dead cell viability dyes, and analyzed by FACS. To quantify the number of live target cells present in the wells, 20 μl of CountBright absolute counting beads were added to the wells immediately before acquisition. To assess the specificity of killing, the cells were gated on the violet cell trace-labeled population. The survival rate of target cells was calculated as follows: target survival rate = (R1 / R2) * 100 (where R1 = absolute number of live target cells in the presence of effector cells and antibody, R2 = number of live cells only (cultured without effector cells or test antibody)).

[0201] Human CD8+ T cells were gated as CD45+ / CD14- / CD4- / CD8+. Cynomolgus CD8+ T cells were gated as CD45+ / CD20- / CD14- / CD4- / CD8+, and T cell activation was reported as the percentage of CD25+ or CD69+ T cells among total CD8+ T cells.

[0202] EC50 values for target cell survival and T cell activation were calculated using four-parameter non-linear regression analysis of Prism software.

[0203] The anti-BCMA×anti-CD3 bispecific antibodies were tested for their ability to activate resting human and cynomolgus T cells to kill a panel of BCMA-expressing cells with different surface BCMA levels. Resting human T cells were used as effector cells, and REGN5458 mediated the killing of five different BCMA cell lines with EC 50 values ranging from 7.07×10 -10 M to 3.45×10 -11 M. REGN5459 showed the killing of the same five cell lines with EC50 values ranging from 1.66×10 -9 M to 1.06×10 -10 M. As measured by upregulation of CD25 in CD8+ T cells, the EC 50 for T cell activation was similar to the EC 50 for killing. Moderate T cell activation was observed in the presence of the 1-arm CD3 isotype control mAb17664D, but only in the U266 cell line. No cytotoxicity was observed with the isotype controls tested.

[0204] BCMA×CD3-mediated killing by cynomolgus T cells was tested only in the MM cell line H929. The EC 50 of cytotoxicity mediated by REGN5458 and REGN5459 was 2.34×10 -11 and 6.92×10 -11It was. No cytotoxicity or T cell activation was observed with the isotype control antibody mAb15260 using human or cynomolgus effector cells. The results are shown in Tables 15A, 15B, and 16 below. [Table 16] [Table 17] [Table 18]

[0205] Example 9: FACS Cytotoxicity Assay for Autologous T Cell-Mediated Killing of Multiple Myeloma Blasts in the Presence of Anti-BCMA × Anti-CD3 Bispecific Antibodies To monitor specific killing of multiple myeloma cells by flow cytometry, bone marrow mononuclear cells (BMMC) from multiple myeloma patients were seeded on human stromal cells (HS5) and allowed to rest overnight at 37°C. Separately, peripheral blood mononuclear cells (PBMC) from a matched patient were thawed and lymphocytes were enriched by depleting adherent cells. The cells were cultured overnight at 37°C in supplemented RPMI medium at 1×10 6 cells / mL. The next day, the BMMC were co-cultured at 37°C with serial 10-fold dilutions of non-adherent cell-depleted naive PBMC and BCMA×CD3 bispecific or 1-arm CD3 isotype control (starting concentration 66.7 nM) on stromal cells (HS5). On days 3, 4, or 7, the cells were removed from the cell culture plates and analyzed by FACS. To assess the specificity of killing, multiple myeloma cells were gated as single, live, CD90-negative (excluding stromal cells), CD2-negative, and CD56-positive. CD45 was low in multiple myeloma cells in most samples except MM455. For calculation of the adjusted survival rate, the percentage of live target cells was reported as follows: Adjusted survival rate = (R1 / R2)*100, where R1 = percentage of live target cells (%) in the presence of the antibody and R2 = percentage of live target cells (%) in the absence of the test antibody.

[0206] T cells were gated as CD2 positive, CD56 negative, and CD4 positive or CD8 positive. T cell activation was reported as the percentage of CD25+ CD4 or CD8 T cells out of the total CD4 or CD8 T cells.

[0207] The BCMA×CD3 bispecific antibody was tested for its ability to redirect the killing of primary multiple myeloma blasts by autologous donor PBMCs. The maximum BCMA×CD3-mediated cytotoxicity of primary MM blasts ranged from 52% to 96%, and for REGN5458, the EC50 was in the range of 9.89×10 -11 M to 3.67×10 -9 M, and for REGN5459, it was in the range of 4.96×10 -10 M to 7.94×10 -8 M. T cell activation was measured by assessing the upregulation of CD25 on CD8+ T cells. The EC50 of T cell activation was 3.23×10 -9 to 1.69×10 -10 . Moderate cytotoxicity and T cell activation were observed with the 1-arm CD3 (no target binding) isotype control. The results are shown in Tables 17A and 17B below.

Table 19

Table 20

[0208] Example 10: Anti-BCMA×anti-CD3 bispecific antibody prevents the growth of BCMA-expressing tumors (NCI-H929) in vivo in a xenograft tumor model To determine the in vivo efficacy of the BCMA×CD3 bispecific antibody (Ab), xenograft studies were performed. Immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were injected with 10×10 6 individual BCMA-expressing NCI-H929 multiple myeloma cells and 0.5×10 6A mixture with individual human peripheral blood mononuclear cells (PBMCs) was transplanted subcutaneously. Mice (n = 7 per group) were immediately administered PBS vehicle control, irrelevant anti-FelD1 bivalent isotype control Ab (REGN2759), CD3-binding control bispecific Ab (mAb17664D), BCMA×CD3 (G; REGN5458) bispecific Ab, or BCMA×CD3 (G20; REGN5459) bispecific antibody at a dose of 4 mg / kg. The mice were administered the Ab twice a week for a total of 3 weeks, and tumor growth was evaluated over 40 days. BCMA + Tumors grew similarly in vehicle, isotype control, and CD3-binding control-treated mice, but both of the tested BCMA×CD3 Abs prevented tumor growth in vivo.

[0209] Syngeneic tumor transplantation and measurement: NSG mice were transplanted subcutaneously with a mixture of 10×10 6 individual BCMA-expressing NCI-H929 multiple myeloma cells and 0.5×10 6 individual PBMCs derived from normal donors. Mice (n = 7 per group) were immediately administered PBS vehicle control, irrelevant anti-FelD1 bivalent isotype control Ab (REGN2759), CD3-binding control bispecific Ab (mAb17664D), BCMA×CD3 (G; REGN5458) bispecific Ab, or BCMA×CD3 (G20; REGN5459) bispecific antibody at a dose of 4 mg / kg. The mice were administered the Ab twice a week for a total of 3 weeks. Tumor growth was measured twice a week using calipers throughout the duration of the experiment. The mice were sacrificed on day 40 after tumor transplantation.

[0210] Calculation of syngeneic tumor growth and inhibition: To determine tumor volume with an external caliper, the maximum major axis (length in mm) and maximum minor axis (width in mm) were determined. Based on the caliper measurements, the tumor volume was calculated using the formula: volume (mm 3 ) = (length × width 2 ) / 2.

[0211] The BCMA×CD3 bispecific Ab inhibited BCMA in vivo in a xenograft tumor model +The growth of NCI-H929 tumors was prevented. The results are shown in Table 18 below.

Table 21-1

Table 21-2

[0212] Example 11: The anti-BCMA × anti-CD3 bispecific antibody prevents the growth of BCMA-expressing tumors (NCI-H929) in a dose-dependent manner in a xenogeneic in vivo tumor model To determine the in vivo efficacy of the anti-BCMA × anti-CD3 bispecific antibody (Ab), xenograft studies were performed. Immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were subcutaneously implanted with a mixture of 10 × 10 6 BCMA-expressing NCI-H929 human multiple myeloma cells and 0.5 × 10 6 human peripheral blood mononuclear cells (PBMCs) isolated from normal healthy donors. Subsequently, mice (n = 7 per group) were immediately administered PBS vehicle control, CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 4 mg / kg, CD3-binding control bispecific Ab (G20; REGN4460) at a dose of 4 mg / kg, BCMA × CD3 (G; REGN5458) bispecific Ab at either a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg, or BCMA × CD3 (G20; REGN5459) bispecific Ab at either a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. The mice were administered these Abs twice a week for a total of 7 doses, and tumor growth was evaluated over 60 days. BCMA + NCI-H929 tumors grew similarly in mice treated with vehicle and CD3-binding controls, but both tested anti-BCMA × anti-CD3 Abs prevented tumor growth in an in vivo dose-dependent manner.

