Methods of treating multiple myeloma with bispecific Anti-BCMA×Anti-CD3 antibodies

Abstract

To provide a method for treating BCMA+ cancer.SOLUTION: B-cell maturation antigen (BCMA) is expressed on malignant plasma cells. The present invention provides a method for treating multiple myeloma using bispecific antibodies (bsAbs) that bind to both BCMA and CD3 and activate T cells via the CD3 complex in the presence of BCMA-expressing tumor cells. In certain embodiments, the bispecific antigen-binding molecules of the present invention are capable of inhibiting the growth of tumors expressing BCMA.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Sequence Listing Reference This application incorporates by reference the Sequence Listing filed in computer readable format 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 their use. [Background technology]

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

[0004] CD3 is a homodimeric or heterodimeric antigen expressed on T cells in conjunction with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed from the dimeric association of two of four distinct chains: epsilon, zeta, delta, and gamma. CD3 dimeric configurations include gamma / epsilon, delta / epsilon, and zeta / zeta. Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby triggering T cell activation in a manner similar to TCR engagement by peptide-loaded MHC molecules. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes, including T cell activation. Furthermore, bispecific antibodies capable of binding to CD3 and a target antigen have been proposed for therapeutic uses, including targeting T cell immune responses to tissues and cells expressing the target antigen.

[0005] Multiple myeloma patients who are resistant to multiple classes of therapy have reduced overall survival rates (triple and quadruple refractory: 9.2 months, quintuple refractory: 5.6 months). Non-Patent Document 2. Antigen binding molecules that target BCMA, including bispecific antigen binding molecules that bind to both BCMA and CD3, may be useful in therapeutic settings where specifically targeting BCMA-expressing cells and T cell-mediated killing is desired. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Tai et al.,Immunotherapy,7(11):1187-1199,2015 [Non-patent document 2] Gandhi U. et al., Leukemia 33:2266-2275,2013 Summary of the Invention

[0007] In one aspect, the present invention provides a method for detecting a cellular marker, comprising: (a) detecting a cellular marker, as measured by an in vitro FACS binding assay; Sea urchin, EC<approx. 100 nM50 and (b) a first antigen-binding domain that specifically binds to human B-cell maturation antigen (BCMA) on target tumor cells, the first antigen-binding domain having a binding domain of about 10 as measured by an in vitro FACS binding assay. -6 EC less than M 50 and a second antigen-binding domain (D2) that specifically binds to human CD3, having the following structure:

[0008] In some cases, the bispecific antigen-binding molecule comprises about 10 -9 EC less than M 50 Activating T cells in vitro with the bispecific antigen-binding molecule. -9 EC less than M 50 In some cases, the bispecific antigen-binding molecule mediates in vitro T cell killing of tumor cell lines expressing BCMA. -8 EC less than M 50 In some embodiments, the bispecific antigen-binding molecule interacts with amino acid residues 1-43 of BCMA as 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 another B cell disorder characterized in part by having B cells that express BCMA. In some cases, the bispecific antigen-binding molecule inhibits the growth 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 0.001mg / kg, 0.002mg / kg, 0.003mg / kg, 0.004mg / kg, 0.005mg / kg, 0.006mg / kg, 0.007mg / kg, 0.008mg / kg, 0.009mg / kg, 0.01mg / kg, 0.02mg / kg, 0.03mg / kg, 0.04mg / kg, 0.05mg / kg, 0.06mg / kg, 0.07mg / kg, 0.08mg / kg, 0.09mg / kg, 0.1mg / kg, 0.2mg / kg, 0.3mg / kg, 0.4mg / kg, 0.5mg / kg, 0.6mg / kg, 0.7mg / kg, 0.8mg / kg, 0. ... In some embodiments, the dose is administered to a patient in need thereof at least twice a week for at least seven 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 embodiments, the bispecific antigen-binding molecule inhibits the growth 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 growth 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) comprised 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) comprised within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 82. In some embodiments, the second antigen-binding domain comprises (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 100, (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 102, and (c) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 104. In some embodiments, the second antigen-binding domain 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 second antigen-binding domain comprises (a) HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 92, 94, 96, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively, or (b) HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 100, 102, 104, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively. In some cases, 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) a first antigen-binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 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, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively, and (b) a second antigen-binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 92, 94, 96, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively. In some cases, 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.

[0015] In another aspect, the present invention provides (a) HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, respectively, and (b) HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88 and (b) a second antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 100, 102, and 104, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the sequences of SEQ ID NOs: 84, 86, and 88, respectively. In some cases, 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.

[0016] In another aspect, the present invention provides an isolated bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that specifically binds to human BCMA and comprises a CDR 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 a CDR 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. In some cases, 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. In some cases, the first antigen-binding domain comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain 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, respectively. In some cases, 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. In some cases, the second antigen-binding domain comprises the CDRs of 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 for 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 for 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 may be bispecific antibodies. In some cases, the bispecific antibody comprises a human IgG heavy chain constant region. In some embodiments, the human IgG heavy chain constant region is of isotype IgG1. In some embodiments, the human IgG heavy chain constant region is of isotype IgG4. In various embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype. In some embodiments, the bispecific antibody comprises a first heavy chain comprising a constant region comprising the amino acid sequence of SEQ ID NO: 130. In some embodiments, 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 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 a bispecific antigen-binding molecule (e.g., a bispecific antibody) as 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 a bispecific antigen-binding molecule (e.g., a bispecific antibody) as 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 present invention provides a method of inhibiting the growth of a plasma cell neoplasm in a subject, the method comprising administering to the subject an isolated bispecific antigen-binding molecule or a pharmaceutical composition comprising the bispecific antigen-binding molecule, as described above or herein. Optionally, the plasma cell neoplasm is multiple myeloma. Optionally, the method further comprises administering a second therapeutic agent or treatment 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), obendamustine (Treanda), or any other known to be effective in treating a plasma cell neoplasm 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 may be an immunomodulatory agent. In certain embodiments, the second therapeutic agent may be a proteasome inhibitor, including bortezomib (Velcade), carfilzomib (Kyprolis), and ixazomib (Ninlaro). In certain embodiments, the second therapeutic agent may be a histone deacetylase inhibitor, such as panobinostat (Farydak). In certain embodiments, the second therapeutic agent may 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 present invention provides a method of treating a patient suffering from multiple myeloma or another BCMA-expressing B-cell malignancy, comprising administering to the subject an isolated bispecific antigen-binding molecule, or a pharmaceutical composition comprising the bispecific antigen-binding molecule, as described above or herein. In some cases, the BCMA-expressing B-cell malignancy is selected from the group consisting of Waldenstrom's macroglobulinemia, Burkitt's lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin's lymphoma. In some cases, the method further comprises administering a second therapeutic agent. In some embodiments, the second therapeutic agent comprises an anti-tumor agent (chemotherapeutic agent), a DNA alkylating agent, an immunomodulator, a proteasome inhibitor, a histone deacetylase inhibitor, radiation therapy, stem cell transplantation, an immunomodulator, 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 the other 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 may be selected from a PD-1 inhibitor such as pembrolizumab (Keytruda), nivolumab (Opdivo), or cemiplimab (REGN2810). In certain embodiments, the checkpoint inhibitor may be selected from a PD-L1 inhibitor, such as atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi). In certain embodiments, the checkpoint inhibitor may be selected from a CTLA-4 inhibitor, such as ipilimumab (Yervoy). Other combinations that can be used in conjunction 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, comprising administering to the subject an isolated bispecific antigen-binding molecule, or a pharmaceutical composition comprising same, as described above or herein, in combination with an anti-PD-1 antibody or antigen-binding fragment thereof. In some cases, 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 x 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 treating BCMA-expressing tumors.

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

[0028] The present invention further includes the use of a bispecific antigen-binding molecule as described above or herein in the manufacture of a medicament for treating a disease or disorder associated with BCMA expression. In some cases, the disease or disorder is cancer. In some embodiments, the cancer is multiple myeloma. The present invention further includes a bispecific antigen-binding molecule (e.g., a bispecific antibody) for use in treating a 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 a subject an antibody comprising a first heavy chain and common light chain pair comprising a first antigen-binding domain that specifically binds to human B-cell maturation antigen (BCMA) and a second heavy chain and common light chain pair that specifically binds to human CD3. and a second heavy chain and common light chain pair comprising a second antigen-binding domain that specifically binds to the first antigen-binding domain, wherein the first antigen-binding domain comprises three heavy chain complementarity-determining regions (CDRs) and three light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, 84, 86, and 88, respectively, and the second antigen-binding domain comprises three heavy chain CDRs and three light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 92, 94, 96, 84, 86, and 88, respectively, and the bispecific antibody is administered to the subject at a dose of 1 mg at least once a week. In some cases, the bispecific antibody is administered at a dose of at least 1 mg every week or two weeks, at least 1.5 mg every week or two weeks, at least 2.0 mg every week or two weeks, at least 2.5 mg every week or two weeks, at least 3.0 mg every week or two weeks, at least 3.5 mg every week or two weeks, at least 4 mg every week or two weeks, at least 5 mg every week or two weeks, at least 6 mg every week or two weeks, at least 7 mg every week or two weeks, at least 8 mg every week or two weeks, at least 9 mg every week or two weeks, at least 10 mg every week or two weeks, at least 15 mg every week or two weeks, at least 16 mg every week or two weeks, at least 18 mg every week or two weeks, at least 19 mg every week or two weeks, at least 20 mg every week or two weeks, at least 21 mg every week or two weeks, at least 22 mg every week or two weeks, at least 23 mg every week or two weeks, at least 24 mg every week or two weeks, at least 25 mg every week or two weeks, at least 26 mg every week or two weeks, at least 27 mg every week or two weeks, at least 28 mg every week or two weeks, at least 29 mg every week or two weeks, at least 30 mg every week or two weeks, at least 31 mg every week or two weeks, at least 32 mg every week or two weeks, at least 33 mg every week or two weeks, at least 34 mg every week or two weeks, at least 35 mg every week or two weeks, at least 36 mg every week or two weeks, at least 37 mg every week or two weeks, at least 38 mg every week or two weeks, at least 39 mg every week or two weeks, at least 40 mg every week or two weeks, at least 41 mg every week or two weeks, at least 42 mg every week or two weeks, at least 43 mg every week or two weeks, at least 44 mg every 20 mg at least every week or two weeks, 25 mg at least every week or two weeks, 30 mg at least every week or two weeks, 35 mg at least every week or two weeks, 40 mg at least every week or two weeks, 45 mg at least every week or two weeks, 50 mg at least every week or two weeks, 55 mg at least every week or two weeks, 60 mg at least every week or two weeks, 65 mg at least every week or two weeks, 70 mg at least every week or two weeks, 75 mg at least every week or two weeks, 80 mg at least every week or two weeks, 85 mg at least every week or two weeks, 90 mg at least every week or two weeks, 95 mg at least every week or two weeks,The subject is administered a dose of at least 100 mg every week or two weeks, at least 150 mg every week or two weeks, at least 200 mg every week or two weeks, at least 250 mg every week or two weeks, at least 300 mg every week or two weeks, at least 350 mg every week or two weeks, at least 400 mg every week or two weeks, at least 450 mg every week or two weeks, at least 500 mg every week or two weeks, at least 550 mg every week or two weeks, at least 600 mg every week or two weeks, at least 650 mg every week or two weeks, at least 700 mg every week or two weeks, at least 750 mg every week or two weeks, at least 800 mg every week or two weeks, at least 850 mg every week or two weeks, or at least 900 mg every week or two weeks.

[0030] 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: 90, 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: 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 first heavy chain and common light chain pair comprising a first antigen-binding domain that specifically binds human B-cell maturation antigen (BCMA) and a second heavy chain and common light chain pair comprising a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises three heavy chain complementarity-determining regions (CDRs) and three light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, 84, 86, and 88, respectively; and the second antigen-binding domain comprisesThe bispecific antibody comprises three heavy chain CDRs and three light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 100, 102, 104, 84, 86, and 88, respectively, and is administered to a subject at a dose of 1 mg at least once a week. In some cases, the bispecific antibody is administered at a dose of at least 1 mg every week or two weeks, at least 1.5 mg every week or two weeks, at least 2.0 mg every week or two weeks, at least 2.5 mg every week or two weeks, at least 3.0 mg every week or two weeks, at least 3.5 mg every week or two weeks, at least 4 mg every week or two weeks, at least 5 mg every week or two weeks, at least 6 mg every week or two weeks, at least 7 mg every week or two weeks, at least 8 mg every week or two weeks, at least 9 mg every week or two weeks, at least 10 mg every week or two weeks, at least 15 mg every week or two weeks, at least 20 mg every week or two weeks, at least 25 mg every week or two weeks, at least 30 mg every week or two weeks 35mg, 40mg at least every week or two weeks, 45mg at least every week or two weeks, 50mg at least every week or two weeks, 55mg at least every week or two weeks, 60mg at least every week or two weeks, 65mg at least every week or two weeks, 70mg at least every week or two weeks, 75mg at least every week or two weeks, 80mg at least every week or two weeks, 85mg at least every week or two weeks, 90mg at least every week or two weeks, 95mg at least every week or two weeks, 100mg at least every week or two weeks, 150mg at least every week or two weeks, 200mg at least every week or two weeks, 250mg at least every week or two weeks, 300mg, 350mg at least every week or two weeksThe subject is administered a dose of 400 mg at least every week or two weeks, 450 mg at least every week or two weeks, 500 mg at least every week or two weeks, 550 mg at least every week or two weeks, 600 mg at least every week or two weeks, 650 mg at least every week or two weeks, 700 mg at least every week or two weeks, 750 mg at least every week or two weeks, 800 mg at least every week or two weeks, 850 mg at least every week or two weeks, or 900 mg at least every week or two weeks.

[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 herein, the bispecific anti-BCMA x anti-CD3 antibody may be administered in a dosing regimen comprising a split primary dose. In some embodiments, the bispecific anti-BCMA x anti-CD3 antibody is administered to the subject at a dose of 1 mg at least weekly. In some embodiments, the bispecific anti-BCMA x anti-CD3 antibody is administered to the subject at a dose of 3 mg to 900 mg at least weekly. In some cases, the bispecific antibody is administered at a dose of 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 7 mg at least once a week. 7mg at least every week or two weeks, 8mg at least every week or two weeks, 9mg at least every week or two weeks, 10mg at least every week or two weeks, 15mg at least every week or two weeks, 20mg at least every week or two weeks, 25mg at least every week or two weeks, 30mg at least every week or two weeks, 35mg at least every week or two weeks, 40mg at least every week or two weeks, 45mg at least every week or two weeks, 50mg at least every week or two weeks, 55mg at least every week or two weeks, 60mg at least every week or two weeks, 65mg at least every week or two weeks, 70mg at least every week or two weeks, 75mg at least every week or two weeks, 80mg at least every week or two weeks, 85mg at least every week or two weeks, 90mg at least every week or two weeks g, 95 mg at least every week or two weeks, 100 mg at least every week or two weeks, 150 mg at least every week or two weeks, 200 mg at least every week or two weeks, 250 mg at least every week or two weeks, 300 mg at least every week or two weeks, 350 mg at least every week or two weeks, 400 mg at least every week or two weeks, 450 mg at least every week or two weeks, 500 mg at least every week or two weeks, 550 mg at least every week or two weeks, 600 mg at least every week or two weeks, 650 mg at least every week or two weeks, 700 mg at least every week or two weeks, 750 mg at least every week or two weeks, 800 mg at least every week or two weeks, 850 mg at least every week or two weeks, or 900 mg at least every week or two weeks.

[0034] In any of the methods described above or discussed herein, the BCMA+ cancer may be multiple myeloma and the subject being administered the anti-BCMA x 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 receiving the anti-BCMA x anti-CD3 bispecific antibody has previously been treated with an anti-CD38 antibody therapy. In some cases, the anti-CD38 antibody is daratumumab or isatuximab.

