Methods for treating multiple myeloma using bispecific anti-BCMA x anti-CD3 antibodies

Abstract

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

Description

[0001] Reference to a sequence listing

[0002] This application incorporates by reference a sequence listing submitted in computer readable form as File 10702WO01-Sequence, which was created on December 4, 2020 and contains 81,956 bytes. Technical Field

[0003] The present invention relates to bispecific antigen binding molecules (eg, bispecific antibodies) that bind BCMA and CD3 and methods of using the same. Background Art

[0004] B-cell maturation antigen (BCMA), also known as TNFRSF17 or CD269, is a type III transmembrane protein that lacks a signal peptide and contains a cysteine-rich extracellular domain. BCMA, together with closely related proteins, promotes B-cell survival at different stages of development. BCMA is expressed exclusively in cells of the B-cell lineage, specifically in the interfollicular regions of germinal centers, as well as in 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 elevated 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 Macroglobulinemia, Burkitt's lymphoma, and diffuse large B-cell lymphoma. Tai et al., Immunotherapy, 7(11): 1187-1199, 2015.

[0005] CD3 is a homodimer or heterodimer antigen expressed on T cells that binds to the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed by the dimerization association of two of the following four different chains: ε, ζ, δ and γ. The CD3 dimer arrangement includes γ / ε, δ / ε and ζ / ζ. Antibodies against CD3 have been shown to aggregate CD3 on T cells, thereby causing T cell activation in a manner similar to the participation of TCR by MHC molecules loaded with peptides. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes involving T cell activation. In addition, bispecific antibodies capable of binding CD3 and target antigens have been proposed for therapeutic uses involving targeting T cell immune responses to tissues and cells expressing target antigens.

[0006] The overall survival rate of patients with multiple myeloma refractory to multiple treatments is reduced (triple and quadruple refractory: 9.2 months, quintuple refractory: 5.6 months). Gandhi U. et al., Leukemia 33: 2266-2275, 2013. Antigen binding molecules targeting BCMA, including bispecific antigen binding molecules that bind both BCMA and CD3, will be useful in therapeutic settings where specific targeting of cells expressing BCMA and T cell-mediated killing are desired. Summary of the invention

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

[0008] In some cases, the bispecific antigen binding molecule is present in less than about 10 -9 EC of M 50 In vitro activation of T cells. In some cases, the bispecific antigen binding molecules mediate in vitro T cell killing of BCMA-expressing tumor cell lines, EC 50 Less than about 10 -9 M. In some cases, the bispecific antigen binding molecules mediate in vitro autologous T cell killing of primary myeloma cells expressing BCMA, EC 50 Less than about 10 -8 M. In some embodiments, the bispecific antigen binding molecule interacts with amino acid residues 1 to 43 of BCMA as shown in SEQ ID NO:115.

[0009] In some cases, the target tumor cell is a plasma cell. In some cases, the target tumor cell is from a patient with multiple myeloma, or from another B cell disorder, which is characterized in part by having a B cell expressing BCMA. In some cases, the bispecific antigen binding molecule inhibits the proliferation of tumor cells expressing BCMA at a dosage of about 0.04 mg / kg to about 4.0 mg / kg. In some cases, the dosage is 0.04 mg / kg, 0.4 mg / kg or 4 mg / kg. In some cases, the dosage is about 0.001 mg / kg, 0.002 mg / kg, 0.003 mg / kg, 0.004 mg / kg, 0.005 mg / kg, 0.006 mg / kg, 0.007 mg / kg, 0.008 mg / kg, 0.009 mg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0. 7mg / kg, 0.8mg / kg, 0.9mg / kg, 1mg / kg, 1.1mg / kg, 1.2mg / kg, 1.3mg / kg, 1.4mg / kg, 1.5mg / kg, 1.6mg / kg, 1.7mg / kg, 1.8mg / kg, 1.9mg / kg, 2mg / kg, 3mg / kg, 4mg / kg, 5mg / kg, 6mg / kg, 7mg / kg, 8mg / kg, 9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg. In some embodiments, the dosage is applied to patients in need at least twice a week for at least seven times. In some embodiments, the dosage is applied to patients in need at least weekly. In some embodiments, the dosage is applied to patients at least every two weeks. In some embodiments, the dose is administered to the patient at least every four weeks. In some cases, the bispecific antigen binding molecule inhibits the proliferation of BCMA+ tumor cells selected from myeloma cells, lymphoma cells, and leukemia cells. In some cases, the bispecific antigen binding molecule inhibits the proliferation of BCMA+ tumor cells selected from H929 cells, MOLP-8 cells, and OPM cells.