[0213] Xenograft tumor implantation and measurement: NSG mice were implanted with 10 × 106 Individual BCMA-expressing NCI-H929 multiple myeloma cells were transplanted subcutaneously with a mixture of 0.5 × 10 6 PBMCs derived from normal healthy donors. Mice (n = 7 per group) were immediately administered PBS vehicle control, CD3-binding control bispecific Ab (G; mAb17664D), CD3-binding control bispecific Ab (G20; REGN4460), BCMA×CD3 (G; REGN5458) bispecific Ab, or BCMA×CD3 (G20; REGN5459) bispecific Ab. mAb17664D and REGN4460 were administered at 4 mg / kg, while REGN5458 and REGN5459 were administered at either 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. The mice were administered the Abs at a total of 7 doses twice a week. Tumor growth was measured twice a week using calipers throughout the duration of the experiment.

[0214] Calculation of heterologous tumor growth and inhibition: To determine tumor volume with an external caliper, the maximum major axis (length in mm) and the maximum minor axis (width in mm) were determined. Based on the caliper measurements, the tumor volume was calculated using the formula: volume (mm 3 ) = (length × width 2 ) / 2.

[0215] The BCMA×CD3 bispecific Ab prevented the growth of BCMA + NCI-H929 tumors in a dose-dependent manner in this heterologous in vivo tumor model. The results are shown in Table 19 below and in Figures 1 and 2.

Table 22-1

Table 22-2

Table 22-3

Table 22-4

Table 22-5

[0216] Example 12: The anti-BCMA × anti-CD3 bispecific antibody reduced the size of BCMA-expressing tumors (NCI-H929) established in a dose-dependent manner in a xenogeneic in vivo tumor model and prevented their growth. To determine the in vivo efficacy of the anti-BCMA × anti-CD3 bispecific antibody (Ab), xenograft studies were performed. Immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were subcutaneously implanted with a mixture of 10 × 10 6 BCMA-expressing NCI-H929 human multiple myeloma cells and 0.5 × 10 6 human peripheral blood mononuclear cells (PBMCs) isolated from normal healthy donors. Tumors were allowed to grow and establish for 5 days until they reached a size of approximately 70 mm 3 . On day 5, mice (n = 7 - 8 per group) were administered PBS vehicle control, CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 4 mg / kg, CD3-binding control bispecific Ab (G20; REGN4460) at a dose of 4 mg / kg, BCMA × CD3 (G; REGN5458) bispecific Ab at either a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg, or BCMA × CD3 (G20; REGN5459) bispecific Ab at either a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. These Abs were administered to the mice twice a week for a total of 7 doses, and tumor growth was evaluated over 55 days. BCMA + NCI-H929 tumors grew similarly in mice treated with vehicle and CD3-binding controls, but both BCMA × CD3 Abs tested reduced the established tumors in a dose-dependent manner in vivo and prevented tumor growth.

[0217] Xenograft tumor implantation and measurement: NSG mice were implanted with 10 × 10 6 BCMA-expressing NCI-H929 multiple myeloma cells and 0.5 × 10 6A mixture with individual PBMCs was transplanted subcutaneously. The tumors grew and established for 5 days until they reached a size of approximately 70 mm 3 and then, on day 5, mice (n = 7 - 8 per group) were immediately administered PBS vehicle control, CD3-binding control bispecific Ab (G; mAb17664D), CD3-binding control bispecific Ab (G20; REGN4460), BCMA×CD3 (G; REGN5458) bispecific Ab, or BCMA×CD3 (G20; REGN5459) bispecific Ab. mAb17664D and REGN4460 were administered at 4 mg / kg, while REGN5458 and REGN5459 were administered at either 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. The mice were administered the Abs at a total of 7 doses twice a week. Tumor growth was measured twice a week using calipers throughout the duration of the experiment.

[0218] Calculation of heterologous tumor growth and inhibition: To determine tumor volume with an external caliper, the maximum major axis (length in mm) and maximum minor axis (width in mm) were determined. Based on the caliper measurements, the tumor volume was calculated using the formula: volume (mm 3 ) = (length × width 2 ) / 2.

[0219] The anti-BCMA×anti-CD3 bispecific antibody established the size of BCMA + NCI-H929 tumors in a dose-dependent manner and prevented their growth in this heterologous in vivo tumor model. The results are shown in Table 20 below and Figures 3 and 4.

Table 23 - 1

Table 23 - 2

Table 23 - 3

Table 23 - 4

[0220] Example 13: The anti-BCMA × anti-CD3 bispecific antibody prevents the growth of BCMA-expressing tumors (MOLP-8) in a dose-dependent manner in a xenogeneic in vivo tumor model. To determine the in vivo efficacy of the anti-BCMA × anti-CD3 bispecific antibody (Ab), xenograft studies were performed. Immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were subcutaneously implanted with a mixture of 5 × 10 6 individual BCMA-expressing MOLP-8 human multiple myeloma cells and 1 × 10 6 individual human peripheral blood mononuclear cells (PBMCs) isolated from normal healthy donors. Subsequently, mice (n = 7 per group) were immediately administered PBS vehicle control, CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 4 mg / kg, CD3-binding control bispecific Ab (G20; REGN4460) at a dose of 4 mg / kg, BCMA × CD3 (G; REGN5458) bispecific Ab at either a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg, or BCMA × CD3 (G20; REGN5459) bispecific Ab at either a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. The mice were administered these Abs twice a week for a total of 7 doses, and tumor growth was evaluated over 56 days. BCMA + MOLP-8 tumors grew similarly in mice treated with vehicle and CD3-binding controls, but both of the tested BCMA × CD3 Abs prevented tumor growth in an in vivo dose-dependent manner.

[0221] Xenograft tumor implantation and measurement: NSG mice were implanted with 5 × 10 6 individual BCMA-expressing MOLP-8 multiple myeloma cells and 1 × 10 6The mixture with individual PBMCs was transplanted subcutaneously. Mice (n = 7 per group) were immediately administered PBS vehicle control, CD3-binding control bispecific Ab (G; mAb17664D), CD3-binding control bispecific Ab (G20; REGN4460), BCMA×CD3 (G; REGN5458) bispecific Ab, or BCMA×CD3 (G20; REGN5459) bispecific Ab. mAb17664D and REGN4460 were administered at 4 mg / kg, while REGN5458 and REGN5459 were administered at either 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. The mice were administered the Abs at a total of 7 doses, twice a week. Tumor growth was measured twice a week with calipers throughout the duration of the experiment.

[0222] Calculation of xenograft tumor growth and inhibition: To determine tumor volume with an external caliper, the maximum major axis (length in mm) and the maximum minor axis (width in mm) were determined. Based on the caliper measurements, the tumor volume was calculated by the formula: volume (mm 3 ) = (length × width 2 ) / 2.

[0223] The anti-BCMA×anti-CD3 bispecific antibody prevented the growth of BCMA + MOLP-8 tumors in a dose-dependent manner in this xenograft in vivo tumor model. The results are shown in Table 21 below and Figures 5 and 6.

Table 24-1

Table 24-2

Table 24-3

Table 24-4

Table 24-5

[0224] Example 14: The anti-BCMA x anti-CD3 bispecific antibody delays the growth of BCMA-expressing tumors (MOLP-8) in a xenographic in vivo tumor model. To determine the in vivo efficacy of the anti-BCMA x anti-CD3 bispecific antibody (Ab), xenograft studies were performed. On day 11, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were injected intraperitoneally with 4 x 10 6 human peripheral blood mononuclear cells (PBMCs) from a normal healthy donor. On day 0, mice were injected intravenously with 2 x 10 6 BCMA + MOLP-8 human multiple myeloma tumor cells engineered to also express firefly luciferase (MOLP-8-luciferase cells). Next, mice (n = 5 per group) were immediately administered a CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 4 mg / kg or a BCMA x CD3 (G; REGN5458) bispecific Ab at a dose of 4 mg / kg. These Abs were administered to the mice two more times on days 3 and 7 for a total of three doses. Tumor growth was evaluated over 48 days by measuring the bioluminescence (BLI) of the tumors in anesthetized animals. As a positive control, a mouse group (n = 5) was administered only MOLP-8-luciferase cells and no PBMCs or antibodies. To measure the background BLI level, a mouse group (n = 5) was not treated and received no tumors, PBMCs, or antibodies. The BCMA + MOLP-8-luciferase tumors grew progressively in CD3-binding control-treated mice, but treatment with the BCMA x CD3 Ab REGN5458 delayed tumor growth in vivo.

[0225] Xenograft tumor implantation and measurement: On day 11, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were injected intraperitoneally with 5 x 10 6 human PBMCs from a normal healthy donor. On day 0, mice were injected with 2 x 10 6 BCMA+ MOLP-8-luciferase cells were administered intravenously. Subsequently, mice (n = 5 per group) were immediately administered a CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 4 mg / kg or a BCMA×CD3 (G; REGN5458) bispecific Ab at a dose of 4 mg / kg. The mice were administered these Abs two more times on days 3 and 7, for a total of three doses. Tumor growth was evaluated over 48 days by measuring tumor BLI in anesthetized animals. As a positive control, a mouse group (n = 5) was administered only MOLP-8-luciferase cells, and no PBMC or antibody was administered. To measure the background BLI level, a mouse group (n = 5) was not treated and received no tumor, PBMC, or antibody.