[0036] In any of the methods described above or herein, the BCMA+ cancer can be multiple myeloma, and the subject receiving the anti-BCMA x anti-CD3 bispecific antibody has previously been treated with a proteasome inhibitor or an immunomodulatory agent. In some cases, the proteasome inhibitor is bortezomib, carfilzomib, or ixazomib. In some cases, 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 may be a patient with a multiple myeloma immune subtype 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 herein, the subject is at least triple refractory to previous therapy (i.e., has progressed after at least three previous lines of therapy). In some cases, the subject is quadruple refractory to previous therapy. In some cases, the subject is quintuple refractory to previous therapy.

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

[0042] In some embodiments of the dosing regimen, the bispecific antibody comprises a first heavy chain and common light chain pair comprising a first antigen-binding domain that specifically binds human B-cell maturation antigen (BCMA) and a second heavy chain and common light chain pair comprising a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises three heavy chain complementarity-determining regions (CDRs) and three light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, 84, 86, and 88, respectively, and the second antigen-binding domain comprises three heavy chain CDRs and three light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 92, 94, 96, 84, 86, and 88, respectively. In some embodiments, 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. 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: 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 first heavy chain and common light chain pair comprising a first antigen-binding domain that specifically binds to human B-cell maturation antigen (BCMA) and a second heavy chain and common light chain pair 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 comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, 84, 86, and 88, respectively, and the second antigen-binding domain comprises three heavy chain CDRs and three light chain CDRs comprising the amino acid sequences of SEQ ID NOs: 100, 102, 104, 84, 86, and 88, respectively. In some cases, the first heavy chain comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 98, 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.

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

[0045] In any of the various embodiments of the dosing regimen, the subject has previously been treated with an anti-CD38 antibody therapy, a proteasome inhibitor, or an immunomodulatory agent. In some cases, the anti-CD38 antibody is daratumumab or isatuximab. In some cases, the proteasome inhibitor is The tumor inhibitor is bortezomib, carfilzomib, or ixazomib. In some cases, 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 treatment. In some cases, the subject is quadruple refractory or quintuple refractory to previous treatment.

[0048] In various embodiments, any of the features or components of the embodiments described above or discussed herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value described above or discussed herein can be combined with another associated value described above or discussed herein to recite a range where those values ​​represent the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure.

[0049] Other embodiments will be apparent from a review of the detailed description that follows. [Brief explanation of the drawings]

[0050] [Figure 1] Figure 1 shows preventative dose-dependent tumor inhibition of BCMA-expressing NCI-H929 human multiple myeloma tumor cells in vivo by the anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459, respectively. NCI-H929 cells express high levels of BCMA. [Figure 2] Figure 1 shows preventative dose-dependent tumor inhibition of BCMA-expressing NCI-H929 human multiple myeloma tumor cells in vivo by the anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459, respectively. NCI-H929 cells express high levels of BCMA. [Figure 3] Figure 1 shows therapeutic dose-dependent tumor inhibition of established BCMA-expressing NCI-H929 human multiple myeloma tumor cells in vivo by the anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459, respectively. NCI-H929 cells express high levels of BCMA. [Figure 4]Figure 1 shows therapeutic dose-dependent tumor inhibition of established BCMA-expressing NCI-H929 human multiple myeloma tumor cells in vivo by the anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459, respectively. NCI-H929 cells express high levels of BCMA. [Figure 5] Figure 1 shows preventative dose-dependent tumor inhibition of BCMA-expressing MOLP-8 human multiple myeloma tumor cells in vivo by the anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459, respectively. MOLP-8 cells express moderate levels of BCMA. [Figure 6] Figure 1 shows preventative dose-dependent tumor inhibition of BCMA-expressing MOLP-8 human multiple myeloma tumor cells in vivo by the anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459, respectively. MOLP-8 cells express moderate levels of BCMA. [Figure 7] Figure 1 shows therapeutic reduction of established tumor burden of BCMA-expressing OPM-2 human multiple myeloma tumor cells in vivo by anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459 compared to controls. OPM-2 cells express low levels of BCMA. DETAILED DESCRIPTION OF THE INVENTION

[0051] Before the present invention is described, it is to be understood that the particular methods and experimental conditions described may vary. It is understood that the invention is not limited to such methods and conditions. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be 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 those skilled in the art to which this invention belongs.As used herein, the term "about" when used in relation to a specific listed numerical value means that this value can vary by 1% or less from the listed value.For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

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

[0054] definition As used herein, the term "CD3" refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR) and consisting of a homodimer or heterodimer formed from the association of two of the four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma. Human CD3-epsilon comprises the amino acid sequence set forth in SEQ ID NO: 116, human CD3-delta comprises the amino acid sequence set forth in SEQ ID NO: 117, human CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 118, and CD3-gamma comprises the amino acid sequence set forth in SEQ ID NO: 119. All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human form of the respective protein, polypeptide, or protein fragment unless specifically identified as being derived from a non-human species. Thus, the term "CD3" refers to human CD3 unless specifically identified as being derived from a non-human species, e.g., "mouse CD3," "simian CD3," etc.

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

[0056] As used herein, the phrase "cell surface-expressed CD3" refers to one or more CD3 proteins expressed on the surface of a cell in vitro or in vivo, where at least a portion of the CD3 protein is exposed on the extracellular face of the cell membrane and is accessible to the antigen-binding portion of an antibody. "Cell surface-expressed CD3" includes CD3 proteins contained within functional T cell receptors in the cell membrane. The phrase "cell surface-expressed CD3" includes CD3 proteins expressed as part of homodimers or heterodimers on the surface of a cell (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The phrase "cell surface-expressed CD3" also refers to a CD3 chain (e.g., CD3-epsilon, CD3-delta, or CD3) expressed by itself on the surface of a cell, without other CD3 chain types. -gamma). Alternatively, "cell surface-expressed CD3" can include or consist of CD3 protein expressed on the surface of a cell that normally expresses CD3 protein. Alternatively, "cell surface-expressed CD3" can include or consist of CD3 protein expressed on the surface of a cell that does not normally express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

[0057] As used herein, the term "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 regulating B cell maturation and differentiation into immunoglobulin-producing plasma cells. The amino acid sequence of human BCMA is set forth in SEQ ID NO: 115 and can also be found in GenBank accession number NP_001183.2.

[0058] As used herein, "antibodies that bind BCMA" or "anti-BCMA antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize BCMA.

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

[0060] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., BCMA or CD3). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains, inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules that consist of four polypeptide chains, two heavy (H) chains and two light (L) chains, inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V). H The heavy chain constant region comprises three domains: C H 1. C H 2, and C H 3. Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L 1) V H Area and V LThe regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of an 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. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0061] The term "antibody" as used herein also includes antigen-binding fragments of complete antibody molecules. "Antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and similar terms, as used herein, include naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind antigens to form complexes. Antibody-binding fragments of antibodies can be derived from complete antibody molecules using any suitable standard techniques, such as, for example, proteolytic or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques to, for example, suitably encode one or more variable and / or constant domains. Alternatively, codons can be introduced to 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) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, 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] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. H Domain is V L In the antigen-binding fragment associated with the domain, V H Domains and V L The domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be a dimer, with the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H Domain or V L It may also include a domain.

[0064] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the 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 LIn any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may be linked to each other and / or to one or more monomeric V H Or V L The variable domain and constant domain configurations may comprise homodimers or heterodimers (or other multimers) of any of the variable domain and constant domain configurations listed above in non-covalent association with the domains (e.g., via disulfide bonds).

[0065] Like intact antibody molecules, antibody-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope 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 antibodies of the present invention using routine techniques available in the art.

[0066] The antibodies of the present invention may function through 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 nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, thereby resulting in lysis of the target cells. CDC and ADCC can be measured using assays that are well known and available in the art. (See, e.g., 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 an antibody is important for the antibody's ability 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 x anti-CD3 bispecific antibody of the invention is a human antibody. The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include, for example, amino acid residues in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). 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., a mouse) have been grafted onto human framework sequences.

[0068] The antibodies of the present invention may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described below), antibodies isolated from a recombinant combinatorial human antibody library (described below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. 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, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V and V regions of the recombinant antibody. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but which 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 contains a stable four-chain construct of approximately 150-160 kDa in which dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, forming approximately 75-80 kDa molecules consisting of covalently linked light and heavy chains (half antibodies). These forms have been extremely difficult to separate, even after affinity purification.

[0070] The frequency of the second form in various intact IgG isotypes is dependent on the presence of the second form in the hinge region of the antibody. This is due to, but is not limited to, structural differences associated with isotypes. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention provides a method for the reduction of the occurrence of the second form by modifying the hinge region, C H 2 area, or C H Antibodies with one or more mutations in three regions are included, which may be desirable, for example, to improve the yield of a desired antibody form in production.

[0071] The antibody of the present invention may be an isolated antibody. As used herein, an "isolated antibody" refers to 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 it naturally occurs or is naturally produced, is an "isolated antibody" for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. 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 material and / or chemicals.

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

[0073] The anti-BCMA or anti-BCMA x anti-CD3 antibodies disclosed 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 compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, 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, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce numerous antibodies and antibody-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within a domain are mutated back to the residue 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 residue is found within the first 8 amino acids of FR1, or the mutated residue is found within the last 8 amino acids of FR4, or the mutated residue is found only in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in 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 antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antibody-binding fragments obtained by this general method are included within the scope of the present invention.

[0074] The present invention also includes anti-BCMA or anti-BCMA x anti-CD3 antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-BCMA or anti-BCMA x anti-CD3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, 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, LCVR, and / or CDR amino acid sequences set forth in Tables 1 and 3 herein, or the 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 a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes may include saccharide, phosphoryl, or sulfonyl moieties on an antigen.

[0076] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% nucleotide sequence identity of the nucleotide bases as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain cases, 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 term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned using a program such as GAP or BESTFIT with a predetermined gap weight, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, 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 with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other in conservative substitutions, the percent sequence identity or degree of similarity may 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, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids with 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; and (6) acidic side chains: aspartic acid and glutamic acid. and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has 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 that has a non-negative value in the PAM250 log-likelihood matrix.

[0078] Sequence similarity for polypeptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the best overlapping regions between the query and search sequences (Pearson (2000) (see above)). Another preferred algorithm for 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, with 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 mutations The anti-CD3 antibodies disclosed herein contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy chain variable domain compared to the corresponding germline sequences.

[0080] The present 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 in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes collectively referred to herein as "germline mutations"), and which have weaker or no detectable binding to the CD3 antigen.

[0081] Furthermore, antibodies of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residues in 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, weaker or reduced binding affinity, improved or enhanced pharmacokinetic properties, reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner, taking into account the guidelines of this disclosure, are encompassed within the scope of the present invention.

[0082] The present invention also includes antigen-binding molecules comprising antigen-binding domains with HCVR and / or CDR amino acid sequences 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 term "substantial identity" or "substantially identical" means that two amino acid sequences, when optimally aligned using predefined gap weights, such as by the programs GAP or BESTFIT, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may 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, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331.

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

[0084] Antibody binding properties As used herein, the term "binding," in the context of the binding of either an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to a predetermined antigen, such as, for example, a cell surface protein or 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, binding affinities are typically about 10, as determined by surface plasmon resonance (SPR) techniques, e.g., on a BIAcore 3000 instrument, using an antigen as the ligand and an antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or antiligand). -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 K below M D Cell-based binding strategies, such as fluorescence-activated cell sorting (FACS) binding assays, are also routinely used, and FACS data correlate 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, an antibody or antigen-binding protein of the invention has a K that is at least 10-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). D Affinity corresponding to value According to the present invention, the antibody binds to a specific antigen or cell surface molecule (receptor) having a K value that is 10 times lower than that of a non-specific antigen. D Although antibody affinities corresponding to values ​​can be considered as undetectable binding, such antibodies can be paired with a second antigen-binding arm to produce bispecific antibodies of the invention.

[0087] "K D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. D There is an inverse relationship between K and binding affinity, and therefore, K D The smaller the value, the higher, i.e., stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, i.e., a smaller K DConversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction, i.e., a larger K D In some situations, a higher binding affinity (or K) of a particular molecule (e.g., an antibody) to its interaction partner molecule (e.g., antigen X) compared to the binding affinity of the molecule (e.g., an antibody) to another interaction partner molecule (e.g., antigen Y) can be used. D ) is larger than K D A smaller K value (lower, or weaker, affinity) D The binding affinity is expressed as a binding ratio determined by dividing by the binding affinity (higher, or stronger, affinity), e.g., 5-fold or 10-fold higher binding affinity as the case may be.

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

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

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

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

[0092] In one embodiment, the reduction in binding is determined by measuring the EC2 concentration that allows half-maximal binding to target cells. 50 It can be defined as an increase in antibody concentration.

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

[0094] Bispecific antigen binding molecules The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide, or may contain antigen-binding domains specific for two or more target polypeptides. For example, Tut See, e.g., t et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-BCMA monospecific or anti-BCMA x anti-CD3 bispecific antibodies of the invention can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be operatively linked (e.g., by chemical conjugation, 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 phrase "anti-CD3 antibody" or "anti-BCMA antibody" herein is intended to include both monospecific anti-CD3 or anti-BCMA antibodies, as well as bispecific antibodies comprising a CD3-binding arm and a BCMA-binding arm. Thus, the present invention includes bispecific antibodies in which one immunoglobulin arm binds to human CD3 and the other immunoglobulin arm is specific for human BCMA. The CD3-binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences set forth in Table 3 herein, or the 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 weakly binds to human CD3 and induces human T cell activation. In other embodiments, the CD3-binding arm weakly binds to human CD3 and induces tumor-associated antigen-expressing cell killing in the context of a bispecific or multispecific antibody. In other embodiments, the CD3-binding arm weakly binds to or associates with human and cynomolgus monkey (monkey) CD3, yet the binding interaction is not detectable by in vitro assays known in the art. The BCMA-binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences set forth in Table 1 herein.

[0097] According to certain exemplary embodiments, the present invention comprises bispecific antigen-binding molecules that specifically bind to CD3 and BCMA. Such molecules may be referred to herein as, for example, "anti-BCMA x anti-CD3" or "anti-CD3 / anti-BCMA," or "anti-CD3 x BCMA" or "CD3 x 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 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 bispecific antigen-binding molecules described above that specifically bind to CD3 and BCMA have a K of greater than about 40 nM as measured in an in vitro affinity binding assay. D The anti-CD3 antigen-binding molecule may include an anti-CD3 antigen-binding molecule that binds to CD3 with a weak binding affinity exhibiting a

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

[0101] As used herein, the expression "bispecific antigen-binding molecule" refers to a molecule comprising at least one "Bispecific antigen-binding domain" refers to a protein, polypeptide, or molecular complex comprising one antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain in a bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, either 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 a first and a second antigen-binding domain, the CDRs of the first antigen-binding domain may be 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, an isolated bispecific antigen-binding molecule comprises a first antigen-binding domain comprising (a) three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) comprised 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) comprised within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 82. In some cases, 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. In some cases, 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. 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.

[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) comprised 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) comprised within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 82. In some cases, the second antigen-binding domain comprises (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 100, (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 102, and (c) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 104. In some cases, the second antigen-binding domain 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 second antigen-binding domain comprises (a) HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 92, 94, 96, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively, or (b) HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 100, 102, 104, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively. In some cases, 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. nothing.

[0105] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises (a) a first antigen-binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively, and (b) a second antigen-binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 92, 94, 96, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively. In some cases, 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, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively, and (b) a second antigen-binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 100, 102, 104, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively. In some cases, 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 a CDR 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 a CDR 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. In some cases, 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. In some cases, the first antigen-binding domain comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain 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, respectively. In some cases, 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. In some cases, the second antigen-binding domain comprises the CDRs of 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, 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.

[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, 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.

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

[0111] The first and second antigen-binding domains may be directly or indirectly linked to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first and second antigen-binding domains 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 the bispecific antigen-binding molecule. As used herein, a "multimerization domain" is any polymer, protein, polypeptide, peptide, or amino acid capable of associating with a second multimerization domain of the same or similar structure or configuration. For example, a multimerization domain may be linked to a second multimerization domain of the same or similar structure or configuration. H A non-limiting example of a multimerizing component is a polypeptide comprising the Fc portion of an immunoglobulin (C H 2-C H 3 domains), for example the Fc domain of IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0112] The bispecific antigen-binding molecules of the present invention typically comprise two multimerization domains, e.g., 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, e.g., IgG1 / IgG1, IgG2 / IgG2, or IgG4 / IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, or IgG2 / IgG4.

[0113] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing 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 comprising or consisting of a leucine zipper, a helix loop motif, or a coiled-coil motif.

[0114] Any bispecific antibody format or technology can be used to generate the bispecific antigen-binding molecules of the invention. For example, an antibody or fragment thereof having a first antigen-binding specificity can be coupled to one or more other molecules, such as another antibody or antibody fragment, having a second antigen-binding specificity. The bispecific antigen-binding molecules can be operably linked (e.g., by chemical bonding, genetic fusion, non-covalent bonding, or otherwise) to the fruiting body to generate bispecific antigen-binding molecules. Certain exemplary bispecific formats that can be used in the context of the present invention include, for example, scFv-based 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), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab. 2 These include, but are not limited to, bispecific formats (for a review of the aforementioned formats, see, e.g., 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, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring Fc domain. For example, the present invention includes bispecific antigen-binding molecules containing one or more modifications in the Fc domain that result in a modified Fc domain with modified binding interactions (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a C H 2 or C HThe FcRn-binding domain contains modifications in three regions that increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome at a pH of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or at positions 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or at positions 250 and / or 428, or at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), 307 and / or 308 modifications (e.g., 308F or 308P).

[0116] The present invention is the first C H 3 domain and second Ig C H Also included are bispecific antigen-binding molecules comprising three domains, a first and a second Ig C H The three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking that amino acid difference. H The 3 domain binds to protein A and the second Ig C H The 3 domain contains mutations that reduce or abolish Protein A binding, such as the H95R modification (according to IMGT exon numbering, H435R according to EU numbering). H3 may further include a Y96F modification (according to IMGT, Y436F according to EU). See, e.g., U.S. Patent No. 8,586,713. H Additional modifications that may be found within 3 include D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I by EU) and N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I by EU) and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU). 22I).

[0117] In certain embodiments, the Fc domain may be a chimera that combines Fc sequences from two or more immunoglobulin isotypes. For example, the chimeric Fc domain may be a chimeric Fc domain that combines Fc sequences from human IgG1, human IgG2, or human IgG4 C. H C derived from 2 regions H Part or all of the 2 sequence, and C derived from human IgG1, human IgG2, or human IgG4 H The chimeric Fc domain may comprise some or all of the three sequences. The chimeric Fc domain may also comprise a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region. A specific example of a chimeric Fc domain that may be comprised in any of the antigen-binding molecules described herein is a chimeric Fc domain that comprises, from the N-terminus to the C-terminus, a chimeric Fc domain consisting of [IgG4 C H Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein comprises, from the N-terminus to the C-terminus, [IgG1 CH 1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any of the antigen-binding molecules of the present invention are described in U.S. Publication No. 2014 / 0243504, published August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, may have altered Fc receptor binding, which in turn affects Fc effector function.

[0118] Sequence variants The antibodies and bispecific antigen-binding molecules of the present invention 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 compared to the corresponding germline sequences from which the individual antigen-binding domains were derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The antigen-binding molecules of the present invention may comprise an antigen-binding domain derived from any of the exemplary amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can easily produce numerous antibodies and antibody-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll framework and / or CDR residues within the domain are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence; for example, only the mutated residues are found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues are found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antigen-binding domain was originally derived). Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR regions; for example, certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antigen-binding domains containing one or more germline mutations may have improved binding specificity, increased binding affinity, or improved or enhanced biological properties of antagonists or agonists. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained by this general method can be readily tested for one or more desired properties, such as reduced immunogenicity (optionally).

[0119] pH dependent binding The present invention includes anti-BCMA antibodies and anti-BCMA x anti-CD3 bispecific antigen-binding molecules with pH-dependent binding properties. For example, anti-BCMA antibodies of the present invention may exhibit reduced binding to BCMA at acidic pH compared to neutral pH. Alternatively, anti-BCMA antibodies of the present invention may exhibit enhanced binding to BCMA at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​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, and 5.0. As used herein, the term "neutral pH" refers to a pH of 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, "reduced binding at acidic pH compared to neutral pH" refers to the K of an antibody that binds to its antigen at neutral pH. D K value of an antibody that binds to its antigen at acidic pH D For example, an antibody or antigen-binding fragment thereof may have an acidic / neutral K of about 3.0 or greater. D When a ratio is presented, for purposes of the present invention, an antibody or antigen-binding fragment thereof may be considered to exhibit "reduced binding to BCMA at acidic pH compared to neutral pH." In certain exemplary embodiments, the acidic / neutral K of an antibody or antigen-binding fragment of the invention D The ratio can be 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 with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for reduced (or enhanced) binding to a specific antigen at acidic pH compared to neutral pH. Furthermore, modification of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For example, by substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, an antibody can be obtained that has reduced antigen binding at acidic pH compared to neutral pH.

[0122] Antibodies containing Fc variants According to certain embodiments of the present invention, there are provided anti-BCMA antibodies and anti-BCMA x 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, e.g., at acidic pH compared to neutral pH. For example, the present invention provides 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. H 2 or C H Antibodies containing mutations in the FcRn-3 region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0) can result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, a modification at position 250 (e.g., E or Q), at positions 250 and 428 (e.g., L or F), at positions 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or a modification at positions 250 and / or 428, or at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification is 428L. (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), 307 and / or 308 modifications (e.g., 308F or 308P). All positions are indicated with EU numbering.

[0123] For example, the present invention includes anti-BCMA antibodies, and anti-BCMA x anti-CD3 bispecific antigen binding molecules, comprising an Fc domain comprising one or more 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 foregoing Fc domain mutations, and other mutations in 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 provides high affinity (e.g., subnanomolar K D The present invention includes antibodies and antigen-binding fragments thereof that bind to human BCMA having a nucleotide sequence (a value).

[0125] According to certain embodiments, the present invention provides a method for producing a medicament having a K of less than about 5 nM as measured using surface plasmon resonance, e.g., the assay format defined in Example 4 herein. DIn certain embodiments, the antibodies or antigen-binding fragments of the invention have a K 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 when measured using surface plasmon resonance, e.g., an assay format as defined in Example 4 herein, or a substantially similar assay. D The present invention relates to bispecific antigen-binding molecules (e.g., bispecific antigen-binding molecules having a K of less than about 25 pM when measured using surface plasmon resonance, e.g., an assay format as defined in Example 4 herein, or a substantially similar assay). D binds to human BCMA with a K of less than about 170 pM D The present invention relates to a bispecific antibody that binds to monkey BCMA and has the formula:

[0126] The present invention also includes antibodies and antigen-binding fragments thereof that bind to BCMA with a dissociation half-life (t) of greater than about 10 minutes or greater than about 125 minutes when measured by surface plasmon resonance at 25° C., e.g., using an assay format defined in Example 4 herein, or in a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the invention bind to BCMA with a t 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 when measured by surface plasmon resonance at 25° C., e.g., using an assay format defined in Example 4 herein, or in a substantially similar assay. The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies) that bind to BCMA in more than about 10 minutes as measured by surface plasmon resonance at 25°C, e.g., using an assay format defined in Example 4 herein, or in a substantially similar assay.

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

[0128] The present invention also includes anti-BCMA x 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 tumor growth of established tumors in immunodeficient mice bearing human multiple myeloma xenografts (see, e.g., Examples 10-15); and (c) suppressing tumor growth of syngenic melanoma and colon cancer cells engineered to express human BCMA in immunocompetent 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 therapeutic situation and the particular targeting properties desired. In some cases, low affinity includes a K of greater than 300 nM, greater than 500 nM, or greater than 1 μM. D or EC 50 The present invention also includes antibodies that bind to CD3 with undetectable affinity (e.g., as measured by surface plasmon resonance assays). 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 in which one arm binds to CD3 and another 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, low, 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 resulting adverse side effects associated therewith.

[0130] The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies) that can simultaneously bind to human CD3 and human BCMA. The binding arm that interacts with cells expressing CD3 may have weak to undetectable binding as measured in a suitable in vitro binding assay. The extent to which a bispecific antigen-binding molecule binds to cells expressing CD3 and / or BCMA can be assessed 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 that express CD3, but do not express BCMA (e.g., Jurkat) and / or BCMA-expressing cells.

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

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

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

[0135] The present invention includes anti-BCMA x anti-CD3 bispecific antigen binding molecules that can deplete or reduce tumor antigen-expressing cells in a subject (see, e.g., Examples 8-16, or substantially similar assays). For example, according to certain embodiments, anti-BCMA x anti-CD3 x anti-CD3 bispecific antigen-binding molecules are provided, wherein a single or multiple administration of 0.04 mg / kg, 0.4 mg / kg, or 4 mg / kg of the bispecific antigen-binding molecule to a subject causes a reduction in the number of BCMA-expressing cells in the subject (e.g., suppression or inhibition of tumor growth in the subject).

[0136] Epitope mapping and related techniques The epitopes on CD3 and / or BCMA bound by the antigen-binding molecules of the present invention 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 may 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 herein, the term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have two or more epitopes. Thus, different antibodies may bind to different regions of an antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes can include sugar, phosphoryl, or sulfonyl moieties on an antigen.

[0137] Various techniques known to those skilled in the art can be used to determine whether an antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring, NY, USA). Routine cross-blocking assays, alanine scanning mutation analysis, and peptide blot analysis as described in (Reineke, 2004, Methods) (Harb., NY) Mol Biol 248:443-463) and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9:487-496). Another method that can be used to identify the amino acids in a polypeptide with which an antibody antigen-binding domain interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest and then binding an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to cause hydrogen-deuterium exchange in 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, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography of antigen / antibody complexes may also be used for epitope mapping purposes.

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

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

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

[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 ways. In the first way, the reference antigen-binding molecule is allowed to bind to the BCMA protein (or CD3 protein) under saturating conditions, and then the binding of the test antibody to the BCMA (or CD3) molecule is evaluated. In the second way, In this method, the test antibody is allowed to bind to BCMA (or CD3) molecules under saturating conditions, and then the binding of the reference antigen-binding molecule to BCMA (or CD3) molecules is evaluated. In both directions, if only the first (saturating) antigen-binding molecule can bind to BCMA (or CD3) molecules, it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to BCMA (or CD3). As will be appreciated by those skilled in the art, an antibody that competes for binding with a reference antigen-binding molecule does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[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 positioned relative to one another to produce a bispecific antigen-binding molecule of the present invention using conventional methods. (A discussion of exemplary bispecific antibody formats that can be used to construct bispecific antigen-binding molecules of the present invention is provided elsewhere herein.) In certain embodiments, one or more individual components (e.g., heavy and light chains) of a multispecific antigen-binding molecule of the present invention are derived from a chimeric, humanized, or fully human antibody. Methods for producing such antibodies are well known in the art. For example, one or more heavy and / or light chains of a bispecific antigen-binding molecule of the present invention can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generation technology), a high-affinity chimeric antibody against a specific antigen (e.g., CD3 or BCMA) is first isolated with a human variable region and a mouse constant region. Antibodies are characterized and selected for desirable characteristics including affinity, selectivity, epitope, etc. The murine constant regions are replaced with the desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules of the invention.

[0144] Genetically engineered animals can be used to generate human bispecific antigen-binding molecules. For example, genetically modified mice can be used that are unable to rearrange and express endogenous mouse immunoglobulin light chain variable sequences, and the mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to mouse kappa constant genes at the endogenous mouse kappa locus. Such genetically modified mice can be used to produce fully human bispecific antigen-binding molecules containing two different heavy chains associated with an identical light chain containing variable domains derived from one of two different human light chain variable region gene segments (see, for example, US2011 / 0195454). "Fully human" refers to an antibody, or antigen-binding fragment thereof, or immunoglobulin domain that contains amino acid sequences encoded by DNA derived from human sequences across the entire length of each polypeptide of the antibody, or antigen-binding fragment thereof, or immunoglobulin domain. In some instances, the fully human sequence is derived from an endogenous human protein. In other instances, the fully human protein or protein sequence includes a chimeric sequence in which each component sequence is derived from a human sequence. Without being bound by any theory, chimeric proteins or sequences are generally designed to minimize the creation of immunogenic epitopes at the junctions of the component sequences, for example, compared to any wild-type human immunoglobulin region or domain.

[0145] biological equivalent The present invention encompasses antigen-binding molecules having amino acid sequences that differ from those of the exemplary molecules disclosed herein but that retain the ability to bind to CD3 and / or BCMA. Such variant antibodies contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but have essentially equivalent biological activity to the described bispecific antigen-binding molecules. It exhibits biological activity.

[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 biologically equivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives that do not show significant differences in absorption rate and extent when administered at the same molar dose, either in a single dose or multiple doses, under similar experimental conditions. Some antigen-binding proteins may be considered equivalents or pharmaceutical alternatives if their absorption extent is equivalent but their absorption rate is not, and such differences in absorption rate are intentional and reflected in the label, are not essential for achieving effective body drug concentrations for long-term use, and are not considered medically significant for the particular drug product tested.

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

[0148] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched one or more times compared to therapy continued without switching between the reference product and the biological product without an expected increase in the risk of adverse effects, including clinically significant changes in immunogenicity or reduced efficacy.

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

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

[0151] Biologically equivalent variants of the exemplary bispecific antigen-binding molecules shown herein can be constructed, for example, by making 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 unnecessary or incorrect intramolecular disulfide bridges during renaturation. 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 characteristics of the molecule, for example, mutations that eliminate or remove glycosylation.

[0152] Species selectivity and species cross-reactivity According to certain embodiments of the present invention, antigen-binding molecules that bind to human CD3 but not to CD3 from other species are provided. Antigen-binding molecules that bind to human BCMA but not to BCMA from other species are also provided. The present 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, human CD3 and / or human BCM Antigen-binding molecules that bind to 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 are provided. For example, in certain exemplary embodiments of the present invention, bispecific antigen-binding molecules are provided that include 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 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 Formulation and Administration The present invention provides pharmaceutical compositions comprising the antigen-binding molecules of the present invention. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in formularies 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 (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA complexes, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0155] The dose of an antigen-binding molecule administered to a patient may vary depending on the patient's age and size, target disease, pathological condition, route of administration, etc. Preferred doses 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, it may be advantageous to administer the bispecific antigen-binding molecule of the present invention intravenously in a single dose of typically 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. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective doses and schedules for administering bispecific antigen-binding molecules can be determined empirically; for example, the patient's progress can be monitored by periodic evaluation and the dose adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed 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 compositions of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa), 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 a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device facilitates application when delivering the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and easily replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0158] Numerous reusable pen and autoinjector delivery devices have utility for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include the 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(TM)(Novo Nordisk, Copenhagen, Denmark), BD(TM) pen(Becton Dickinson, Franklin Lakes, NJ), OPTIPEN(TM), OPTIPEN PRO(TM), OPTIPEN Examples of disposable pen delivery devices that are suitable for use in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), the SURECLICK auto-injector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA pen (Abbott Labs, Abbott Park, IL).