[0226] Measurement of xenograft tumor growth: Tumor burden was measured using BLI imaging. Mice were injected IP with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after this injection, BLI imaging of the mice was performed under isoflurane anesthesia using a Xenogen IVIS system. Image collection was performed at a field of view in D, a subject height of 1.5 cm, and a medium binning level of the autoexposure time determined by Living Image software. The BLI signal was extracted using Living Image software: The region of interest was traced around each cell mass, and the photon intensity was recorded as p / s / cm2 / sr.

[0227] The anti-BCMA×anti-CD3 bispecific antibody REGN5458 retarded the growth of BCMA + MOLP-8-luciferase tumors in this xenograft in vivo tumor model. The results are shown in Table 22 below.

Table 25-1

Table 25-2

[0228] Example 15: The anti-BCMA × anti-CD3 bispecific antibody reduces the tumor (OPM-2) burden to background levels in vivo To determine the in vivo efficacy of the anti-BCMA × anti-CD3 bispecific antibody (Ab), a xenograft tumor study was conducted. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were intravenously administered 2 × 10 6 individuals of BCMA + OPM-2 human multiple myeloma tumor cells engineered to also express firefly luciferase (OPM-2-luciferase cells). On day 10, the mice were intraperitoneally injected with 4 × 10 6 individuals of human peripheral blood mononuclear cells (PBMCs) from normal healthy donors. On day 21, the mice (n = 5 per group) were administered a CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 0.4 mg / kg, a BCMA × CD3 (G; REGN5458) bispecific Ab at 0.4 mg / kg, or a BCMA × CD3 (G20; REGN5459) bispecific Ab at 0.4 mg / kg. The mice were administered these Abs two more times on days 25 and 28 for a total of three doses. Tumor growth was evaluated over 61 days by measuring the tumor bioluminescence (BLI) of anesthetized animals. As a positive control, a mouse group (n = 5) was administered only OPM-2-luciferase cells and no PBMCs or antibodies. To measure the background BLI level, a mouse group (n = 5) was not treated and received no tumors, PBMCs, or antibodies. BCMA + OPM-2-luciferase tumors grew gradually in mice treated with the CD3-binding control, but BCMA × CD3 Ab treatment with REGN5458 and REGN5459 reduced the tumor burden to background levels in the majority of animals.

[0229] Xenograft tumor implantation and measurement: On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl2×10 of the / SzJ(NSG) mice were designed to also express firefly luciferase (OPM-2-luciferase cells). 6 BCMA + OPM-2 human multiple myeloma tumor cells were intravenously administered. On day 10, 4×10 6 human peripheral blood mononuclear cells (PBMCs) from normal healthy donors were injected intraperitoneally into the mice. On day 21, mice (n = 5 per group) were administered CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 0.4 mg / kg, BCMA×CD3 (G; REGN5458) bispecific Ab at 0.4 mg / kg, or BCMA×CD3 (G20; REGN5459) bispecific Ab at 0.4 mg / kg. These Abs were administered to the mice two more times on days 25 and 28 for a total of three doses. Tumor growth was evaluated over 61 days by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, a mouse group (n = 5) was administered only OPM-2-luciferase cells and no PBMCs or antibodies. To measure the background BLI level, a mouse group (n = 5) was not treated and received no tumors, PBMCs, or antibodies.

[0230] Measurement of xenograft tumor growth: Tumor burden was measured using BLI imaging. Mice were injected IP with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after this injection, BLI imaging of the mice was performed under isoflurane anesthesia using a Xenogen IVIS system. Image collection was performed at a medium binning level with a field of view in D, a subject height of 1.5 cm, and an automatically determined exposure time by Living Image software. The BLI signal was extracted using Living Image software: The region of interest was traced around each cell mass, and the photon intensity was recorded as p / s / cm2 / sr.

[0231] BCMA +OPM-2-luciferase tumors grew gradually in mice treated with CD3-binding control, but BCMA×CD3 Ab treatment with REGN5458 and REGN5459 reduced tumor burden to background levels in the majority of animals. The results are shown in Table 23 below and in Figure 7.

Table 26-1

Table 26-2

Table 26-3

[0232] Example 16: BCMA×CD3 bispecific antibodies inhibit the growth of syngeneic tumors in vivo in a dose-dependent manner To determine the in vivo efficacy of anti-BCMA×anti-CD3 bispecific antibodies (Abs), syngeneic tumor studies were performed in mice expressing human CD3. C57BL / 6 mice expressing human CD3deg were used instead of mouse CD3deg (CD3-humanized mice), and 0.5×10 6 individual B16 melanoma cells engineered to express full-length human BCMA (B16 / BCMA cells) or 1×10 6 individual MC38 colon cancer cells engineered to express full-length human BCMA (MC38 / BCMA) were subcutaneously implanted. Mice (n = 7 per group) were then immediately administered CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 0.4 mg / kg or BCMA×CD3 (G; REGN5458) bispecific Ab at doses of 0.4 mg / kg or 0.04 mg / kg. These Abs were administered to the mice three times in total, at 4 and 7 days, and tumor growth was evaluated throughout the experiment. B16 / BCMA tumors and MC38 / BCMA tumors grew in CD3-binding control-treated mice, but BCMA×CD3 REGN5458 was able to inhibit the growth of both tumor strains in vivo in a dose-dependent manner.

[0233] Orthotopic tumor transplantation and measurement: Instead of mouse CD3δ (CD3-humanized mice), C57BL / 6 mice expressing human CD3δ were engineered to express full-length human BCMA (B16 human / BCMA cells), and 0.5×10 6 either B16F10 melanoma cells or 1×10 6 MC38 colon cancer cells engineered to express full-length human BCMA (MC38 / BCMA) were subcutaneously transplanted. Subsequently, mice (n = 7 per group) were immediately administered a CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 0.4 mg / kg, or a BCMA×CD3 (G; REGN5458) bispecific Ab at doses of 0.4 mg / kg or 0.04 mg / kg. These Abs were administered to the mice two more times on days 4 and 7 for a total of three doses, and tumor growth was evaluated throughout the experiment.

[0234] Calculation of syngenic tumor growth and inhibition: To determine tumor volume with an external caliper, the maximum major axis (length in mm) and maximum minor axis (width in mm) were determined. Based on the caliper measurements, tumor volume was calculated using the formula: volume (mm 3 ) = (length × width 2 ) / 2.

[0235] B16 / BCMA tumors and MC38 / BCMA tumors grew in CD3-binding control-treated mice, but BCMA×CD3 REGN5458 was able to suppress the growth of both tumor strains in a dose-dependent manner in vivo. The results are shown in Table 24 below.

Table 27-1

Table 27-2

[0236] Example 17: Epitope mapping of REGN5458 binding to BCMA by hydrogen-deuterium exchange H / D exchange epitope mapping by mass spectrometry (HDX-MS) was performed to determine the amino acid residues of BCMA (recombinant human BCMA, amino acid sequence of SEQ ID NO: 115) that interact with REGN5458 (BCMA×CD3 bispecific antibody). A general description of the H / D exchange method can be found, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259, and Engen and Smith (2001) Anal.Chem.73:256A-265A.

[0237] The HDX-MS experiments were performed on an integrated HDX / MS platform consisting of a Leaptec HDX PAL system for deuterium labeling and quenching, a Waters Acquity M-Class (Auxiliary Solvent Manager) for sample digestion and loading, a Waters Acquity M-Class (μBinary Solvent Manager) for analytical gradient, and a Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

[0238] The labeling solution was prepared as PBS buffer in D2O at pD 7.0 (10 mM phosphate buffer, 140 mM NaCl, and 3 mM KCl, equivalent to pH 7.4 at 25 °C). For deuterium labeling, 10 μL of hBCMA.hFc (REGN2746, 54.5 μM; hBCMA.hFc (Ag-Ab complex) pre-mixed with REGN5458 at a molar ratio of 1:2 or SEQ ID NO: 120) was incubated at 20 °C with 90 μL of D2O labeling solution at various time points during replication (e.g., non-deuterated control = 0 seconds, deuterium-labeled for 5 minutes and 10 minutes). The deuteration reaction was quenched by adding 100 μL of pre-cooled quench buffer (0.5 M TCEP-HCl, 8 M urea, and 1% formic acid) to each sample and incubating at 20 °C for 5 minutes. Next, the quenched samples were injected into a Waters HDX Manager for online pepsin / protease XIII digestion. The digested peptides were separated on a C8 column (1.0 mm × 50 mm, NovaBioassays) with a gradient from 10% to 32% B (mobile phase A: 0.5% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile) over 13 minutes. The eluted peptides were analyzed by LC-MS / MS or LC-MS mode on a Q Exactive HF mass spectrometer.