[0159] In certain circumstances, pharmaceutical compositions 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 Press, Boca Raton, Florida. In yet another embodiment, a sustained-release system can be placed in the vicinity of the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other sustained-release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0160] The injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, and intramuscular injection, drip infusion, etc. These injectable preparations may be prepared by publicly known methods. For example, the injectable preparations may be prepared by, for example, conventionally used injectable preparations. The antibody or its salt may be prepared by dissolving, suspending, or emulsifying it in a suitable sterile aqueous or oily medium. Aqueous media for injection include, for example, saline, glucose-containing isotonic solutions, and other adjuvants. These may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injections prepared in this manner are preferably filled into suitable ampoules.

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

[0162] Therapeutic Uses of Antigen-Binding Molecules The present invention includes methods comprising administering to a subject in need thereof a therapeutic composition comprising an anti-BCMA antibody or 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 phrase "subject in need thereof" refers to a human or non-human animal that exhibits one or more symptoms or signs of cancer (e.g., a subject that exhibits a tumor or a subject that is afflicted with any of the cancers described herein below), or who would otherwise 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 comprising them) are useful, inter alia, for the treatment of any disease or disorder in which stimulating, activating, and / or targeting an immune response is beneficial. In particular, the anti-BCMA antibodies or anti-BCMA x anti-CD3 bispecific antigen-binding molecules of the present invention can be used to treat, prevent, and / or ameliorate 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 BCMA-expressing cells in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. BCMA-expressing cells 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 including multiple myeloma or other B-cell or plasma cell cancers, such as Waldenstrom's macroglobulinemia, Burkitt's lymphoma, and diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin's lymphoma. According to certain embodiments of the present invention, anti-BCMA antibodies or anti-BCMA x anti-CD3 bispecific antibodies are useful for treating patients suffering from multiple myeloma. According to other related embodiments of the present invention, methods are provided comprising administering the anti-BCMA antibodies or anti-BCMA x anti-CD3 bispecific antigen binding molecules disclosed herein to a patient suffering from multiple myeloma. 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. You can check whether it is working properly.

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

[0166] According to certain aspects, the present invention provides methods 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 methods for treating multiple myeloma, comprising administering to a patient an anti-BCMA antibody or an anti-BCMA x anti-CD3 bispecific antigen binding molecule 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 methods 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 administered in combination with or in combination with the antigen-binding molecules of the present invention include, for example, anti-tumor agents (e.g., chemotherapeutic agents including melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), obendamustine (Treanda), or any other known to be effective in treating plasma cell neoplasms 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 may be an immunomodulatory agent. In certain embodiments, the second therapeutic agent may be a proteasome inhibitor, including bortezomib (Velcade), carfilzomib (Kyprolis), or ixazomib (Ninlaro). In certain embodiments, the second therapeutic agent may be a histone deacetylase inhibitor, such as panobinostat (Farydak). In certain embodiments, the second therapeutic agent may 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 may be beneficially administered in combination with the antigen-binding molecules of the 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, and IL-18, or cytokine inhibitors comprising their respective receptors. Pharmaceutical compositions of the invention (e.g., pharmaceutical compositions comprising the anti-BCMA x anti-CD3 bispecific antigen-binding molecules disclosed herein) may also be administered as part of a therapeutic 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 tumor cells and the other arm that binds to an antigen on T cells, antibody-drug conjugates, bispecific antibodies conjugated to anti-tumor agents, checkpoint inhibitors such as those targeting PD-1 or CTLA-4, or combinations thereof. In certain embodiments, the checkpoint inhibitor is pembrolizumab (Keytruda), nivolumab (Opdivo). In certain embodiments, the checkpoint inhibitor may be selected from a PD-1 inhibitor such as rifampin (RI), or cemiplimab (REGN2810). In certain embodiments, the checkpoint inhibitor may be selected from a PD-L1 inhibitor such as atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi). In certain embodiments, the checkpoint inhibitor may be selected from a CTLA-4 inhibitor 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 therapeutic combinations 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, where 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 with antivirals, 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 also includes radiation therapy and / or conventional chemotherapy.

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

[0170] The present invention includes pharmaceutical compositions in which the antigen-binding molecules of the invention are co-formulated with one or more additional therapeutically active ingredients described elsewhere herein.

[0171] Dosing regimen According to certain embodiments of the 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 method according to this aspect of the invention comprises sequentially administering multiple doses of the antigen-binding molecule of the invention to the subject. As used herein, "sequentially administering" means that each dose of the antigen-binding molecule is administered to the subject at different time points, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The invention includes methods comprising sequentially administering to a patient a single primary dose of the 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, a "primary dose" refers to the dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" refers to the dose administered after the primary dose, and a "tertiary dose" refers to the dose administered after the secondary dose. The primary, secondary, and tertiary doses may all contain the same amount of antigen-binding molecule, but generally may differ from each other in terms of administration frequency. However, in certain embodiments, the amount of antigen-binding molecule contained in the primary, secondary, and / or tertiary dose differs from each other (e.g., 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 beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis. In any of the embodiments, The primary dose (e.g., the first weekly dose) can be divided into two doses administered on separate days (e.g., consecutive days) that are no more than three days apart. In any of the embodiments, the first nominal dose (i.e., the secondary dose) can be divided into two doses administered on separate days (e.g., consecutive days) that are no more than three days apart. For example, if the primary or secondary dose is 6 mg, the dose can be divided into two 3 mg doses that are administered on consecutive days, or on separate days that are no more than three days apart. In various embodiments, the dose (e.g., administered once weekly as a single dose or 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, g, 31mg, 32mg, 33mg, 34mg, 35mg, 36mg, 37mg, 38mg, 39mg, 40mg, 41mg, 42mg, 43mg, 44mg, 45mg, 46mg, 47mg, 48mg, 49mg, 50 mg, 51mg, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 7 0mg, 71mg, 72mg, 73mg, 74mg, 75mg, 76mg, 77mg, 78mg, 79mg, 80mg, 81mg, 82mg, 83mg, 84mg, 85mg, 86mg, 87mg, 88mg, 89mg , 90mg, 91mg, 92mg, 93mg, 94mg, 95mg, 96mg, 97mg, 98mg, 99mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 205mg, 210mg, 215mg, 220 mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, 300mg,305mg、310mg、315mg、320mg、325mg、330mg、335mg、340mg、345mg、350mg、355mg、360mg、365mg、370mg、375mg、380mg、385mg、390mg、395mg、400mg、405mg g、410mg、415mg、420mg、425mg、430mg、435mg、440mg、445mg、450mg、455mg、460mg、465mg、470mg、475mg、480mg、485mg、490mg、495mg、500mg、510mg、520mg mg、530mg、540mg、550mg、560mg、570mg、580mg、590mr、600mg、610mg、620mg、630mg、640mg、650mg、660mg、670mg、680mg、690mg、700mg、710mg、720mg、7 30mg、740mg、750mg、760mg、770mg、780mg、790mg、800mg、810mg、820mg、830mg、840mg、850mg、860mg、870mg、880mg、890mg、900mg、910mg、920mg、930mg 940mg、950mg、960mg、970mg、980mg、990mg、1000mg、1.5g、2g、2.5g、3g、3.5g、4g、4.5g、5g、5.5g、6g、6.5g、7g、7.5g、8g、8.5g、9g、9.5g、10g、or moreかるか、or at least 1mg、2mg、3mg、4mg、5、mg、6mg、7mg、8mg、9mg、10mg、11mg、12mg、13mg、14mg、15mg、16mg、17mg、18mg、19mg、20mg、21mg、22mg、23mg、24mg、 25mg、26mg、27mg、28mg、29mg、30mg、31mg、32mg、33mg、34mg、35mg、36mg、37mg、38mg、39mg、40mg、41mg、42mg、43mg、44mg、45mg、46mg、47mg、48mg、49mg、 50mg、51mg、52mg、53mg、54mg、55mg、56mg、57mg、58mg、59mg、60mg、61mg、62mg、63mg、64mg、65mg、66mg、67mg、68mg、69mg、70mg、71mg、72mg、73mg、74mg<h2 style=";text-align:left;direction:ltr">75mg<h2 style=";text-align:left;direction:ltr"> 、76mg、77mg、78mg、79mg、80mg、81mg、82mg、83mg、84mg、85mg、86mg、87mg、88mg、89mg、90mg、91mg、92mg、93mg、94mg、95mg、96mg、97mg、98mg、99mg、100mg mg、105mg、110mg、115mg、120mg、125mg、130mg、135mg、140mg、145mg、150mg、155mg、160mg、165mg、170mg、175mg、180mg、185mg、190mg、195mg、200mg、2 05mg、210mg、215mg、220mg、225mg、230mg、235mg、240mg、245mg、250mg、255mg、260mg、265mg、270mg、275mg、280mg、285mg、290mg、295mg、300mg、305mg 、310mg、315mg、320mg、325mg、330mg、335mg、340mg、345mg、350mg、355mg、360mg、365mg、370mg、375mg、380mg、385mg、390mg、395mg、400mg、405mg、410mg mg、415mg、420mg、425mg、430mg、435mg、440mg、445mg、450mg、455mg、460mg、465mg、470mg、475mg、480mg、485mg、490mg、495mg、500mg、510mg、520mg、5 30mg、540mg、550mg、560mg、570mg、580mg、590mr、600mg、610mg、620mg、630mg、640mg、650mg、660mg、670mg、680mg、690mg、700mg、710mg、720mg、730mg 、740mg、750mg、760mg、770mg、780mg、790mg、800mg、810mg、820mg、830mg、840mg、850mg、860mg、870mg、880mg、890mg、900mg、910mg、920mg、930mg、940mg mg、950mg、960mg、970mg、980mg、990mg、1000mg、1.5g、2g、2.5g、3g、3.5g、4g、4.5g、5g、5.5g、6g、6.5g、7g、7.5g、8g、8.5g、9g、9.5g、10g、or above.Any of these amounts can be used to define the ranges of the primary, secondary, or tertiary doses discussed herein and are within the scope of this disclosure. In some embodiments, all doses are administered as a single dose (e.g., a single infusion), including doses administered in weeks 1 and 2 of the dosing regimen. For example, a primary dose of 1 mg to 5 mg can be administered as a single dose in week 1, a secondary dose of 3 mg to 400 mg can be administered as a single dose in week 2, and a tertiary dose of 50 mg to 800 mg can be administered as a single dose in week 3, and thereafter during the weekly dosing portion of the dosing regimen. In another example, a primary dose of 5 mg can be administered as a single dose in week 1, a secondary dose of 25 mg can be administered as a single dose in week 2, and a tertiary dose of 50 mg to 800 mg can be administered as a single dose in week 3, and thereafter during the weekly dosing portion of the dosing regimen. In some cases, the dosing schedule may include administration thereafter (eg, after 12 to 16 weeks) every two weeks, every three weeks, once a month, etc.

[0173] In certain exemplary embodiments of the invention, each secondary and / or tertiary dose is 1 to 26 (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, The phrase "immediately preceding dose" as used herein refers to a dose of an antigen-binding molecule in a multiple administration series that is administered to a patient prior to administration of the immediately following dose with no intervening doses.

[0174] The method according to this aspect of the invention involves the use of antigen-binding molecules (e.g., antibodies specific for BCMA and CD3). The method may include administering any number of secondary and / or tertiary doses of an anti-BCMA antibody or bispecific antigen-binding molecule (specifically binding to a BCMA antibody or bispecific antigen-binding molecule) to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, 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., two, three, four, five, six, seven, eight, or more) tertiary doses are administered to the patient.

[0175] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding 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 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The administration frequency may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical testing.

[0176] Diagnostic Uses of Antibodies The anti-BCMA antibodies of the present invention may also be used to detect and / or measure BCMA, or BCMA-expressing cells, in a sample, e.g., for diagnostic purposes. For example, an anti-BCMA antibody or a fragment thereof may be used to diagnose a condition or disease characterized by aberrant expression of BCMA (e.g., overexpression, underexpression, lack of expression, etc.). An exemplary diagnostic assay for BCMA may include, for example, contacting a sample obtained from a patient with an anti-BCMA antibody of the present invention, where the anti-BCMA antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-BCMA antibody may be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be 3 H, 14 C. 32P, 35 S or 125 The antibody may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Another exemplary diagnostic use of the anti-BCMA antibodies of the invention is 89 For the purpose of non-invasive identification and tracking of tumor cells in a subject, such as Zr-desferrioxamine labeling. 89 Zr-labeled antibodies (e.g., positron emission tomography (PET) imaging) are included. (See, e.g., 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.) Certain 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 assays of the present invention include any tissue or fluid sample obtainable from a patient that contains a detectable amount of BCMA protein or a fragment thereof under normal or pathological conditions. Generally, the level of BCMA in a particular 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 to first 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 BCMA-related disease (e.g., a tumor containing BCMA-expressing cells) or condition. [Example]

[0178] The following examples are presented to provide those of ordinary skill 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 of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[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 genetically modified mice containing DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions with human BCMA antigen.

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

[0181] Chimeric antibodies against BCMA with human variable regions and mouse constant regions were first isolated. The antibodies were characterized and selected for desirable characteristics, 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. While the constant region selected can vary depending on the specific application, the high-affinity antigen-binding and target specificity characteristics 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 selected anti-BCMA antibodies of the invention. The corresponding nucleic acid sequence identifiers are set forth in Table 2. [Table 1] [Table 2]

[0183] Example 2: Generation of anti-CD3 antibodies 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 bispecific anti-BCMA x anti-CD3 antibodies according to the invention. Table 3 shows the amino acid sequence identifiers of the heavy and light chain variable regions 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 preparing bispecific antibodies according to the 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 bispecific antigen-binding molecules that bind to CD3 and BCMA; such bispecific antigen-binding molecules are also referred to herein as "anti-BCMA x anti-CD3 or anti-CD3 x BCMA or anti-BCMA x anti-CD3 bispecific molecules." The anti-BCMA portion of the anti-BCMA x 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. The simultaneous binding of BCMA on tumor cells and CD3 on T cells promotes direct killing (cytolysis) of the targeted tumor cells by the activated T cells.

[0185] Bispecific antibodies comprising an anti-BCMA-specific binding domain and an anti-CD3-specific binding domain were constructed using standard methods, where the anti-BCMA antigen-binding domain and the anti-CD3 antigen-binding domain each comprise a distinct HCVR paired with a common LCVR. In the exemplified bispecific antibodies, these molecules were constructed utilizing a heavy chain from an anti-CD3 antibody, a 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, bispecific antibodies can be constructed utilizing a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-BCMA antibody, and an antibody light chain that is promiscuous or known to pair effectively with various heavy chain arms. [Table 5]

[0186] Table 6 shows the amino acid sequence identifiers for the bispecific anti-BCMA x 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) comprising the amino acid sequence of SEQ ID NO: 126, a second heavy chain (comprising a second antigen-binding domain) comprising the amino acid sequence of SEQ ID NO: 127, and a common light chain comprising the amino acid sequence of SEQ ID NO: 129. The first heavy chain of bsAb25441D bispecific antibody (REGN5458) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 130. The second heavy chain of bsAb25441D bispecific antibody (REGN5458) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 131. , comprising a constant region comprising the amino acid sequence of SEQ ID NO: 131. The common light chain of bsAb25441D bispecific antibody (REGN5458) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 132.

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

[0189] Example 4: Surface plasmon resonance derived binding affinities and kinetic constants of anti-BCMA antibodies and anti-BCMA x anti-CD3 bispecific antibodies The equilibrium dissociation constants (K) of hBCMA.mmh (SEQ ID NO: 106) binding to purified anti-BCMA mAb and anti-BCMA x anti-CD3 bispecific mAb DThe binding activity (RI) was determined using a real-time surface plasmon resonance biosensor with a Biacore4000 instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39) to capture purified anti-BCMA mAb and anti-BCMA x anti-CD3 bispecific mAb. All Biacore binding studies were performed in a buffer consisting of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v 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 monkey BCMA expressed with a C-terminal myc-myc-hexahistidine tag (monkey BCMA-MMH; SEQ ID NO: 110) were prepared in HBS-ET running buffer (90 to 1.11 nM, 3-fold dilutions).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 monkey BCMA expressed with a C-terminal mFc tag (monkey BCMA-MFC; SEQ ID NO: 112) were prepared in HBS-ET running buffer (30 to 0.37 nM, 3-fold dilutions), or 30 nM BCMA expressed with a C-terminal mFc tag (mouse BCMA-MFC; SEQ ID NO: 114) were prepared. Antigen samples were then injected over the surface-captured anti-BCMA and anti-BCMA x anti-CD3 bispecific mAbs at a flow rate of 30 μL / min. Antibody-reagent binding was monitored for 5 min while dissociation was monitored for 10 min in HBS-ET running buffer. All binding kinetic experiments were performed at 25°C. Kinetic binding (k) was calculated by fitting real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve-fitting software. a ) rate constant and kinetic dissociation (k d The binding-dissociation equilibrium constant (K D ) and 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 invention have a K in the range of 1.06 nM to 3.56 nM. D As shown in Table 8, the antibody bound to human BCMA-MMH with a sigma-positive antibody titer of 1000. At 25°C, all anti-BCMA antibodies of the invention have a K in the range of 22.3 pM to 10 pM. D As shown in Table 9, at 25°C, the two anti-BCMA antibodies of the invention bound to human BCMA-MFC with K values ​​ranging from 38.8 nM to 49.92 nM. D As shown in Table 10, at 25°C, the four anti-BCMA antibodies of the invention bound to monkey BCMA-MMH with K values ​​ranging from 148 pM to 14.7 nM. D As shown in Table 11, at 25°C, the four anti-BCMA antibodies of the invention bound to monkey BCMA-MFC with K values ​​ranging from 677 pM to 18.8 nM. D The antibody bound to mouse BCMA-MFCs with a value of 0.01. [Table 7] [Table 8] [Table 9] [Table 10] [Table 11]

[0191] Example 5: FACS binding of anti-BCMA x anti-CD3 bispecific antibodies to human and cynomolgus CD3-expressing cells Flow cytometry analysis was used to identify human and cynomolgus monkey CD3 (Jurkat The binding of BCMAxCD3 bispecific antibodies to BCMAxCD3 cells, mfCD3-engineered Jurkat cells, primary human CD8+ and cynomolgus monkey CD8+ T cells was determined. Briefly, 1e05 cells / well were incubated on ice for 30 minutes in the presence of a FACS wash using block (PBS + 1% filtered FBS + 5% mouse serum) with serial dilutions of BCMAxCD3 and control antibodies. After incubation, cells were washed twice with cold FACS wash (PBS + 1% filtered FBS) and bound antibodies were detected by incubating with an anti-human secondary antibody conjugated with Alexa647 for an additional 30 minutes on ice. Wells containing no antibody or secondary antibody only served as controls. To detect monkey and human T cells, a cocktail of human and cynomolgus monkey cross-reactive antibodies against CD4, CD8, and CD16 was added to the anti-human secondary antibody. After incubation, cells were washed, resuspended in 200 μL of cold PBS containing 1% filtered FBS, and analyzed by BD FACS Canto II. Cells were analyzed by flow cytometry. Cells were gated by FSC-A to select singlet events, followed by side scatter and forward scatter to select live events. For monkey T cells, additional gating was performed on CD8+ / CD16- cells.

[0192] EC50 values ​​for FACS binding were calculated using a four-parameter nonlinear regression analysis in Prism software.

[0193] Jurkat cells are a human CD3 expressing T cell lymphoblastoid cell line. REGN5458 had a median EC50 of 1.50 x 10, respectively. -8 M and 3.20 x 10 -8 REGN5459 bound to human CD3 on Jurkat cells and primary human CD8+ T cells bearing M. Binding of REGN5459 was weak to human CD3, with a median EC50 of 5.58 x 10 on Jurkat cells. -7 M and 4.71 × 10 for primary human CD8 T cells. -6Using CRISPR / Cas9 technology, Jurkat cell lines were engineered to express cynomolgus monkey CD3ε and CD3δ chains instead of the human versions. The median EC50 for binding of REGN5458 to the mfCD3-engineered Jurkat cell line was 1.51x10 -8 M and its median EC50 for binding to primary cynomolgus monkey 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 a negative isotype control antibody called mAb15260. [Table 12]

[0195] Example 6: FACS binding assay to evaluate cell surface antigen binding capacity The ability of the anti-BCMA x CD3 antibody, mAb25442D, to bind to the surface of BCMA-positive multiple myeloma (NCI-H929, MM.1S, OPM-2, and RPMI-8226), BCMA-positive lymphoma (Raji and Daudi), and BCMA-negative (HEK293) cells was determined by flow cytometry. Cells were harvested from flasks using cell dissociation buffer (Millipore, catalog no. S-004-C) and dissociated at 50% CO2 per well of a 96-well V-bottom plate in staining buffer (PBS, calcium and magnesium free (Irving 9240) + 2% FBS (ATCC 30-2020)). Cells were seeded at a density of 0,000 cells / well. Cells were stained for 30 minutes at 4°C with two-fold serial dilutions of an Alexa647-conjugated anti-BCMA × CD3 antibody (mAb25442D-A647) or an Alexa647-conjugated isotype control with the same CD3-binding arm paired with an irrelevant tumor-targeting arm (isotype-A647). 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 from dead cells. Cells were then washed and fixed for 25 minutes at 4°C using a 50% solution of BD Cytofix (BD, catalog no. 554655) diluted in PBS. Samples were run on an Accuri C6 flow cytometer (BD Biosciences) and analyzed using Flowjo 10.2 (Tree Star). After gating for live and single cells, the mean fluorescence intensity (MFI) was determined, and the MFI value was calculated as the EC 50 A four-parameter logistic equation was used on a 10-point response curve to calculate EC values, which were plotted in Graphpad Prism. The zero condition for each dose-response curve was also included in the analysis as a series of two-fold serial dilutions, represented as the lowest dose. The signal-to-noise ratio (S / N) was determined by taking the ratio of mAb25442D-A647 MFI to Isotype-A647 MFI (Table 13). The S / N for mAb25442D-A647 ranged from 2 to 470, and the EC 50 The values ​​ranged from 27 to 83 nM. No detectable binding was observed in HEK293 cells. [Table 13]

[0196] Example 7: T cell activation via bispecific anti-BCMA x anti-CD3 antibodies in the presence of BCMA-expressing cells The activity of the anti-BCMA x anti-CD3 bispecific antibody was assessed in a Jurkat / NFATLuc reporter bioassay utilizing several cell lines with different levels of BCMA surface expression. Jurkat cells were engineered to express an NFAT-luciferase reporter (Jurkat / NFATLuc.3C7). 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 μl of assay medium (RPMI medium containing 10% FBS and 1% P / S / G) in a Thermo Nunclon Delta 96-well white microwell plate (Thermo Scientific, catalog number 136102). BCMA x CD3 bispecific antibody (mAb25441D or Three-fold serial dilutions of either mAb25442D or bivalent anti-BCMA antibody (mAb21581) were immediately added in 50 μL of assay buffer. Plates were gently agitated and incubated for 4–6 hours in a 37°C, 5% CO2 incubator. NFAT-luciferase activity was determined using Promega One-Glo (Cat. No. E6130) and a Perkin Elmer Envision plate reader. RLU was calculated as EC 50 To calculate values, a four-parameter logistic equation was used on the 12-point response curves, which were plotted in GraphPad Prism. The no-antibody treatment condition for each dose-response curve was also included in the analysis as a series of 3-fold serial 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 ranging from 0.61 nM to 2.1 nM and an S:N ratio ranging from 8 to 123. mAb25442D activated Jurkat / NFATLuc cells in the presence of BCMA-expressing cells with an EC50 ranging from 2.6 nM to 11 nM and an S:N ratio ranging from 7 to 120. The BCMA × CD3 bispecific mAb25441D, with its high-affinity CD3-binding arm, was consistently more potent than mAb25442D, with its low-affinity CD3-binding arm, while the S:N ratios were 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 below in Tables 14A and 14B. [Table 14] [Table 15]

[0198] Example 8: FACS-based cytotoxicity assay to assess T cell-mediated killing of BCMA-expressing multiple myeloma cells in the presence of anti-BCMA x 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 with a titration of APC-conjugated anti-hBCMA-19F2 antibody. Cells and beads were incubated at RT for 30 min. After incubation, cells and beads were washed three times, 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 anti-BCMA 19F2 ABC for 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 resting human or cynomolgus T cells was determined by flow cytometry. Briefly, human or cynomolgus peripheral blood mononuclear cells (PBMCs) were cultured at 1 × 10 6 Cells were seeded at 1000 cells / mL in supplemented RPMI (human) or X-Vivo (cynomolgus monkey) medium and incubated overnight at 37°C to enrich for lymphocytes by depleting adherent macrophages, dendritic cells, and some monocytes. The following 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 4:1) and serial dilutions of BCMA x CD3 bispecific or control antibodies. After 48–72 hours, cells were removed from the cell culture plate, stained with a cocktail of phenotypic antibodies and live / dead cell viability dyes, and analyzed by FACS. To quantify the number of live target cells present in each well, 20 μl of CountBright absolute counting beads were added to each well immediately before acquisition. To assess the specificity of killing, cells were gated on the Violet CellTrace-labeled population. Target cell viability was calculated as follows: target viability = (R1 / R2) * 100, where R1 = absolute number of viable target cells in the presence of effector cells and antibody, and R2 = number of viable target cells only (cultured without effector cells or test antibody).

[0201] Human CD8+ T cells were gated as CD45+ / CD14- / CD4- / CD8+. Cynomolgus monkey 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 nonlinear regression analysis in Prism software.

[0203] The anti-BCMA x anti-CD3 bispecific antibody was tested for its ability to activate resting human and cynomolgus monkey T cells to kill a panel of BCMA-expressing cells differing in surface BCMA levels. Using resting human T cells as effector cells, REGN5458 mediated EC 50 The value is 7.07 x 10 -10 M~3.45×10 -11 M range. REGN5459 demonstrated killing of the same five cell lines with an EC50 value of 1.66 x 10 -9 M~1.06×10 -10 M. EC for T cell activation, as measured by upregulation of CD25 on CD8+ T cells. 50 ECs die 50 Moderate T cell activation was observed in the presence of the one-arm CD3 isotype control mAb 17664D, but only in the U266 cell line. No cytotoxicity was observed with any of the isotype controls tested.

[0204] BCMAxCD3-mediated killing by cynomolgus monkey T cells was only tested in the MM cell line H929. EC of cytotoxicity mediated by REGN5458 and REGN5459 50 are 2.34×10 -11 and 6.92 × 10 -11No cytotoxicity or T cell activation was observed with the isotype control antibody mAb15260 using human or cynomolgus monkey 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 primary multiple myeloma blast cells in the presence of anti-BCMA x anti-CD3 bispecific antibodies To monitor specific killing of multiple myeloma cells by flow cytometry, bone marrow mononuclear cells (BMMCs) from multiple myeloma patients were seeded onto human stromal cells (HS5) and rested overnight at 37°C. Separately, peripheral blood mononuclear cells (PBMCs) from matched patients were thawed and cultured at 1 × 10 for lymphocyte enrichment by depletion of adherent cells. 6 BMMCs were cultured overnight at 37°C in supplemented RPMI medium at 1000 cells / mL. The next day, BMMCs were cocultured at 37°C with adherent cell-depleted naive PBMCs and serial 10-fold dilutions of BCMA x CD3 bispecific or one-arm CD3 isotype control (starting concentration 66.7 nM) on stromal cells (HS5). On days 3, 4, or 7, cells were removed from the cell culture plate and analyzed by FACS. To assess killing specificity, multiple myeloma cells were gated as single, live, CD90-negative (excluding stromal cells), CD2-negative, and CD56-positive. CD45 expression was low in multiple myeloma cells in most samples, except for MM455. For calculation of adjusted viability, the percentage of live target cells was reported as follows: adjusted viability = (R1 / R2) * 100, where R1 = the percentage (%) of live target cells in the presence of antibody, and R2 = the percentage (%) of live target cells in the absence of 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 among total CD4 or CD8 T cells.

[0207] BCMAxCD3 bispecific antibodies were tested for their ability to redirect killing of primary multiple myeloma blasts by autologous donor PBMCs. Maximal BCMAxCD3-mediated cytotoxicity of primary MM blasts ranged from 52 to 96%, with an EC50 of 9.89 x 10 for REGN5458. -11 M~3.67×10 -9 M range, and for REGN5459 is 4.96 x 10 -10 M~7.94×10 -8 M. T cell activation was measured by assessing the upregulation of CD25 on CD8+ T cells. The EC50 for T cell activation was 3.23 x 10 -9 ~1.69×10 -10 Moderate cytotoxicity and T cell activation was 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 x anti-CD3 bispecific antibodies prevent the growth of BCMA-expressing tumors (NCI-H929) in vivo in a xenogeneic tumor model To determine the in vivo efficacy of a BCMA x CD3 bispecific antibody (Ab), a xenogeneic tumor study was performed in immunodeficient NOD.Cg-Prkdc mice. scid Il2rg tm1Wjl / SzJ(NSG) mice, 10 × 10 6 BCMA-expressing NCI-H929 multiple myeloma cells and 0.5 x 10 isolated from normal donors 6Mice (n = 7 per group) were subcutaneously implanted with a mixture of 1000 human peripheral blood mononuclear cells (PBMCs). A PBS vehicle control, an irrelevant anti-FelD1 bivalent isotype control Ab (REGN2759), a CD3-binding control bispecific Ab (mAb17664D), a BCMA x CD3 (G; REGN5458) bispecific Ab, and a BCMA x CD3 (G; REGN5458) bispecific Ab were administered. The bispecific Ab or the BCMAxCD3 (G20; REGN5459) bispecific antibody was immediately administered at a dose of 4 mg / kg. Mice were treated with Ab twice weekly for a total of 3 weeks, and tumor growth was assessed over a 40-day period. + Tumors grew similarly in vehicle-, isotype control-, and CD3-binding control-treated mice, but both BCMAxCD3 Abs tested prevented tumor growth in vivo.

[0209] Syngeneic tumor implantation and measurement: NSG mice were implanted with 10 x 10 6 BCMA-expressing NCI-H929 multiple myeloma cells and 0.5 × 10 cells derived from a normal donor 6 A mixture of 1000 PBMCs was subcutaneously implanted into mice (n = 7 per group). Mice (n = 7 per group) were immediately treated with PBS vehicle control, an irrelevant anti-FelD1 bivalent isotype control Ab (REGN2759), a CD3-binding control bispecific Ab (mAb17664D), a BCMAxCD3 (G; REGN5458) bispecific Ab, or a BCMAxCD3 (G20; REGN5459) bispecific Ab at a dose of 4 mg / kg. Mice were treated with Abs twice weekly for a total of 3 weeks. Tumor growth was measured twice weekly using calipers for the duration of the experiment. Mice were sacrificed 40 days after tumor implantation.

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

[0211] BCMA × CD3 bispecific Ab inhibits BCMA in vivo in xenogeneic tumor models. +The results are shown in Table 18 below. [Table 21-1] [Table 21-2]

[0212] Example 11: Anti-BCMA x anti-CD3 bispecific antibodies prevent 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 an anti-BCMA x anti-CD3 bispecific antibody (Ab), a xenogeneic tumor study was performed in immunodeficient NOD.Cg-Prkdc mice. scid Il2rg tm1Wjl / SzJ(NSG) mice, 10 × 10 6 BCMA-expressing NCI-H929 human multiple myeloma cells and 0.5 x 10 isolated from normal healthy donors 6 Mice (n=7 per group) were then subcutaneously implanted with a mixture of 1000 human peripheral blood mononuclear cells (PBMCs). Mice (n=7 per group) were then injected with PBS vehicle control, CD3-binding control bispecific Ab (G; mAb17664D). Mice were immediately administered a CD3-binding control bispecific Ab (G20; REGN4460) at a dose of 4 mg / kg, a BCMA x CD3 (G; REGN5458) bispecific Ab at doses of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg, or a BCMA x CD3 (G20; REGN5459) bispecific Ab at doses of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. Mice were administered these Abs twice weekly for a total of seven doses, and tumor growth was assessed over a 60-day period. + NCI-H929 tumors grew similarly in mice treated with vehicle and CD3-binding controls, but both anti-BCMA x anti-CD3 Abs tested prevented tumor growth in a dose-dependent manner in vivo.