[0239] LC-MS / MS data of the non-deuterated BCMA samples were searched against a database containing BCMA and its randomized sequences using the Byonic search engine (Protein Metrics). The search parameters (ELN) were set as default using non-specific enzyme digestion and human glycosylation as common variable modifications. The list of identified peptides was then imported into HDX Workbench software (version 3.3) to calculate the deuterium incorporation of each peptide detected by LC-MS from all deuterated samples. For a given peptide, deuterium incorporation (D) and the percentage of deuterium incorporation (D (%)) were calculated using the centroid mass (intensity-weighted average mass) at each time point.

Number

[0240] A total of eight peptides from hBCMA.hFc were identified from both hBCMA.hFc alone and hBCMA.hFc complexed with the REGN5458 sample, representing 100% sequence coverage of hBCMA. The average standard deviation (SD) of all peptides was evaluated to be 1.4% (detailed calculations were defined in ELN and Pascal, BD et al (2012) Journal of the American Society for Mass Spectrometry 23(9):1512-1521). Thus, any peptide showing a different percentage of D incorporation value exceeding 4.2% (3 times the average SD) was defined as being significantly protected. In the case of hBCMA.hFc, the peptide corresponding to amino acids 1-43 of SEQ ID NO: 106 (MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNA; SEQ ID NO: 121) was significantly protected by REGN5458. The protection of these residues by REGN5458 was confirmed using hBCMA.mmH (REGN2744, amino acid sequence of SEQ ID NO: 106).

Table 28

[0241] Example 18: FACS Binding Assay of BCMA×CD3 Bispecific Antibody and Additional BCMA Antibodies against Multiple Myeloma Cell Lines after Overnight Incubation with Anti-BCMA Antibody Flow cytometry analysis was used to determine the effect of overnight incubation of multiple myeloma cell lines with anti-BCMA antibodies on the surface BCMA levels. MM cell lines (H929, Molp8, U266, and MM1.S) were washed twice and cultured at 37°C in R10 medium (RPMI + 10% FBS + pen / strep / glut) containing 66.7 or 667 nM anti-BCMA antibody, DAPT (γ-secretase inhibitor), or medium only. After 18 hours, the wells were washed with cold FACS wash (PBS + 1% filtered FBS) and resuspended in 667 nM of the same anti-BCMA antibody in cold staining buffer (Miltenyi 130-091-221) for 30 minutes on ice. After incubation, the cells were washed twice with cold FACS wash (PBS + 1% filtered FBS) and the bound antibody was detected by further incubating with the appropriate anti-human secondary antibody (anti-hIgG or anti-HIS) for 30 - 45 minutes on ice. After incubation, the cells were washed and resuspended in 200 μL of cold PBS containing 1% filtered FBS and analyzed by flow cytometry on a BD FACS Canto II. The fold increase in staining was calculated by dividing the MFI of the stained cells incubated overnight with BCMA ab or DAPT by the MFI of the stained cells incubated overnight with medium only.

[0242] BCMA is rapidly cleaved from the cell surface by the enzyme γ-secretase. Overnight incubation with a γ-secretase inhibitor such as DAPT prevents cleavage of BCMA and increases the level of BCMA on the cell surface. Tables 26 - 29 report the fold increase in the median fluorescence intensity (MFI) of BCMA in cells incubated overnight with anti-BCMA antibody or DAPT compared to cells incubated with medium only. Overnight incubation with DAPT resulted in a 2.3 - 4-fold, 2.4 - 8.6-fold, 5.3 - 9.0-fold, and 11.9-fold increase in the BCMA levels detected by anti-BCMA antibodies (BCMA×CD3 bispecific R5458, parental BCMA antibody mAb15281, and other in-house BCMA antibodies) in H929, Molp8, U266, and MM.1S, respectively.

[0243] Notably, incubation of MM cell lines overnight with 66.7 or 667 nM REGN5458 or the parental bivalent anti-BCMA antibody mAb21581 was observed to similarly increase the levels of surface BCMA detected by FACS, suggesting that binding of the anti-BCMA antibody prevents cleavage of BCMA by gamma secretase. Antibodies induced different increases in surface BCMA by cell line, with further magnification in Molp8 and MM1S cells compared to H929 or U266. This phenomenon was not limited to REGN5458 as it was also observed with other in-house BCMA antibodies. [Table 29] [Table 30] [Table 31] [Table 32]

[0244] Example 19: Autologous T cell-mediated killing of human and cynomolgus plasma cells in the presence of BCMA×CD3 bispecific antibodies Enriched CD138 by unstimulated autologous T cells + Specific killing of human or cynomolgus plasma cells was evaluated by flow cytometry. Human or cynomolgus bone marrow aspirates and blood were provided within 24 hours of collection. CD138 + plasma cells were enriched from bone marrow by positive selection using the EasySep Human CD138 + Positive Selection Kit according to the manufacturer's instructions. PBMCs from whole blood were separated by density gradient centrifugation. PBMCs were labeled with 1 μM Vybrant CFDA-SE fluorescent tracer dye. After labeling, 1×10 4 cells of enriched CD138 +Plasma cells were seeded in 96-well round-bottom plates at an E:T ratio of 10:1 with Vybrant CFDA-SE-labeled PBMCs, and serial dilutions of REGN5458, a CD3-binding control bsAb, or a BCMA-binding control mAb in complete medium at 37 °C for 72 hours. At the end of the culture, viable CD138 + plasma cells were analyzed by flow cytometry using a fixable LIVE / DEAD dye and a plasma cell-specific cell surface marker. The viability was normalized to the control condition (plasma cells in the presence of PBMCs only). T cell activation was evaluated by flow cytometry. Activation was reported as the percentage of CD2 + / CD4 + or CD2 + / CD8 + / CD16 - T cells. The percentage of T cell activation was normalized to the control condition (plasma cells in the presence of PBMCs only).

[0245] In vitro studies evaluated the effect of REGN5458 or negative controls (BCMA-binding control mAb or CD3-binding control bsAb) on primary human and cynomolgus monkey T cell activation and autologous plasma cell cytotoxicity. The EC 50 values for cytotoxicity and T cell activation percentages for each donor are summarized in Table 30.

[0246] REGN5458 mediated the cytotoxicity of primary human plasma cells from Donors 1 and 2 in a concentration-dependent manner in the presence of autologous T cells, with EC 50 values of 42.8 pM and 191 pM, respectively, and maximum percentages of cytotoxicity of 91% and 89%, respectively. In parallel, REGN5458 mediated T cell activation in a concentration-dependent manner in the presence of human plasma cells from Donors 1 and 2, with EC + values for CD8 50 T cell activation of 214 pM and 860 pM, respectively, and maximum percentages of CD8 + T cell activation of 2% and 36%, respectively. Cytotoxicity of plasma cells in both donors and CD8+ An increase in T cell activation was observed at nanomolar concentrations of the CD3-binding control. No effect on cytotoxicity or T cell activation was observed with the BCMA-binding control at any of the concentrations tested with either donor.

[0247] REGN5458 mediated cytotoxicity of primary cynomolgus plasma cells in a concentration-dependent manner in both donors, with an EC 50 that was calculated for donor 1, but an EC 50 could not be determined for donor 2. In both donors, treatment with REGN5458 increased plasma cell cytotoxicity (the maximum percentage of cytotoxicity in donors 1 and 2 was 94% and 91%, respectively). In parallel, REGN5458 mediated T cell activation in a concentration-dependent manner in the presence of cynomolgus plasma cells from donors 1 and 2, with EC + values for CD4 50 T cell activation of 28.1 nM and 18.1 nM, respectively, and EC + values for CD8 50 T cell activation of 22.4 nM and 76.7 nM. The resulting maximum percentage of T cell activation was 9% and 16% in CD4 + T cells and 12% and 17% in CD8 + T cells in donors 1 and 2, respectively.

[0248] Target cell death was not observed with the BCMA-binding control at any concentration tested with any of the cell lines evaluated. Some target cell death and T cell activation in the presence of plasma cells from donor 2 were observed with the nanomolar concentration of the CD3-binding control.

Table 33

[0249] Example 20: Anti-BCMA × anti-CD3 bispecific antibody acts synergistically with anti-PD-1 antibody to enhance antitumor effect in vivo To determine whether a BCMA×CD3 bispecific antibody (Ab) synergizes with PD-1 blockade to provide an excellent anti-tumor effect in vivo, a syngeneic tumor study was conducted in mice expressing human CD3. The results indicate that the combination of REGN5458 and PD-1 blockade provides an anti-tumor effect superior to that of REGN5458 or PD-1 blockade alone.