[0213] Xenograft tumor transplantation and measurement: NSG mice were implanted with 10 x 10 6 BCMA-expressing NCI-H929 multiple myeloma cells and 0.5 x 10 derived from normal healthy donors 6 A mixture of mAb and PBMCs was subcutaneously implanted into mice (n = 7 per group). 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 x CD3 (G; REGN5458) bispecific Ab, or BCMA x 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. Mice were administered Abs twice weekly for a total of seven doses. Tumor growth was measured twice weekly using calipers for the duration of the experiment.

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

[0215] The BCMAxCD3 bispecific Ab inhibited BCMA in a dose-dependent manner in this heterogeneous in vivo tumor model. + The results are shown in Table 19 below and illustrated in Figures 1 and 2. [Table 22-1] [Table 22-2] [Table 22-3] [Table 22-4] [Table 22-5]

[0216] Example 12: Anti-BCMA x anti-CD3 bispecific antibodies reduce the size and prevent the growth of established BCMA-expressing tumors (NCI-H929) in a dose-dependent manner in a xenogeneic in vivo tumor model To determine the in vivo efficacy of an anti-BCMA x anti-CD3 bispecific antibody (Ab), a xenogeneic tumor study was performed in immunodeficient NOD.Cg-Prkdc mice. scid Il2rg tm1Wjl / SzJ(NSG) mice, 10 × 10 6 BCMA-expressing NCI-H929 human multiple myeloma cells and 0.5 x 10 isolated from normal healthy donors 6 A mixture of human peripheral blood mononuclear cells (PBMCs) was implanted subcutaneously. Tumors were approximately 70 mm in size. 3 The mice were allowed to grow and establish for 5 days until they were viable. Then, 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 x CD3 (G; REGN5458) bispecific Ab at either 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg, or BCMA x CD3 (G20; REGN5459) bispecific Ab at either 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. These Abs were administered twice weekly for a total of seven doses, and tumor growth was assessed over a 55-day period. + NCI-H929 tumors grew similarly in mice treated with vehicle and CD3-binding controls, but both BCMAxCD3 Abs tested shrank established tumors and prevented tumor growth in a dose-dependent manner in vivo.

[0217] Xenograft tumor transplantation and measurement: NSG mice were implanted with 10 x 10 6BCMA-expressing NCI-H929 multiple myeloma cells and 0.5 x 10 derived from normal healthy donors 6 The mixture of 1000 cells and 1000 PBMCs was implanted subcutaneously. The tumors were approximately 70 mm in size. 3 Tumors were allowed to grow and establish for 5 days until tumor size reached 100%. 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 x CD3 (G; REGN5458) bispecific Ab, or BCMA x 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. Mice were administered Abs twice weekly for a total of seven doses. Tumor growth was measured twice weekly using calipers for the duration of the experiment.

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

[0219] The anti-BCMA × anti-CD3 bispecific antibody demonstrated a dose-dependent response to BCMA in this heterogeneous in vivo tumor model. + The results are shown in Table 20 below and in Figures 3 and 4. [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4]

[0220] Example 13: Anti-BCMA x anti-CD3 bispecific antibodies prevent 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 an anti-BCMA x anti-CD3 bispecific antibody (Ab), a xenogeneic tumor study was performed in immunodeficient NOD.Cg-Prkdc mice. scid Il2rg tm1Wjl / SzJ(NSG) mice, 5 × 10 6 1 x 10 BCMA-expressing MOLP-8 human multiple myeloma cells isolated from normal healthy donors 6 The mixture was subcutaneously implanted with human peripheral blood mononuclear cells (PBMCs). Mice (n=7 per group) were then 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 x CD3 (G; REGN5458) bispecific Ab at a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg, or BCMA x CD3 (G20; REGN5459) bispecific Ab at a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. Mice were administered these Abs twice a week for a total of seven doses, and tumor growth was assessed over a 56-day period. BCMA + MOLP-8 tumors in mice treated with vehicle and CD3-binding control Although similarly grown, both BCMAxCD3 Abs tested prevented tumor growth in a dose-dependent manner in vivo.

[0221] Xenograft tumor implantation and measurement: NSG mice were implanted with 5 x 10 6 BCMA-expressing MOLP-8 multiple myeloma cells and 1 x 10 derived from a normal healthy donor 6A mixture of mAb and PBMCs was subcutaneously implanted into mice (n = 7 per group). 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 x CD3 (G; REGN5458) bispecific Ab, or BCMA x 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. Mice were administered Abs twice weekly for a total of seven doses. Tumor growth was measured twice weekly by caliper for the duration of the experiment.

[0222] Calculation of heterogeneous tumor growth and inhibition: To determine tumor volume with external calipers, the maximum major axis (length in mm) and maximum transverse axis (width in mm) were determined. Based on the caliper measurements, tumor volume was calculated using the formula: Volume (mm 3 )=(length x width 2 ) / 2.

[0223] The anti-BCMA × anti-CD3 bispecific antibody inhibited BCMA in a dose-dependent manner in this heterogeneous in vivo tumor model. + The results are shown in Table 21 below and in Figures 5 and 6. [Table 24-1] [Table 24-2] [Table 24-3] [Table 24-4] [Table 24-5]

[0224] Example 14: Anti-BCMA x anti-CD3 bispecific antibodies slow the growth of BCMA-expressing tumors (MOLP-8) in a xenographic in vivo tumor model. To determine the in vivo efficacy of an anti-BCMA x anti-CD3 bispecific antibody (Ab), a xenogeneic tumor study was performed. scid Il2rg tm1Wjl / SzJ(NSG) mice were cultured with 4 × 10 6 On day 0, mice were injected intraperitoneally with 2 × 10 human peripheral blood mononuclear cells (PBMCs). On day 0, mice were injected with 2 × 10 PBMCs engineered to also express firefly luciferase (MOLP-8-luciferase cells). 6 BCMA + MOLP-8 human multiple myeloma tumor cells were administered intravenously. Mice (n = 5 per group) were then immediately administered either 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. Mice were administered these Abs twice more on days 3 and 7, for a total of three doses. Tumor growth was assessed over 48 days by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, a group of mice (n = 5) received only MOLP-8-luciferase cells and no PBMCs or antibody. To measure background BLI levels, a group of mice (n = 5) was left untreated and did not receive tumor, PBMCs, or antibody. BCMA + MOLP-8-luciferase tumors grew progressively in CD3-binding control-treated mice, but BCMAxCD3 Ab treatment with REGN5458 slowed tumor growth in vivo.

[0225] Xenograft tumor implantation and measurement: On day 11, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were intraperitoneally injected with 5 × 10 6 On day 0, mice were injected with 2 x 10 human PBMCs. 6 BCMA+ MOLP-8-luciferase cells were administered intravenously. Mice (n = 5 per group) were then immediately administered either 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. Mice were administered these Abs twice more on days 3 and 7, for a total of three doses. Tumor growth was assessed over 48 days by measuring tumor BLI in anesthetized animals. As a positive control, a group of mice (n = 5) received only MOLP-8-luciferase cells, but no PBMCs or antibody. To measure background BLI levels, a group of mice (n = 5) was left untreated and did not receive tumor, PBMCs, or antibody.

[0226] Measurement of Heterogeneous Tumor Growth: Tumor burden was measured using BLI imaging. Mice were IP injected with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after the injection, BLI imaging of the mice was performed under isoflurane anesthesia using a Xenogen IVIS system. Image collection was performed with a field of view of 1000 nm, a subject height of 1.5 cm, and a medium binning level with automatic exposure time determined by Living Image software. BLI signal was extracted using Living Image software: regions of interest were drawn around each cell volume, and photon intensity was recorded as p / s / cm2 / sr.

[0227] The anti-BCMA x anti-CD3 bispecific antibody REGN5458 demonstrated BCMA in this heterogeneous in vivo tumor model. + MOLP-8-luciferase slowed tumor growth. The results are shown in Table 22 below. [Table 25-1] [Table 25-2]

[0228] Example 15: Anti-BCMA x anti-CD3 bispecific antibody reduces tumor (OPM-2) burden Reduce to background levels in vivo To determine the in vivo efficacy of an anti-BCMA x anti-CD3 bispecific antibody (Ab), a xenogeneic tumor study was performed. On day 0, immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were transfected with 2 × 10 cells engineered to also express firefly luciferase (OPM-2-luciferase cells). 6 BCMA + Mice were intravenously administered 4 × 10 OPM-2 human multiple myeloma tumor cells from normal healthy donors on day 10. 6 Human peripheral blood mononuclear cells (PBMCs) were injected intraperitoneally. On day 21, 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 x CD3 (G; REGN5458) bispecific Ab at 0.4 mg / kg, or a BCMA x CD3 (G20; REGN5459) bispecific Ab at 0.4 mg / kg. Mice were administered these Abs twice more on days 25 and 28, for a total of three doses. Tumor growth was assessed over a 61-day period by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, a group of mice (n = 5) received only OPM-2-luciferase cells and no PBMCs or antibodies. To measure background BLI levels, a group of mice (n = 5) was left untreated and did not receive tumor, PBMCs, or antibodies. BCMA + OPM-2-luciferase tumors grew slowly in mice treated with the CD3-binding control, but BCMAxCD3 Ab treatment with REGN5458 and REGN5459 reduced 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 tm1Wjl / SzJ(NSG) mice were transfected with 2 × 10 cells engineered to also express firefly luciferase (OPM-2-luciferase cells). 6 BCMA + Mice were intravenously administered 4 × 10 OPM-2 human multiple myeloma tumor cells from normal healthy donors on day 10. 6 Human peripheral blood mononuclear cells (PBMCs) were injected intraperitoneally. On day 21, 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 x CD3 (G; REGN5458) bispecific Ab at 0.4 mg / kg, or a BCMA x CD3 (G20; REGN5459) bispecific Ab at 0.4 mg / kg. Mice were administered these Abs twice more on days 25 and 28, for a total of three doses. Tumor growth was assessed over a 61-day period by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, a group of mice (n = 5) received only OPM-2-luciferase cells and no PBMCs or antibodies. To measure background BLI levels, a group of mice (n = 5) was left untreated and did not receive tumor, PBMCs, or antibodies.

[0230] Measurement of Heterogeneous Tumor Growth: Tumor burden was measured using BLI imaging. Mice were IP injected with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after the injection, BLI imaging of the mice was performed under isoflurane anesthesia using a Xenogen IVIS system. Image collection was performed with a field of view of 1000 nm, a subject height of 1.5 cm, and a medium binning level with automatic exposure time determined by Living Image software. BLI signal was extracted using Living Image software: regions of interest were drawn around each cell volume, and photon intensity was recorded as p / s / cm2 / sr.

[0231] BCMA +OPM-2-luciferase tumors grew slowly in mice treated with the CD3 binding control, but BCMAxCD3 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: BCMAxCD3 bispecific antibodies suppress syngeneic tumor growth in vivo in a dose-dependent manner To determine the in vivo efficacy of the anti-BCMA x anti-CD3 bispecific antibody (Ab), syngeneic tumor studies were performed in mice expressing human CD3. C57BL / 6 mice expressing human CD3deg instead of mouse CD3deg (CD3-humanized mice) were injected with 0.5 x 10 B16 / BCMA cells engineered to express full-length human BCMA. 6 1 × 10 B16 melanoma cells or 1 × 10 engineered to express full-length human BCMA (MC38 / BCMA) 6 MC38 colon cancer cells were subcutaneously implanted into mice. Mice (n = 7 per group) were then immediately administered either a CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 0.4 mg / kg or a BCMA x CD3 (G; REGN5458) bispecific Ab at doses of 0.4 mg / kg or 0.04 mg / kg. Mice were administered two more doses of these Abs on days 4 and 7 for a total of three doses, and tumor growth was assessed throughout the experiment. While B16 / BCMA and MC38 / BCMA tumors grew in CD3-binding control-treated mice, BCMA x CD3 REGN5458 was able to suppress the growth of both tumor lines in a dose-dependent manner in vivo.

[0233] Implantation and measurement of syngeneic tumors: 0.5 x 10 cells engineered to express full-length human BCMA (B16 human / BCMA cells) into C57BL / 6 mice expressing human CD3deg instead of mouse CD3deg (CD3-humanized mice). 6 1 x 10 B16F10 melanoma cells or 1 x 10 engineered to express full-length human BCMA (MC38 / BCMA) 6 MC38 colon cancer cells were subcutaneously implanted into mice (n=7 per group). Mice (n=7 per group) were then immediately administered a CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 0.4 mg / kg or a BCMA x CD3 (G; REGN5458) bispecific Ab at a dose of 0.4 mg / kg or 0.04 mg / kg. Mice were administered these Abs twice more on days 4 and 7, for a total of three doses, and tumor growth was assessed throughout the experiment.

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

[0235] While B16 / BCMA and MC38 / BCMA tumors grew in CD3 conjugate control-treated mice, BCMAxCD3 REGN5458 was able to suppress the growth of both tumor lines 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 x CD3 bispecific antibody). For a general description of H / D exchange methods, see, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259, and Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0237] 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 gradients, and a Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

[0238] Labeling solution was prepared in PBS buffer in DO at pH 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) premixed with SEQ ID NO:120 or REGN5458 at a 1:2 molar ratio) was incubated with 90 μL of DO labeling solution at 20°C at various time points in replicates (e.g., non-deuterated control = 0 s, 5 min, and 10 min deuterium-labeled). The deuteration reaction was quenched by adding 100 μL of pre-chilled quench buffer (0.5 M TCEP-HCl, 8 M urea, and 1% formic acid) to each sample and incubating at 20°C for 5 min. The quenched sample was then injected into a Waters HDX Manager for online pepsin / Protease XIII digestion. Digested peptides were separated on a C8 column (1.0 mm x 50 mm, NovaBioassays) with a 13-minute gradient from 10% to 32% B (mobile phase A: 0.5% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). Eluted peptides were analyzed by Q Exactive HF mass spectrometry in LC-MS / MS or LC-MS mode.

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

number

[0240] A total of eight peptides from hBCMA.hFc were identified from both the hBCMA.hFc alone and the hBCMA.hFc complexed with REGN5458 samples, representing 100% sequence coverage of hBCMA. The mean standard deviation (SD) of all peptides was estimated to be 1.4% (detailed calculations defined in ELN and Pascal, B.D. et al. (2012) Journal of the American Society for Mass Spectrometry 23(9):1512-1521). Thus, a different proportion of peptides with D uptake values ​​greater than 4.2% (three times the mean SD) were identified. Any peptide that exhibited significant protection was defined as 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. 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 BCMAxCD3 bispecific antibodies and additional BCMA antibodies on multiple myeloma cell lines after overnight incubation with anti-BCMA antibodies Flow cytometry analysis was used to determine the effect of overnight incubation of multiple myeloma cell lines with anti-BCMA antibodies on 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 (gamma secretase inhibitor), or medium alone. After 18 hours, 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, cells were washed twice with cold FACS wash (PBS + 1% filtered FBS) and bound antibodies were detected by incubating with the appropriate anti-human secondary antibody (anti-hIgG or anti-HIS) on ice for an additional 30-45 minutes. After incubation, cells were washed, resuspended in 200 µL of cold PBS containing 1% filtered FBS, and analyzed by flow cytometry on a BD FACS Canto II. Fold-increase in staining was calculated by dividing the MFI of stained cells previously incubated overnight with BCMA antibody or DAPT by the MFI of stained cells incubated overnight in medium alone.