[0250] Syngeneic tumor transplantation and measurement: C57BL / 6 mice expressing human CD3deg were subcutaneously transplanted with 1×10 6 individual MC38 colon cancer cells engineered to express full-length human BCMA (MC38 / BCMA), instead of mouse CD3deg (CD3-humanized mice). Tumors were allowed to establish for 3 days, at which point mice (n = 6 or 7 per group) were administered either 4 mg / kg of a surrogate anti-mouse PD-1 antibody (clone RPM1-14), or 4 mg / kg of an isotype control Ab (clone 2A3), along with 0.4 mg / kg of a CD3-binding control bispecific Ab (G; H4sH17664D), and BCMA×CD3 (G; REGN5458) at a dose of 0.04 mg / kg or 0.24 mg / kg. The specific treatment groups are shown in Table 31 below.

Table 34

[0251] These Abs were administered to the mice at a total of 3 doses, 2 more times on days 7 and 11, and tumor growth was evaluated throughout the experiment.

[0252] Calculation of syngenic tumor growth and inhibition: To determine tumor volume using an external caliper, the maximum major axis (length in mm) and maximum minor axis (width in mm) were determined. Based on the caliper measurements, tumor volume was calculated using the formula: volume (mm 3 ) = (length × width 2 ) / 2.

[0253] The results show that the combination of REGN5458 and PD-1 blockade provides a superior anti-tumor effect compared to REGN5458 or PD-1 blockade alone. In particular, the results show that on day 24 (the last day on which data were collected for all treatment groups), the combination of the BCMA×CD3 bispecific antibody and the anti-PD-1 antibody resulted in a statistically significant synergistic treatment effect in inhibiting tumor growth (Table 32, 0.04 mg / kg of BCMA×CD3 and 4 mg / kg of anti-PD-1). On day 24, using two-way ANOVA, p<0.0001 between (i) the combination of REGN5458 (0.04 mg / kg)+isotype and REGN5458 (0.04 mg / kg)+anti-PD-1 antibody (group 3 vs. group 4), (ii) the combination of REGN5458 (0.24 mg / kg)+isotype and REGN5458 (0.24 mg / kg)+anti-PD-1 antibody (group 5 vs. group 6), and (iii) anti-PD-1 and the combination of REGN5458 (0.04 mg / kg)+anti-PD-1 antibody (group 2 vs. group 6). On day 24, using two-way ANOVA, p = 0.0005 between anti-PD-1 and the combination of REGN5458 (0.04 mg / kg)+anti-PD-1 antibody (group 2 vs. group 4). Increasing the dose of the BCMA×CD3 bispecific antibody (0.24 mg / kg) in combination with PD-1 blockade resulted in tumor inhibition comparable to that of the low-dose bispecific antibody and PD-1 blockade in this experiment. The demonstrated synergistic effect with the low-dose bispecific antibody is advantageous because the use of low doses reduces the risk of adverse side effects. Similarly, the combination of the BCMA×CD3 bispecific antibody and the anti-PD-1 antibody showed a synergistic treatment effect at both doses (0.04 mg / kg and 0.24 mg / kg) of the bispecific antibody in the number of mice without tumors at the end of the experiment (day 28), as shown in Table 33.

Table 35-1

Table 35-2

Table 35-3

Table 35-4

Table 36

[0254] Example 21: The anti-BCMA × anti-CD3 bispecific antibody acts synergistically with the anti-PD-1 antibody to enhance the antitumor effect in vivo. Similar results were obtained in a second experiment that was identical to that discussed above in Example 20, except that the number of mice per group was 10 and the high dose of BCMA × CD3 REGN5458 was 0.4 mg / kg. The specific treatment groups in the second experiment are shown in Table 34 below.

Table 37

[0255] The results show that the combination of REGN5458 and PD-1 blockade provides an antitumor effect superior to that of REGN5458 or PD-1 blockade alone. In particular, the results showed that on day 21 (the last day when data for all treatment groups were collected), the combination of the BCMA×CD3 bispecific antibody and the anti-PD-1 antibody resulted in a synergistic therapeutic effect in inhibiting tumor growth (Table 35, 0.04 mg / kg of BCMA×CD3 and 4 mg / kg of anti-PD-1). On day 21, using two-way ANOVA, p<0.0001 was found between (i) the combination of REGN5458 (0.04 mg / kg) + isotype and REGN5458 (0.04 mg / kg) + anti-PD-1 antibody (group 3 vs. group 4), (ii) anti-PD-1 and the combination of REGN5458 (0.04 mg / kg) + anti-PD-1 antibody (group 2 vs. group 4), and (iii) anti-PD-1 and the combination of REGN5458 (0.4 mg / kg) + anti-PD-1 antibody (group 2 vs. group 6). As discussed above in Example 20, increasing the dose of the BCMA×CD3 bispecific antibody (0.4 mg / kg) in combination with PD-1 blockade resulted in tumor inhibition comparable to that of the low-dose bispecific antibody in combination with PD-1 blockade in this experiment. The demonstrated synergistic effect with the low-dose bispecific antibody is advantageous because the use of a low dose reduces the risk of adverse side effects. Similarly, the combination of the BCMA×CD3 bispecific antibody and the anti-PD-1 antibody showed a synergistic therapeutic effect at both doses (0.04 mg / kg and 0.4 mg / kg) of the bispecific antibody in terms of the number of mice without tumors at the end of the experiment (day 25), as shown in Table 36.

Table 38-1

Table 38-2

Table 38-3

Table 38-4

Table 39

[0256] Example 22: Method for treating multiple myeloma with an anti-BCMA × anti-CD3 bispecific antibody In patients with relapsed or refractory multiple myeloma who have exhausted all treatment options, including proteasome inhibitors, immunomodulatory drugs, and anti-CD38 antibody therapy, a phase 1 / 2 trial of REGN5458 (an anti-BCMA × anti-CD3 bispecific antibody) was conducted to evaluate its safety, tolerability, preliminary antitumor activity, and pharmacokinetics (PK), and it showed a significant clinical benefit.

[0257] Patients with treatment-refractory and advanced multiple myeloma, including those with extramedullary (outside the bone marrow) and non-secretory (not secreting detectable biomarkers) cancerous plasma cells, are being studied as part of the REGN5458 program.

[0258] In clinical trials of multiple myeloma, treatment evaluation is based on the reduction of myeloma protein levels and the eradication of myeloma cells. The evaluation of the response of myeloma proteins is based on the reduction in the level of monoclonal (M) protein, a biomarker contained in the urine and blood of patients and used to determine the extent of myeloma disease. A partial response (PR) is defined as a reduction of 50% or more in serum / urine M protein, or a reduction of 50% or more in the difference between the free light chain (FLC) levels involved and those not involved, and a reduction of 50% or more in soft tissue plasmacytoma. A very good partial response (VGPR) is defined as a reduction of 90% or more in serum / urine M protein, or a reduction of 90% or more in the difference between the FLC levels involved and those not involved, a reduction of 90% or more in soft tissue plasmacytoma, and the detection of M protein by immunofixation rather than electrophoresis. A complete response (CR) is defined as the negative detection of M protein by immunofixation in serum and urine, the complete disappearance of soft tissue plasmacytoma, and less than 5% plasma cells in the bone marrow aspirate. A stringent complete response is a complete response (as described above) and a normal FLC ratio ( K κ / λ ratio ≤ 4:1 or KAnd in the case of λ patients, it is defined as ≥1:2). Minimal residual disease (MRD), which reflects the eradication of myeloma cells, is measured separately from the M protein, and MRD negativity is defined as the absence of cancer plasma cells in 100,000 bone marrow cells.

[0259] Objective: Both primary and secondary evaluation items are investigated.

[0260] The main objectives of this study are as follows. (1) Phase 1 part of the study: Evaluate safety, tolerability, and dose-limiting toxicity (DLT), and determine the recommended Phase 2 dose regimen (RP2DR) (maximum tolerated dose regimen [MTDR] or biologically effective dose regimen [BEDR]) of REGN5458 as a single-agent therapy in patients with relapsed or refractory multiple myeloma (MM) who have exhausted all treatment options expected to provide a meaningful clinical benefit. The determination of RP2DR is based on a review of non-clinical and all clinical data, including data on safety, pharmacokinetics (PK), PK / PD (pharmacokinetics / pharmacodynamics) relationships, and efficacy. (2) Phase 2 part of the study: Evaluate the preliminary antitumor activity of REGN5458 as measured by the objective response rate (ORR).

[0261] The second objective of the study is as follows (Phase 1 and Phase 2 parts). (1) Evaluate the preliminary antitumor activity of REGN5458 as measured by duration of response (DOR), progression-free survival (PFS), minimal residual disease (MRD) status, and overall survival (OS), (2) Evaluate the (PK) characteristics of REGN5458, (3) Characterize the immunogenicity of REGN5458, (4) Phase 1 part only: Evaluate the preliminary antitumor activity of REGN5458 as measured by ORR, as well as (5) Phase 2 part only: Evaluate the safety and tolerability of REGN5458.