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

[0243] Of note, overnight incubation of MM cell lines 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, demonstrating the efficacy of anti-BCMA antibodies. These results suggest that binding prevents BCMA cleavage by gamma-secretase. The antibody induced an increase in surface BCMA that varied depending on the cell line, with a greater fold increase 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 monkey plasma cells in the presence of BCMAxCD3 bispecific antibodies Enriched CD138 by unstimulated autologous T cells + Specific killing of human or cynomolgus monkey plasma cells was assessed by flow cytometry. Human or cynomolgus monkey bone marrow aspirates and blood were donated within 24 hours of collection. CD138 + Plasma cells were purified using EasySep Human CD138 according to the manufacturer's instructions. + Bone marrow was enriched by positive selection using a positive selection kit. PBMCs were isolated from whole blood by density separation. PBMCs were labeled with 1 μM Vybrant CFDA-SE fluorescent tracking dye. After labeling, 1 × 10 4 Enriched CD138 +Plasma cells were seeded in round-bottom 96-well 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 for 72 hours at 37°C. At the end of culture, viable CD138 + Plasma cells were analyzed by flow cytometry using fixable LIVE / DEAD dye and plasma cell-specific cell surface markers. Viability was normalized to the control condition (plasma cells in the presence of PBMCs only). T cell activation was assessed by flow cytometry. Activation was assessed by CD25-expressing CD2 + / CD4 + or CD2 + / CD8 + / CD16 - Reported as percentage of T cells. 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 a negative control (BCMA-binding control mAb or CD3-binding control bsAb) on primary human and cynomolgus monkey T cell activation and autologous plasma cell cytotoxicity. EC values ​​for each donor were used to evaluate cytotoxicity and T cell activation rates. 50 The values ​​are summarized in Table 30.

[0246] REGN5458 mediated cytotoxicity of primary human plasma cells from donors 1 and 2 in the presence of autologous T cells in a concentration-dependent manner, resulting in EC 50 The values ​​were 42.8 pM and 191 pM, respectively, with maximum percentage 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, resulting in CD8 + ECs in T cell activation 50 The values ​​were 214 pM and 860 pM, respectively, and CD8 + The maximum percentage of T cell activation was 2% and 36%, respectively. Plasma cell cytotoxicity in both donors and CD8 in donor 2 only.+ Increased T cell activation was observed at nanomolar concentrations of the CD3-binding control. No cytotoxicity or effect on T cell activation was observed with the BCMA-binding control at any of the concentrations tested in either donor.

[0247] REGN5458 mediated cytotoxicity of primary cynomolgus monkey plasma cells in a concentration-dependent manner in both donors, with an EC 50 was calculated for donor 1, but EC 50 The effect of REGN5458 on plasma cell cytotoxicity could not be determined. In both donors, treatment with REGN5458 increased plasma cell cytotoxicity (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 monkey plasma cells from donors 1 and 2, resulting in CD4 + EC for T cell activation 50 The values ​​are 28 0.1nM and 18.1nM, and CD8 + EC for T cell activation 50 The values ​​were 22.4 nM and 76.7 nM. The resulting maximum percentage of T cell activation was observed in donors 1 and 2, where CD4 + In T cells, they were 9% and 16%, respectively, and CD8 + In T cells, the rates were 12% and 17%, respectively.

[0248] No target cell killing was observed with the BCMA-binding control at any concentration tested in any of the cell lines evaluated. Some target cell killing and T cell activation in the presence of plasma cells from donor 2 was observed with the CD3-binding control at nanomolar concentrations. [Table 33]

[0249] Example 20: Anti-BCMA x anti-CD3 bispecific antibodies act synergistically with anti-PD-1 antibodies to enhance anti-tumor effects in vivo To determine whether the BCMAxCD3 bispecific antibody (Ab) synergizes with PD-1 blockade to provide superior antitumor efficacy in vivo, a syngeneic tumor study was performed in mice expressing human CD3. The results show that the combination of REGN5458 and PD-1 blockade provides superior antitumor efficacy than either REGN5458 or PD-1 blockade alone.

[0250] Implantation and measurement of syngeneic tumors: 1 x 106 cells engineered to express full-length human BCMA (MC38 / BCMA) into C57BL / 6 mice expressing human CD3deg instead of mouse CD3deg (CD3-humanized mice). 6 MC38 colon cancer cells were implanted subcutaneously. Tumors were allowed to establish for 3 days, at which point mice (n=6 or 7 per group) were treated with either a surrogate anti-mouse PD-1 antibody (clone RPM1-14) at 4 mg / kg or an isotype control Ab (clone 2A3) at 4 mg / kg, along with a CD3-binding control bispecific Ab (G; H4sH17664D) at a dose of 0.4 mg / kg, and BCMA x CD3 (G; REGN5458) at a dose of 0.04 mg / kg or 0.24 mg / kg. Specific treatment groups are shown in Table 31 below. [Table 34]

[0251] Mice were administered two more doses of these Abs on days 7 and 11 for a total of three doses, and tumor growth was assessed throughout the experiment.

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

[0253] The results indicate that the combination of REGN5458 and PD-1 blockade provides superior anti-tumor efficacy than either REGN5458 or PD-1 blockade alone. Notably, the results showed that on day 24 (the last day data was collected for all treatment groups), the combination of a BCMAxCD3 bispecific antibody and an anti-PD-1 antibody resulted in a statistically significant synergistic therapeutic effect in inhibiting tumor growth (Table 32, 0.04 mg / kg BCMAxCD3 and 4 mg / kg anti-PD-1). On day 24, using a two-way ANOVA, p<0.0001 was observed 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) the combination of anti-PD-1 and REGN5458 (0.04 mg / kg) + anti-PD-1 antibody (Group 2 vs. Group 6). On day 24, using a two-way ANOVA, p=0.0005 was observed between the combination of anti-PD-1 and REGN5458 (0.04 mg / kg) + anti-PD-1 antibody (Group 2 vs. Group 4). Increasing the dose of BCMAxCD3 bispecific antibody (0.24 mg / kg) in combination with PD-1 blockade resulted in tumor inhibition comparable to lower doses of bispecific antibody and PD-1 blockade in this experiment. The demonstrated synergy with lower doses of bispecific antibody is advantageous because the use of lower doses reduces the risk of adverse side effects. Similarly, the combination of BCMAxCD3 bispecific antibody with anti-PD-1 antibody demonstrated synergistic therapeutic effects at both doses of bispecific antibody (0.04 mg / kg and 0.24 mg / kg) in terms of the number of tumor-free mice 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: Anti-BCMA x anti-CD3 bispecific antibodies act synergistically with anti-PD-1 antibodies to enhance anti-tumor effects in vivo Similar results were obtained in a second experiment, identical to those discussed above in Example 20, except that the number of mice per group was 10 and the high dose of BCMAxCD3 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 indicate that the combination of REGN5458 and PD-1 blockade provides superior anti-tumor efficacy than either REGN5458 or PD-1 blockade alone. Notably, the results showed that on day 21 (the last day data was collected for all treatment groups), the combination of the BCMAxCD3 bispecific antibody and the anti-PD-1 antibody resulted in a synergistic therapeutic effect in inhibiting tumor growth (Table 35, 0.04 mg / kg BCMAxCD3 and 4 mg / kg anti-PD-1). At day 21, using a two-way ANOVA, p<0.0001 was observed 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 anti-PD-1 and REGN5458 (0.04 mg / kg) + anti-PD-1 antibody (Group 2 vs. Group 4), and (iii) the combination of anti-PD-1 and REGN5458 (0.4 mg / kg) + anti-PD-1 antibody (Group 2 vs. Group 6). As discussed above in Example 20, increasing doses of BCMA×CD3 bispecific antibody (0.4 mg / kg) in combination with PD-1 blockade were comparable to lower doses of bispecific antibody in combination with PD-1 blockade in this experiment. Comparable tumor inhibition was obtained. The demonstrated synergy with low doses of the bispecific antibody is advantageous because the use of lower doses reduces the risk of adverse side effects. Similarly, the combination of the BCMAxCD3 bispecific antibody with the anti-PD-1 antibody demonstrated a synergistic therapeutic effect at both doses of the bispecific antibody (0.04 mg / kg and 0.4 mg / kg) in terms of the number of tumor-free mice 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: Methods of treating multiple myeloma with anti-BCMA x anti-CD3 bispecific antibodies A Phase 1 / 2 study evaluating the safety, tolerability, preliminary antitumor activity, and pharmacokinetics (PK) of REGN5458 (an anti-BCMA x anti-CD3 bispecific antibody) has demonstrated meaningful clinical benefit in patients with relapsed or refractory multiple myeloma who have exhausted all treatment options, including proteasome inhibitors, immunomodulatory agents, and anti-CD38 antibody therapy.

[0257] Patients with hard-to-treat, advanced multiple myeloma that contains extramedullary (outside the bone marrow) and non-secretory (do not secrete detectable biomarkers) cancerous plasma cells are being studied as part of the REGN5458 program.

[0258] In multiple myeloma clinical trials, treatment assessment is based on the reduction of myeloma protein levels and eradication of myeloma cells. Assessment of myeloma protein response is based on the presence of myeloma protein in the patient's urine and blood. , is based on a decrease in the level of monoclonal (M) protein, a biomarker used to determine the extent of myeloma disease. Partial remission (PR) is defined as a 50% or greater decrease in serum / urine M protein, or a 50% or greater decrease in the difference between involved and uninvolved free light chain (FLC) levels, and a 50% or greater decrease in soft tissue plasmacytoma. Very good partial remission (VGPR) is defined as a 90% or greater decrease in serum / urine M protein, or a 90% or greater decrease in the difference between involved and uninvolved FLC levels, a 90% or greater decrease in soft tissue plasmacytoma, and detection of M protein by immunofixation rather than electrophoresis. Complete remission (CR) is defined as negative detection of M protein by immunofixation in serum and urine, complete disappearance of soft tissue plasmacytoma, and less than 5% plasma cells in bone marrow aspirates. Stringent complete remission is defined as the combination of a complete remission (as described above) and a normal FLC ratio ( K / λ ratio ≤ 4:1 or K and ≥1:2 for λ patients). Minimal residual disease (MRD), reflecting eradication of myeloma cells, is measured separately from M protein, and MRD negativity is defined as the absence of cancer plasma cells within 100,000 bone marrow cells.

[0259] Objectives: Both primary and secondary endpoints will be investigated.

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

[0261] The secondary objectives of the study are as follows (Phase 1 and Phase 2 parts): (1) To 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) to evaluate the PK properties of REGN5458; (3) characterize the immunogenicity of REGN5458; (4) Phase 1 portion only: To evaluate the preliminary antitumor activity of REGN5458 as measured by ORR; and (5) Phase 2 portion only: To evaluate the safety and tolerability of REGN5458.

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

[0263] The Phase 2 portion will begin once the RP2DR has been determined to further evaluate preliminary antitumor activity, safety and tolerability, PK properties, and biomarker responses in patients treated with REGN5458 monotherapy.

[0264] Each patient will receive 16 weekly (QW) infusions of REGN5458, followed by 12 booster doses of REGN5458 every two weeks (Q2W), according to their assigned dosing regimen.

[0265] Each patient will receive a primary dose of REGN5458, followed by a nominal dose if the primary dose is well tolerated. The primary dose of REGN5458 will be administered as a divided (split) infusion (preferably on two consecutive days, but no more than three days apart) at the assigned dose. If this primary dose is well tolerated, the patient will receive a higher nominal dose in the second week (preferably on consecutive days, but no more than three days apart) as a split infusion at the assigned dose, and from week three onwards, the nominal dose will be administered as a single infusion.

[0266] The dose escalation scheme provides for an approximately three-fold increase in the nominal dose for each successive dose cohort compared to the nominal dose of the previously evaluated dose cohort. Similarly, it provides for an approximately three-fold increase in the primary dose in Week 1 for each successive dose cohort. However, if, during the dose-limiting toxicity (DLT) observation period, a DLT occurs in one patient or two or more patients in a dose cohort experience a grade 2 or higher adverse event (excluding grade 2 or higher AEs clearly unrelated to the study drug), and the dose regimen is still deemed to be acceptable, the primary and nominal dose escalation will be no more than two-fold (i.e., a 100% increase) of the primary and nominal doses for each 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 deemed to be acceptable, then in the next dose cohort (i.e., DL3), the primary dose 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 twice that of the previous dose cohort.

[0267] Study Duration: The planned duration of the study for each patient will be up to approximately 24 months, including a screening period (up to 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 persistent clinical activity and safety (a total follow-up period of approximately 60 weeks).

[0268] Study population: Phase 1: Following a 4+3 design, up to seven DLT-evaluable patients can be enrolled in each dose cohort. If the dosing regimen at a given dose level (DL) is deemed tolerable and does not exceed the maximum tolerated dose (MTD), enrollment of up to three additional patients can begin at each DL (for a total of up to 10 patients at each DL). The actual sample size of these dose-escalation cohorts will vary depending on the number of observed patients with documented DLTs, the number of DLs performed, and the number of patients who drop out.

[0269] Phase 2: Approximately 10–14 patients evaluable for safety and efficacy. The analysis of these patients will be combined with the analysis of 6–10 patients treated with RP2DR in Phase 1, resulting in a total of 20 patients treated with RP2DR. This study will enroll MM patients who have exhausted all treatment options expected to provide meaningful clinical benefit, either through disease relapse, treatment-resistant disease, or intolerance or refusal of treatment. Furthermore, each patient must have progressed after at least three prior lines of therapy, including an anti-CD38 antibody, a proteasome inhibitor, and an immunomodulatory drug (IMiD). If a patient has previously been treated with an anti-CD38 antibody and has demonstrated refractory disease to both an IMiD and a proteasome inhibitor, they may be eligible for the study even if they have received fewer than three prior lines of therapy. Refractory disease is defined as lack of response or recurrence of MM within 60 days of 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 ECOG 2-part patients with improved ECOG status expected as a result of effective treatment Individual cases of patients with performance status can be discussed with the medical monitor regarding potential enrollment. 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 (e.g., hypercalcemia, renal failure, osteolytic lesions, or anemia) at study entry. 5. Patients must have measurable myeloma either by serum or urine evaluation of monoclonal components or by analysis of serum (FLC) Measurable disease is defined as one or more of the following: Serum M protein ≥ 1 g / dL; b. Urinary M protein ≥ 200 mg / 24 hours, and / or c. An FLC assay containing an abnormal serum FLC ratio or an FLC level of 10 mg / dL or greater -Patients with immunoglobulin A (IgA) myeloma but without measurable M protein may be enrolled if they have elevated quantitative IgA levels and can be followed longitudinally -Patients with non-secretory MM may be considered for enrollment after discussion with the sponsor, including the feasibility of planning response assessment according to IMWG guidelines. 6.Disease progression according to IMWG criteria 7. Patients with MM who have exhausted all treatment options expected to provide meaningful clinical benefit, either through disease relapse, treatment-refractory disease, or intolerance or refusal of treatment, including any of the following: a. Progression or subsequent progression of at least three lines of disease or intolerance to treatment including proteasome inhibitors, IMiDs, and anti-CD38 antibodies; or b. Have disease that has progressed on or after anti-CD38 antibodies and is "double 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 relapse within 60 days of the last treatment. 8. Adequate hematological function as measured by: Platelet count > 50 x 10 9 / L. To meet this platelet eligibility requirement, patients may not have received a platelet transfusion within 7 days. b. ANC>1.0×10 9 / L patients may not have received granulocyte colony-stimulating factor (G-CSF) within 2 days to meet this absolute neutrophil count eligibility requirement. C. Hemoglobin > 8.0 g / dL 9. Adequate liver function defined as: a. Total bilirubin ≤ 1.5 × ULN b. Transaminases (ALT, AST) ≦2.5 × ULN c. Alkaline phosphatase ≤ 2.5 × ULN -Patients with Gilbert syndrome do not need to meet this total bilirubin requirement unless their total bilirubin has changed from their baseline value. 10. Cockcroft-Gault serum creatinine clearance >30 mL / min -Patients with Cockcroft-Gault creatinine clearance who do not meet the eligibility criteria may be eligible for enrollment if their measured creatinine clearance (based on a 24-hour urine collection or other reliable method) is >30 mL / min. 11. If previously treated with CAR T therapy or gene therapy products, patients must have recovered from the toxicities of this therapy 12. A life expectancy of at least 6 months 13. Willingness and ability to comply with clinic visits and study-related procedures, including serial bone marrow evaluations according to protocol schedule. - Bone marrow aspirate and biopsy, or other tissue infiltrated with malignant plasma cells, must be provided at screening for assessment of BCMA levels in the malignant cells, but demonstration of BCMA levels is not required prior to enrollment. 14. Provide informed consent signed by the study patient 15. Ability to understand and complete all study-related questionnaires.