[0262] Study Design: The Phase 1 portion follows a standard 4 + 3 dose-escalation design with a 28-day DLT observation period to evaluate the safety of REGN5458 and select the RP2DR (defined as MTDR or BEDR) of REGN5458 as a single agent.

[0263] The Phase 2 portion is initiated at the time when the RP2DR is determined to further evaluate the preliminary anti-tumor activity, safety and tolerability, PK characteristics, and biomarker response of patients treated with REGN5458 as a single agent.

[0264] Each patient receives a 16-week (QW) infusion of REGN5458 according to the assigned dosing regimen, followed by 12 additional doses of REGN5458 every 2 weeks (Q2W).

[0265] Each patient receives a first dose of REGN5458 and then, if the first dose is well tolerated, the nominal dose. The first dose of REGN5458 is administered as a divided (split) infusion (preferably on 2 consecutive days, but with an interval within 3 days) at the assigned dose. If this first dose is appropriately tolerated, the patient receives a higher nominal dose on Day 14 (preferably on consecutive days, but within 3 days) as a divided infusion at the assigned dose, and from Week 3 onwards, the nominal dose is administered as a single infusion.

[0266] The dose escalation schema provides an increase of approximately threefold in the nominal dose of each successive dose cohort compared to the nominal dose of the previously evaluated dose cohort. Similarly, it provides an increase of approximately threefold in the primary dose in the first week in each successive dosing cohort. However, during the dose-limiting toxicity (DLT) observation period, if DLT occurs in one patient, or if grade 2 or higher adverse events occur in two or more patients in a dose cohort (excluding grade 2 or higher AEs clearly unrelated to the investigational drug), and the dose regimen is still judged to be acceptable, the escalation of the primary and nominal doses will be no more than twofold (i.e., a 100% increase) of the respective primary and nominal doses of the previously evaluated dose cohort. For example, if one DLT is observed in six or seven patients in DL2 (primary dose of 3 mg and nominal dose of 10 mg) and the dose regimen is judged to be acceptable, the primary dose in the next dose cohort (i.e., DL3) will be 6 mg and the nominal dose will be 20 mg, and subsequent dose escalations in successive dose cohorts will be no more than twofold of the previous dose cohort.

[0267] Study period: The planned period of study for each patient will be up to approximately 24 months, including a screening period (maximum 28 days), a treatment period (40 weeks), and a core follow-up period (approximately 24 weeks), followed by an extended follow-up period of approximately 36 weeks to determine permanent clinical activity and safety (a total follow-up period of approximately 60 weeks).

[0268] Study population: Phase 1 portion: According to a 4+3 design, up to seven DLT - evaluable patients can be enrolled in each dose cohort. If the dosing regimen at the designated dose level (DL) is judged to be acceptable and does not exceed the maximum tolerated dose (MTD), additional enrollment of up to three patients per DL can be initiated (a total of up to 10 patients per DL). The actual sample size of these dose escalation cohorts will vary depending on the number of patients in whom DLT is recorded, the number of DLs implemented, and the number of patients who drop out.

[0269] Part of Phase 2: Approximately 10 - 14 patients who can be evaluated for safety and efficacy. The analysis of these patients is combined with the analysis of 6 - 10 patients treated with RP2DR in the Phase 1 part, resulting in a total of 20 patients treated with RP2DR. In this study, MM patients who have exhausted all treatment options expected to provide a meaningful clinical benefit through any of disease recurrence, treatment-resistant disease, or treatment intolerance or refusal are enrolled. Further, each patient needs to have progressed after at least 3 prior treatment lines, including anti-CD38 antibody, proteasome inhibitor, and immunomodulatory drug (IMiD). If a patient has been previously treated with an anti-CD38 antibody and is shown to be refractory to both IMiD and proteasome inhibitor, the patient may be eligible for the study even if less than 3 prior treatment lines have been administered. Refractory disease is defined as lack of response or recurrence of MM within 60 days from the last treatment.

[0270] Inclusion Criteria - Patients must meet the following criteria to be eligible for inclusion in the trial. 1. 18 years of age or older 2. Eastern Cooperative Oncology Group (ECOG) Performance Status ≤ 1 Individual cases of patients with ECOG Performance Status of 2, where improvement in ECOG status is expected as a result of effective treatment, can be discussed with the medical monitor for potential inclusion. 3. Confirmation of the diagnosis of active MM according to the International Myeloma Working Group (IMWG) diagnostic criteria. 4. Patients must have symptomatic myeloma with myeloma-related organ damage or tissue dysfunction (such as hypercalcemia, renal insufficiency, osteolytic lesions, or anemia) at the start of the study. 5. Patients must have measurable myeloma as measured by either serum or urine assessment of monoclonal components or serum free light chain (FLC) analysis. Measurable disease is defined as one or more of the following. a. Serum M protein ≥ 1 g / dL b. Urinary M protein ≥ 200 mg / 24 hours, and / or c. FLC assay with abnormal serum FLC ratio and FLC level ≥ 10 mg / dL - Patients with immunoglobulin A (IgA) myeloma but without measurable M protein may be enrolled if their quantitative IgA levels are elevated and can be followed up long-term - Patients with non-secretory MM may be considered for enrollment after discussion with the sponsor of the trial, including the feasibility of response assessment according to the IMWG guidelines 6. Disease progression according to IMWG criteria 7. MM patients who have exhausted all treatment options expected to provide a meaningful clinical benefit through any of disease recurrence, treatment-refractory disease, or treatment intolerance or refusal, including any of the following: a. At least three lines of prior or subsequent progression, or intolerance to treatments including proteasome inhibitors, IMiDs, and anti-CD38 antibodies, or b. Disease that has progressed during or after anti-CD38 antibody treatment and is "doubly refractory" or treatment-intolerant to proteasome inhibitors and IMiDs. Anti-CD38 antibodies may have been administered alone or in combination with another agent such as a proteasome inhibitor. Refractory disease is defined as lack of response or recurrence within 60 days of the last treatment 8. Appropriate hematological function measured as follows: a. Platelet count > 50 × 10 9 / L. To meet this platelet eligibility requirement, the patient may not have received a platelet transfusion within 7 days b. ANC > 1.0 × 10 9 / L. To meet this absolute neutrophil count eligibility requirement, the patient may not have received granulocyte colony-stimulating factor (G-CSF) within 2 days c. Hemoglobin > 8.0 g / dL 9. Appropriate liver function defined as follows: a. Total bilirubin ≤ 1.5 × ULN b. Transaminases (ALT, AST) ≤ 2.5 × ULN c. Alkaline phosphatase ≤ 2.5 × ULN - Patients with Gilbert's syndrome do not need to meet this total bilirubin requirement unless the total bilirubin changes from the baseline value. 10. Serum creatinine clearance by Cockcroft - Gault > 30 mL / min - If the measured creatinine clearance (based on 24 - hour urine collection or other reliable methods) is > 30 mL / min, patients with a creatinine clearance by Cockcroft - Gault that does not meet the eligibility criteria may be eligible for enrollment. 11. If previously treated with CAR T - therapy or gene therapy products, the patient needs to have recovered from the toxicity of this therapy 12. At least 6 - month life expectancy 13. Ability to attend the clinic and proceed with and comply with study - related procedures, including serial evaluation of bone marrow according to the protocol schedule - Bone marrow aspiration and biopsy, or other tissues infiltrated by malignant plasma cells must be provided for screening for the evaluation of BCMA levels of malignant cells, but demonstration of BCMA levels before enrollment is not required. 14. Provide an informed consent signed by the study patient 15. Ability to understand the study - related questionnaire and fill in all items.