[0271] Exclusion Criteria - Patients who meet any of the following criteria will be excluded from the trial. 1. Presence of plasma cell leukemia, Waldenström's macroglobulinemia (lymphoplasmacytic lymphoma), or POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal proteins, and skin changes) 2. Patients with known MM brain lesions or meningeal involvement with MM (suspected central nervous system (CNS) myeloma must be ruled out by radiographic imaging and / or lumbar puncture, as appropriate) 3. History of neurodegenerative conditions or CNS movement disorders 4. Cardiac ejection fraction <40% by echocardiogram or multi-gated acquisition (MUGA) scan. 5. Ongoing systemic corticosteroid treatment with more than 10 mg prednisone or anti-inflammatory equivalent per day within 72 hours of starting study drug 6. Vaccination with a replication-capable vector within 28 days prior to the first study drug administration Treatment with systemic standard or investigational anti-myeloma therapy within 7.5 half-lives or 28 days prior to the first dose of study 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. Infection requiring hospitalization or treatment with IV anti-infectives within 2 weeks of first dose of study drug 10. Uncontrolled infection with human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV); or other uncontrolled infections a. Patients with HIV who have controlled infection (undetectable viral load on spontaneous or stable antiviral regimen and CD4 count greater than 350 cells / microliter) are allowed. b. Patients with hepatitis B (hepatitis B surface antigen test positive [HepBsAg+]) whose infection is controlled (serum HBV DNA polymerase chain reaction [PCR] below the limit of detection and receiving hepatitis B antiviral therapy) are permitted. c. Patients who are HCV antibody positive (HCV Ab+) and have controlled infection (HCV RNA undetectable by PCR, either spontaneously or in response to a previous successful course of anti-HCV therapy) are allowed. 11. Documented history of severe allergic or acute hypersensitivity reactions due to previous antibody treatment A severe allergic reaction is defined for this purpose as meeting the criteria for CTCAE v5.0 Grade 3 or Grade 4 severity (i.e., characterized by bronchospasm; or life-threatening consequences; or requiring IV intervention, other emergency intervention, or hospitalization for clinical sequelae) or requiring an emergency room visit. 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. Have documented hypersensitivity to both allopurinol and rasburicase 14. History of allogeneic stem cell transplant at any time, or history of autologous stem cell transplant within 12 weeks of starting study treatment 15. Members of the clinical site trial team or their immediate families, unless prior approval by the sponsor is granted 16. Women of childbearing potential (WOCBP) with a positive serum beta-human chorionic gonadotropin (β-hCG) pregnancy test were ineligible for this study. 17. Patients committed to the institution by order issued by either a judicial or administrative authority 18. Pregnant or breastfeeding women 19. Women* or men** of childbearing potential who are unwilling to practice highly effective contraception before the first dose / initiation of first treatment, during the trial, and for at least 6 months after the last dose. *Highly effective contraceptive methods for women include: a. Stable use of combined (estrogen and progestogen-containing) hormonal contraception (oral, intravaginal, transdermal) or progestogen-only hormonal contraception (oral, injectable, implantable) associated with ovulation inhibition for at least two menstrual cycles prior to screening, b. Intrauterine device (IUD), intrauterine hormone-releasing system (IUS) C. Bilateral tubal ligation d. Vasectomized partner (if the male vasectomized partner is the study participant's only sexual partner and the partner has a medical evaluation of the surgical success of the operation) e. and / or sexual abstinence†,‡. Women of childbearing potential are defined as women who are capable of bearing children after menarche and after menopause, unless they are permanently infertile. Permanent methods of contraception include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy. Postmenopausal status is defined as the absence of menstruation for 12 months without an alternative medical cause. High follicle-stimulating hormone (FSH) levels in the postmenopausal range can be used to confirm postmenopausal status in women not using hormonal contraceptives or hormone replacement therapy. However, in the absence of 12 months of amenorrhea, a single FSH measurement is insufficient to determine the occurrence of postmenopausal status. **With the exception of vasectomy (which has been medically evaluated for surgical success), highly effective contraceptive methods for men include condoms or sexual abstinence†,‡. †Sexual abstinence is considered highly effective only if it is defined as abstinence from heterosexual intercourse for the entire duration of the risks associated with the study treatment. The reliability of sexual abstinence must be evaluated in relation to the duration of the clinical trial and the patient's preferred usual lifestyle. ‡Periodic abstinence (calendar, thermo-thermal, postovulatory), withdrawal (withdrawal), spermicide only, and lactational amenorrhea (LAM) are not acceptable methods of contraception. Female condoms and male condoms should not be used together.

[0272] Treatment: REGN5458 for IV infusion will be provided by the sponsor as a liquid in sterile, single-use vials, each containing REGN5458 at a concentration of 10 mg / mL.

[0273] A pharmacist or other qualified individual will be identified at each site and prepared to administer REGN5458.

[0274] For the primary and first nominal doses, treatment will be administered as two separate 4-hour infusions, preferably on two consecutive days, but no more than 3 days apart (e.g., Week 1, Day 1 and Week 1, Day 2). The first nominal dose administered as a single infusion will be administered over 4 hours. If this infusion is well tolerated without any grade CRS or IRR events, then The REGN5458 infusion may be shortened to 2 hours per the investigator's clinical judgment. If this 2-hour REGN5458 infusion is well tolerated without CRS or IRR events of any grade, subsequent REGN5458 infusions may be shortened to 1 hour per the investigator's clinical judgment. Thereafter, each dose of REGN5458 may be administered for the IV infusion period associated with the absence of CRS or IRR events.

[0275] After the patient receives the primary and first nominal dose, treatment may be administered as a single infusion. The investigator may choose to split the dose into two separate infusions over two days (preferably consecutively, but no more than three days apart).

[0276] The dose of REGN5458 each patient receives will depend on their DL cohort assignment. The dose administered in each DL is a fixed dose and is not dependent on the patient's weight or body surface area (BSA).

[0277] Survey of evaluation items: For the Phase 1 portion of the study, the primary endpoints are: (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 (TEAEs) and adverse events of special interest (AESIs) during REGN5458 treatment and up to 14 months after the last dose.

[0278] For the phase 2 portion of the study, the primary endpoint is ORR measured using the International Myeloma Working Group (IMWG) criteria up to 14 months after the last dose.

[0279] Secondary endpoints (Phase 1 and Phase 2) are as follows: (1) Serum REGN5458 concentrations over time, (2) the incidence over time of anti-drug antibodies (ADAs) resulting from treatment with REGN5458; (3) DOR using IMWG criteria up to 14 months after the last dose; (4) PFS measured using IMWG criteria up to 14 months after the last dose; (5) MRD-negative status using IMWG criteria up to 14 months after the last dose, (6) OS up to 14 months after the last dose. (7) Phase 1 portion only - ORR measured using IMWG criteria up to 14 months after the last dose, and (8) Phase 2 portion only - Incidence and severity of TEAEs and AESIs during REGN5458 treatment up to 14 months after the last dose.

[0280] Procedures and Assessment: Screening only: demographics, complete physical examination, height, medical and oncological history, revised International Staging System (ISS) stage (including chromosomal abnormalities and β2-microglobulin), brain magnetic resonance imaging (MRI), echocardiogram or multi-gated acquisition scan (MUGA), HIV / HBV / HCV testing, 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 (AEs), concomitant medications (CMs).

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

[0283] Blood samples will be collected for serum drug concentration analysis and ADA assessment.

[0284] Statistical Plan: Phase 1 portion: Following a 4+3 design, up to seven DLT-evaluable patients can be enrolled in each dose cohort. If the dosing regimen for a given DL is deemed tolerable and does not exceed the MTD, enrollment of up to three additional patients can begin in each DL (for a total of up to 10 patients in each DL). The actual sample size of these dose escalation cohorts will vary depending on the number of observed patients with documented DLTs, the number of DLs administered, and the number of patients who drop out.

[0285] Phase 2 portion: A sample size of 20 patients will be 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 will receive the investigational drug in RP2DR, and these will contribute to a total sample size of 20 patients for analysis. The remaining 10-14 patients evaluable for safety and efficacy will be enrolled in the Phase 2 portion.

[0286] Phase 2 Conditions for Enrollment Pause and Safety Review: To further evaluate tolerability with RP2DR, the cumulative percentage of patients experiencing unacceptable toxicity (cUT) among patients treated with RP2DR from the Phase 1 and Phase 2 portions will be estimated. The stopping limit will be included based on a frequentist interval utilizing the lower limit of the one-sided 80% confidence interval (CI). Enrollment into the Phase 2 portion will be paused if the lower limit of the one-sided 80% CI of the estimated cUT excludes 20%.

[0287] This evaluation will be performed for the first 12 patients treated with RP2DR in the phase 1 and phase 2 portions. The evaluation will be repeated for the first 16 patients treated with RP2DR in the phase 1 and phase 2 portions (i.e., when four additional patients are enrolled). If the cUT rate excludes 20% (i.e., four or more patients of the first 12 patients treated with RP2DR have unacceptable toxicity or five or more patients of the first 16 patients have unacceptable toxicity), further enrollment in the phase 2 portion will be paused. If four patients are observed to have unacceptable toxicity before enrolling 12 patients or five patients are observed to have unacceptable toxicity before enrolling 16 patients, further enrollment in the phase 2 portion will also be paused.

[0288] Preliminary Results: Forty-nine patients (median age 64; 31% were 70 years or older) had previously undergone autologous stem cell transplantation, with 33 patients (67.3%) having received a median of five prior lines of systemic therapy (range, 2-17). Multiple myeloma immune subtypes at study entry 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. Of the 1,000 patients, 30.6% were at least triple-refractory, 30.6% were quadruple-refractory, and 57.1% were quintuple-refractory. Eighty percent of patients were refractory to carfilzomib, and 92% were refractory to pomalidomide. Patients were treated in cohorts receiving REGN5458 at six dose levels, ranging from 3 mg to 96 mg. The median follow-up period was 2.63 months (range, 0.5-13.4). 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, most commonly anemia (22.4%), neutropenia (14.3%), and lymphopenia (12.2%). The most common serious AEs were infections (20.4%) and CRS (12.2%). No patients experienced grade 3 or higher CRS, and fewer than 40% of patients experienced CRS. CRS occurred primarily during the first week of treatment and was 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% across all dose levels (29.2% for dose levels 1-3, 41.2% for dose levels 4 and 5, and 62.5% for dose level 6), with 95% of responders achieving at least a very good partial response (VGPR) and 42.1% having a complete or stringent CR. Four of seven (57%) evaluable patients achieved 10 -5Minimal residual disease (MRD)-negative status was achieved with a sensitivity of . Tumor response, as assessed by immunohistochemistry, 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 greater, 52.6% of responders had a DOR of 6 months or greater, and 36.8% of responders had a DOR of 8 months or greater. The median observed duration of response was 6.01 months. Responses occurred early (most by week 4) and deepened over time. Of responding patients with 6 months or greater follow-up, 83% (10 / 12) had a continuous response for up to 13 months. To date, 74% of responders are receiving ongoing 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. Patients with bone marrow plasmacytosis <50% had an ORR of 71.4%, while patients with bone marrow plasmacytosis ≥50% had an ORR of 9.1%. Of patients with bone marrow plasmacytosis <50%, 35.7% achieved a stringent complete response and 35.7% achieved a very good partial response. Meaningful improvements in overall health status / quality of life were observed at week 4 and maintained through week 24 (to date). Tumor response did not correlate with BCMA expression as assessed by immunohistochemistry. A summary of the observed responses is shown below in Table 37. [Table 40]

[0291] It is therefore contemplated that the bispecific antibodies described herein can be used at doses of at least 3 mg administered once weekly either as divided doses (e.g., weeks 1 and 2) or as a single infusion (e.g., from week 3 onwards) to treat human subjects afflicted with BCMA-expressing cancers such as multiple myeloma, particularly those who are refractory to previous treatments (e.g., triple-, quadruple-, or quintuple-resistant) or who have relapsed after previous treatments.

[0292] The present 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, and all such modifications are intended to fall within the scope of the appended claims.

Claims

1. 1. A pharmaceutical composition comprising a bispecific antibody for treating refractory multiple myeloma in a subject in need thereof, the composition comprising: the bispecific antibody comprises a first antigen-binding domain that specifically binds to human B-cell maturation antigen (BCMA) and a second antigen-binding domain that specifically binds to human CD3; The first antigen-binding domain comprises: (a) a heavy chain variable region (HCVR) comprising three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 68, 70, and 72, respectively; and (b) a light chain variable region (LCVR) comprising three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively; The second antigen-binding domain comprises: (a) an HCVR comprising three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 92, 94, and 96, respectively; and (b) an LCVR comprising three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively; A pharmaceutical composition, wherein the refractory multiple myeloma is triple refractory to an anti-CD38 antibody, a proteasome inhibitor, and an immunomodulatory agent.

2. 2. The pharmaceutical composition of claim 1, wherein the HCVR of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 66 and the LCVR of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

82.

3. 2. The pharmaceutical composition of claim 1, wherein the HCVR of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 90 and the LCVR of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

82.

4. 2. The pharmaceutical composition of claim 1, wherein the HCVR of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 66, the LCVR of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 82, the HCVR of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 90, and the LCVR of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

82.

5. 5. The pharmaceutical composition of claim 4, wherein 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 SEQ ID NO: 127, and a common light chain comprising the amino acid sequence of SEQ ID NO:

129.

6. 1. A pharmaceutical composition comprising a bispecific antibody for treating refractory multiple myeloma in a subject in need thereof, the composition comprising: the bispecific antibody comprises a first antigen-binding domain that specifically binds to human B-cell maturation antigen (BCMA) and a second antigen-binding domain that specifically binds to human CD3; The first antigen-binding domain comprises: (a) a heavy chain variable region (HCVR) comprising three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 68, 70, and 72, respectively; and (b) a light chain variable region (LCVR) comprising three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively; The second antigen-binding domain comprises: (a) an HCVR comprising three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 100, 102, and 104, respectively; and (b) an LCVR comprising three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively; A pharmaceutical composition, wherein the refractory multiple myeloma is triple refractory to an anti-CD38 antibody, a proteasome inhibitor, and an immunomodulatory agent.

7. 7. The pharmaceutical composition of claim 6, wherein the HCVR of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 66 and the LCVR of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

82.

8. The pharmaceutical composition of claim 6, wherein the HCVR of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 98 and the LCVR of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

82.

9. 7. The pharmaceutical composition of claim 6, wherein the HCVR of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 66, the LCVR of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 82, the HCVR of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 98, and the LCVR of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

82.

10. 10. The pharmaceutical composition of claim 9, wherein 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 SEQ ID NO: 128, and a common light chain comprising the amino acid sequence of SEQ ID NO:

129.

11. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the subject has been diagnosed with a multiple myeloma immune subtype selected from immunoglobulin G, immunoglobulin A, lambda light chain, or kappa light chain, or the subject has extramedullary plasmacytoma.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the subject is quadruple-refractory or quintuple-refractory to previous treatment.

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