[0271] Exclusion criteria - Patients who meet any of the following criteria are excluded from the clinical trial. 1. Presence of plasma cell leukemia, Waldenström macroglobulinemia (lymphoplasmacytic lymphoma), or POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes) 2. Patients with known MM brain lesions or meningeal disorders associated with MM (if CNS myeloma is suspected, it is necessary to exclude by X - ray imaging and / or lumbar puncture as needed) 3. History of neurodegenerative conditions or CNS movement disorders 4. Ejection fraction < 40% by echocardiogram or multiple gated acquisition (MUGA) scan. 5. Continuous systemic corticosteroid treatment with prednisone or anti-inflammatory equivalents at more than 10 mg per day within 72 hours from the start of the investigational drug. 6. Vaccination within 28 days before the first administration of the investigational drug with a vector that may be replicative. 7. Treatment with any systemic, standard or investigational anti-myeloma therapy within 5 half-lives or within 28 days before the first administration of the investigational drug, whichever is shorter. 8. Prior treatment with anti-BCMA antibodies (including antibody-drug conjugates or bispecific antibodies) or BCMA-directed CAR T therapy. 9. Hospitalization or treatment with IV anti-infectives for an infection within 2 weeks from the first administration of the investigational drug. 10. Uncontrolled infection due to human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV) infection; or other uncontrolled infections. a. Patients with HIV in which the infection is controlled (undetectable viral load and CD4 count > 350 cells / microliter spontaneously or on a stable anti-viral regimen) are permitted. b. Patients with hepatitis B (hepatitis B surface antigen positive [HepBsAg+]) in which the infection is controlled (serum HBV DNA polymerase chain reaction [PCR] below the detection limit and receiving anti-viral therapy for hepatitis B) are permitted. c. Patients who are HCV antibody positive (HCV Ab+) and have a controlled infection (undetectable HCV RNA by PCR, either spontaneously or according to the previous course of successful anti-HCV therapy) are permitted. 11. A documented history of severe allergic or anaphylactic reactions due to previous antibody treatment. - For this purpose, a severe allergic reaction is defined as meeting the criteria for severity of CTCAE v5.0 grade 3 or grade 4 (i.e., characterized by bronchospasm; or life-threatening consequences; or requiring IV intervention, other emergency intervention, or hospitalization for clinical sequelae), or as requiring a visit to the emergency treatment room. 12. History of hypersensitivity to any compound in the tetracycline antibiotic group (caution due to the possible presence of trace components in the investigational drug material) 13. Confirmed hypersensitivity to both allopurinol and rasburicase 14. History of allogeneic stem cell transplantation at any time, or history of autologous stem cell transplantation within 12 weeks from the start of the test treatment 15. Members of the clinical site trial team or their close relatives, unless pre-approved by the trial sponsor 16. Women with a positive serum beta-human chorionic gonadotropin (β-hCG) pregnancy test and a possibility of childbirth (WOCBP) are ineligible for this study. 17. Patients dedicated to the facility by an order issued by either the judicial or administrative authority 18. Women who are pregnant or breastfeeding 19. Women* or men** who are likely to give birth and do not wish to practice a highly effective contraceptive method at least 6 months before the first administration / start of the first treatment, during the trial, and after the last administration. *Highly effective contraceptive methods for women include: a. Stable use of combined (estrogen and progestogen-containing) hormonal contraception (oral, vaginal, transdermal) or progestogen-only hormonal contraception (oral, injection, implant) related to ovulation inhibition for two or more menstrual cycles before screening, b. Intrauterine device (IUD), intrauterine hormone-releasing system (IUS) c. Bilateral tubal ligation d. Vasectomized partner (when the vasectomized male partner is the only sexual partner of the study participant and the partner has obtained a medical evaluation of the surgical success of the operation) e. and / or sexual abstinence†,‡. Women who are potentially capable of childbearing are defined as fertile women from menarche until menopause, unless permanently infertile. Permanent contraceptive methods include hysterectomy, bilateral salpingectomy, bilateral oophorectomy, etc. The postmenopausal state is defined as the absence of menstruation for 12 months without alternative medical causes. High follicle-stimulating hormone (FSH) levels in the postmenopausal range can be used to confirm the postmenopausal state in women not using hormonal contraceptives or hormone replacement therapy. However, a single FSH measurement is insufficient to determine the occurrence of the postmenopausal state if there has been no amenorrhea for 12 months. **Except for vasectomy (medically evaluated for surgical success), highly effective contraceptive means for men include condoms or sexual abstinence†,‡. †Sexual abstinence is considered a highly effective method only when defined as refraining from heterosexual intercourse throughout the entire period of risk associated with the investigational treatment. The reliability of sexual abstinence needs to be evaluated in relation to the duration of the clinical trial and the patient's preferred normal lifestyle. ‡Periodic abstinence (calendar, symptothermal method, post-ovulation method), withdrawal (coitus interruptus), spermicides only, and lactational amenorrhea method (LAM) are not acceptable contraceptive methods. Female and male condoms should not be used together.

[0272] Treatment: REGN5458 for intravenous infusion is provided to the investigator as a liquid in a sterile disposable vial. Each vial contains REGN5458 at a concentration of 10 mg / mL.

[0273] A pharmacist or other qualified individual is identified at each site and the administration preparation of REGN5458 is carried out.

[0274] For the single dose and the first nominal dose, treatment is administered as two separate 4-hour infusions, preferably on two consecutive days if possible, but with an interval within 3 days (e.g., day 1 and day 2 of week 1). The first nominal dose administered as a single infusion is given over 4 hours. If this infusion is well tolerated without any grade of CRS or IRR event, subsequent REGN5458 infusions may be shortened to 2 hours according to the clinical judgment of the investigator. If this 2-hour REGN5458 infusion is well tolerated without any grade of CRS or IRR event, subsequent REGN5458 infusions may be shortened to 1 hour according to the clinical judgment of the investigator. Thereafter, each dose of REGN5458 can be administered with an IV infusion period associated with the absence of CRS or IRR events.

[0275] Treatment after the patient has received the single dose and the first nominal dose can be administered as a single infusion. The principal investigator can choose to divide the dose into two separate infusions over 2 days (preferably consecutive, but with an interval within 3 days).

[0276] The dose of REGN5458 received by each patient follows the assignment of the DL cohort. The dose administered at each DL is a fixed dose and does not depend on the patient's weight or body surface area (BSA).

[0277] Investigation of evaluation items: In the Phase 1 part, the primary evaluation items of the investigation are as follows. (1) The incidence of DLT from the first administration to the end of the DLT observation period, and (2) The incidence and severity of treatment-emergent adverse events (TEAE) and adverse events of special interest (AESI) during the REGN5458 treatment period and up to 14 months after the last dose.

[0278] In the Phase 2 part, the primary evaluation item of the study is the ORR measured using the International Myeloma Working Group (IMWG) criteria up to 14 months after the last dose.

[0279] The secondary evaluation items are as follows (for the Phase 1 and Phase 2 parts). (1) The concentration of REGN5458 in serum over time, (2) The incidence rate of anti-drug antibodies (ADA) over time due to treatment with REGN5458, (3) DOR using the IMWG criteria up to 14 months after the last dose, (4) PFS measured using the IMWG criteria up to 14 months after the last dose, (5) The proportion of MRD-negative status using the IMWG criteria up to 14 months after the last dose, (6) OS up to 14 months after the last dose (7) Only for the Phase 1 part - ORR measured using the IMWG criteria up to 14 months after the last dose, and (8) Only for the Phase 2 part - The incidence rate and severity of TEAE and AESI during the REGN5458 treatment period up to 14 months after the last dose.

[0280] Procedures and assessments: Screening only: Demographics, complete physical examination, height, medical and oncology history, Revised International Staging System (ISS) stage (including chromosomal abnormalities and β2-microglobulin), brain magnetic resonance imaging (MRI), echocardiogram or multigated acquisition scan (MUGA), HIV / HBV / HCV tests, prothrombin time (PT) (International Normalized Ratio [INR]), and aPTT / PTT.

[0281] Safety: Vital signs, limited physical examination, weight, electrocardiogram, ECOG status, laboratory evaluations, adverse events (AE), concomitant medications (CM).

[0282] Validity: Serum protein electrophoresis (SPEP), urine protein electrophoresis (UPEP), 24-hour urine sample, serum and urine immunofixation, free light chain (FLC) assay, bone marrow aspiration / biopsy, immunoglobulin quantification (immunoglobulin A [IgA], immunoglobulin M [IgM], immunoglobulin G [IgG], immunoglobulin D [IgD], immunoglobulin E [IgE]), evaluation of extramedullary plasmacytoma (if applicable, measurement by clinical examination and / or radiological examination [biopsy is optional]), skeletal evaluation.

[0283] Blood samples are collected for drug concentration analysis in serum and ADA evaluation.

[0284] Statistical plan: Phase 1 part: According to the 4 + 3 design, up to 7 DLT-evaluable patients can be enrolled in each dose cohort. If the designated DL dosing regimen is determined to be acceptable and does not exceed the MTD, additional enrollment of up to 3 patients per DL can be initiated (up to 10 patients in total per DL). The actual sample sizes of these dose-escalation cohorts will vary depending on the number of observed patients in whom DLTs were recorded, the number of DLs implemented, and the number of patients who dropped out.

[0285] Phase 2 part: The sample size of 20 patients is determined based on clinical considerations to further investigate the safety and preliminary antitumor activity of REGN5458 in patients treated with RP2DR. In Phase 1, 6 - 10 patients are dosed with the investigational drug with RP2DR, and these contribute to the total sample size of 20 patients for analysis. The remaining 10 - 14 patients who are evaluable for safety and efficacy are enrolled in the Phase 2 part.

[0286] Conditions for Temporary Suspension of Registration and Phase 2 of Safety Review: To further evaluate the tolerability in RP2DR, estimate the proportion of the cumulative number of patients who experience unacceptable toxicity (cUT) among patients treated with RP2DR in the Phase 1 and Phase 2 portions. The stopping boundary is included based on the frequentist interval that utilizes the lower limit of the one-sided 80% confidence interval (CI). If the lower limit of the one-sided 80% CI of the estimated cUT excludes 20%, the registration to the Phase 2 portion is temporarily suspended.

[0287] This evaluation is performed on the first 12 patients treated with RP2DR in the Phase 1 and Phase 2 portions. The evaluation is repeated for the first 16 patients treated with RP2DR in the Phase 1 and Phase 2 portions (i.e., when 4 additional patients are registered). If the proportion of cUT excludes 20% (i.e., 4 or more patients out of the first 12 patients treated with RP2DR have unacceptable toxicity, or 5 or more patients out of the first 16 patients have unacceptable toxicity), further registration to the Phase 2 portion is temporarily suspended. If it is observed that 4 patients have unacceptable toxicity before registering 12 patients, or if it is observed that 5 patients have unacceptable toxicity before registering 16 patients, further registration to the Phase 2 portion is also temporarily suspended.

[0288] Preliminary results: The median of the 5 previous lines of systemic therapy (range, 2 - 17; 49 patients (median age 64; 31% 70 years or older) with 33 patients (67.3%) had previously received autologous stem cell transplantation. At the start of the trial, multiple myeloma immunological subtypes included immunoglobulin (Ig) G (21 patients, 42.9%), IgA (11 patients, 22.4%), lambda light chain (11 patients, 24.4%), and kappa light chain (6 patients, 12.2%). All patients were refractory to anti - CD38 antibodies, all % were at least triple - refractory, 30.6% were quadruple - refractory, and 57.1% were quintuple - refractory. 80% of patients were refractory to carfilzomib and 92% were refractory to pomalidomide. Patients were treated in a cohort receiving REGN5458 at 3 mg - 96 mg at 6 dose levels. The median follow - up period was 2.63 (range 0.5 - 13.4) months. The majority (63%) had a Revised International Staging System (ISS) stage of II.

[0289] The most common adverse events (AEs) were cytokine release syndrome (CRS; 38.8%), anemia (36.7%), fatigue (34.7%), nausea (31%), fever (31%), and back pain (27%). Grade 3 or higher AEs occurred in 43% of patients, the most common being anemia (22.4%), neutropenia (14.3%), and lymphopenia (12.2%). The most common serious adverse events were due to infections (20.4%) and CRS (12.2%). No patients experienced grade 3 or higher CRS, and less than 40% of patients experienced CRS. CRS mainly occurred in the first week of treatment, grade 1 in 33% of patients and grade 2 in 6% of patients. No correlation was observed between CRS and dose level. No patients experienced grade 3 or higher neurotoxicity.

[0290] The objective response rate (ORR) was 38.8% at all dose levels (29.2% at dose levels 1 - 3, 41.2% at dose levels 4 and 5, and 62.5% at dose level 6), 95% of responders achieved at least a very good partial response (VGPR), and 42.1% had a complete response (CR) or a stringent CR. Four out of seven (57%) evaluable patients achieved minimal residual disease (MRD) negativity at a sensitivity of 10 -5 . When evaluated by immunohistochemistry, tumor response was not affected by BCMA expression in core biopsies. A total of 63.2% of responders had a duration of response (DOR) of 4 months or more, 52.6% of responders had a DOR of 6 months or more, and 36.8% of responders had a DOR of 8 months or more. The median duration of the observed response was 6.01 months. Responses occurred early (mostly by week 4) and deepened over time. Of the responding patients followed for 6 months or more, 83% (10 / 12) showed a continuous response for up to 13 months. To date, 74% of responders are receiving continuous treatment. The ORR for patients with extramedullary plasmacytoma (EMP) was 14.3%, while the ORR for patients without EMP was 45%, including 20% complete or stringent complete responses and 22.5% very good partial responses. The ORR for patients with less than 50% myeloma was 71.4%, while the ORR for patients with 50% or more myeloma was 9.1%. Among patients with less than 50% myeloma, 35.7% achieved a stringent complete response and 35.7% achieved a very good partial response. A significant improvement in overall health status / quality of life was observed at week 4 and maintained until week 24 (to date). When evaluated by immunohistochemistry, tumor response was not correlated with BCMA expression. A summary of the observed responses is shown in Table 37 below.

Table 40

[0291] Accordingly, the bispecific antibodies described herein can be used at a dose of at least 3 mg administered once a week, either as a divided dose (e.g., in the first and second weeks) or a single injection (e.g., after the third week), to treat human subjects suffering from BCMA-expressing cancers such as multiple myeloma, particularly those who are resistant to previous treatments (e.g., triple, quadruple, or quintuple resistant), or who have relapsed after previous treatment.

[0292] The invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

Claims

1. A pharmaceutical composition comprising a bispecific antibody for treating multiple myeloma in a human subject in need of treatment for multiple myeloma, wherein the bispecific antibody comprises a first heavy chain paired with a common light chain comprising a first antigen-binding domain that specifically binds to human B cell maturation antigen (BCMA), and a second heavy chain paired with a common light chain comprising a second antigen-binding domain that specifically binds to human CD3, wherein the first heavy chain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 66 and a human IgG heavy chain constant region, the second heavy chain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 90 and a human IgG heavy chain constant region, and the common light chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82 and a human light chain constant region, the pharmaceutical composition is for use in an administration regimen, the administration regimen comprising: (a) a single administration of 5 mg of the bispecific antibody administered to the subject during the first week of the administration regimen; (b) a single administration of 25 mg of the bispecific antibody administered to the subject during the second week of the administration regimen; and (c) a single administration of 200 mg of the bispecific antibody administered to the subject during the third week of the administration regimen.

2. The pharmaceutical composition according to claim 1, wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 126, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 127, and the common light chain comprises the amino acid sequence of SEQ ID NO:

129.

3. The pharmaceutical composition according to claim 1 or 2, wherein the multiple myeloma is relapsed or refractory multiple myeloma.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the subject has received at least three prior treatments for multiple myeloma.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the subject has received at least four prior treatments for multiple myeloma.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the subject has received at least five prior treatments for multiple myeloma. **Claim 7**: The pharmaceutical composition according to any one of claims 1 to 6, wherein the subject has been previously treated with an anti-CD38 antibody therapy. **Claim 8**: The pharmaceutical composition according to claim 7, wherein the anti-CD38 antibody therapy comprises daratumumab or isatuximab. **Claim 9**: The pharmaceutical composition according to any one of claims 1 to 8, wherein the subject has been previously treated with a proteasome inhibitor. **Claim 10**: The pharmaceutical composition according to claim 9, wherein the proteasome inhibitor comprises bortezomib, carfilzomib, or ixazomib. **Claim 11**: The pharmaceutical composition according to any one of claims 1 to 10, wherein the subject has been previously treated with an immunomodulatory drug (IMiD). **Claim 12**: The pharmaceutical composition according to claim 11, wherein the immunomodulatory drug (IMiD) comprises lenalidomide or pomalidomide. **Claim 13**: The pharmaceutical composition according to any one of claims 1 to 12, wherein the subject is diagnosed with a multiple myeloma immunotype selected from immunoglobulin G, immunoglobulin A, lambda light chain, or kappa light chain. **Claim 14**: The pharmaceutical composition according to any one of claims 1 to 13, wherein the subject has extramedullary plasmacytoma. **Claim 15**: The pharmaceutical composition according to any one of claims 1 to 14, wherein the dosing regimen further comprises a single dose of 200 mg of the bispecific antibody administered once a week to the subject during the once-weekly dosing period of the dosing regimen. **Claim 16**: The pharmaceutical composition according to claim 15, wherein the once-weekly dosing period begins during the third week of the dosing regimen and continues for a total of at least 12 weeks during the once-weekly dosing period of the dosing regimen. **Claim 17**: The pharmaceutical composition according to claim 16, wherein the dosing regimen further comprises a single dose of 200 mg of the bispecific antibody administered to the subject once every two weeks after the once-weekly dosing period of the dosing regimen, and continues for at least 10 weeks during the alternate-week dosing period of the dosing regimen. **Claim 18**: The pharmaceutical composition according to claim 17, wherein the dosing regimen further comprises a single administration of 200 mg of the bispecific antibody administered to the subject once every four weeks after the bi-weekly dosing period of the dosing regimen. **Claim 19**: The pharmaceutical composition according to any one of claims 1 to 18, wherein the human IgG heavy chain constant region is isotype IgG1 or isotype IgG4.

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