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

[0011] In some embodiments, the isolated bispecific antigen binding molecule comprises a first antigen binding domain comprising: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising an amino acid sequence of SEQ ID NO: 66; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising an amino acid sequence of SEQ ID NO: 82. In some cases, the first binding domain of the bispecific antigen binding molecule comprises a HCDR1 comprising an amino acid sequence of SEQ ID NO: 68, a HCDR2 comprising an amino acid sequence of SEQ ID NO: 70, and a HCDR3 comprising an amino acid sequence of SEQ ID NO: 72. In some cases, the first binding domain of the bispecific antigen binding molecule comprises a LCDR1 comprising an amino acid sequence of SEQ ID NO: 84, a LCDR2 comprising an amino acid sequence of SEQ ID NO: 86, and a LCDR3 comprising an amino acid sequence of SEQ ID NO: 88. In some cases, the first antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO:66 and a 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 complementary determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising an amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 98; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising an amino acid sequence of SEQ ID NO: 82. In some cases, the second antigen binding domain comprises: (a) HCDR1 comprising an amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 100; (b) HCDR2 comprising an amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 102; and (c) HCDR3 comprising an amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 104. In some cases, the second antigen binding domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 84, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a 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 NO: 92, 94, 96, respectively; and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NO: 84, 86, 88, respectively; or (b) HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NO: 100, 102, 104, respectively; and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NO: 84, 86, 88, respectively. In some cases, the second antigen binding domain comprises: (a) a HCVR comprising the amino acid sequence of SEQ ID NO:90, and a LCVR comprising the amino acid sequence of SEQ ID NO:82; or (b) a HCVR comprising the amino acid sequence of SEQ ID NO:98, and a 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, and HCDR3 domains, which respectively comprise the amino acid sequences of SEQ ID NOs: 68, 70, and 72, and LCDR1, LCDR2, and LCDR3 domains, which respectively comprise the amino acid sequences of SEQ ID NOs: 84, 86, and 88.

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

[0015] In another aspect, the present invention provides an isolated bispecific antigen-binding molecule comprising: (a) a first antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 68, 70, and 72, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively; and (b) a second antigen-binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 100, 102, and 104, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid 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 a HCVR comprising the amino acid sequence of SEQ ID NO:66 and a LCVR comprising the amino acid sequence of SEQ ID NO:82; and (b) a second antigen binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:98 and a 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 a 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 a 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 a HCVR / LCVR amino acid sequence pair selected from 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 a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain selected from 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 a 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 CDRs of a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 90 / 82 and 98 / 82.

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

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

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

[0020] In another aspect, the present invention provides a pharmaceutical composition comprising a bispecific antibody molecule (eg, a bispecific antibody) as discussed above or herein, and a pharmaceutically acceptable carrier or diluent.

[0021] In another aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a bispecific antigen binding molecule (eg, a bispecific antibody) discussed above or 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 for inhibiting the growth of plasma cell tumors in a subject, including administering to the subject a bispecific antigen binding molecule as described above or as discussed herein or a pharmaceutical composition comprising the bispecific antigen binding molecule. In some cases, plasma cell tumors are multiple myeloma. In some cases, the method also includes administering a second therapeutic agent or treatment regimen. In some embodiments, the second therapeutic agent includes, for example, an anti-tumor agent (e.g., a chemotherapeutic agent, including melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), obendamustine (obendamustine, bendamustine (Treanda)) or any other chemotherapeutic agent known to be effective for treating plasma cell tumors in a subject). In some embodiments, the second therapeutic agent includes a steroid. In some embodiments, the second therapeutic agent includes a targeted therapy comprising thalidomide, lenalidomide and bortezomib, which are approved for the treatment of newly diagnosed patients. For example, lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab and daratumumab are examples of second therapeutic agents for effective treatment of relapsed myeloma. In certain embodiments, the second therapeutic agent is a regimen comprising radiotherapy or stem cell transplantation. In certain embodiments, the second therapeutic agent may be an immunomodulator. In certain embodiments, the second therapeutic agent may be a proteasome inhibitor, comprising bortezomib (Velcade), carfilzomib / Kyprolis, ixazomib / Ninlaro. In certain embodiments, the second therapeutic agent can be a histone deacetylase inhibitor, such as panobinostat / Farydak. In certain embodiments, the second therapeutic agent can be a monoclonal antibody, an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a checkpoint inhibitor, or a combination thereof.

[0025] In another aspect, the invention provides a method of treating a patient suffering from multiple myeloma or another B-cell malignancy expressing BCMA, wherein the method comprises administering to the subject an isolated bispecific antigen binding molecule as described above or herein, or a pharmaceutical composition comprising the bispecific antigen binding molecule. In some cases, the B-cell malignancy expressing BCMA is selected from 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. In some cases, the method also includes administering a second therapeutic agent. In some embodiments, the second therapeutic agent includes an anti-tumor agent (chemotherapeutic agent); DNA alkylating agent; immunomodulator; proteasome inhibitor; histone deacetylase inhibitor; radiotherapy; stem cell transplantation; immunomodulator; monoclonal antibody interacting with an antigen expressed on the surface of a tumor cell; a monoclonal antibody different from the monoclonal antibody described herein, which can interact with different antigens on the surface of plasma cells; a bispecific antibody, one arm of which binds to an antigen on the surface of a tumor cell and the other arm binds to an antigen on a T cell; an antibody drug conjugate; a bispecific antibody conjugated to an anti-tumor agent; a checkpoint inhibitor, such as a checkpoint inhibitor targeting 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 cemipril monoclonal antibody (REGN2810). In certain embodiments, the checkpoint inhibitor may be selected from a PD-L1 inhibitor, such as atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Durvalumab / Imfinzi). In certain embodiments, the checkpoint inhibitor may be selected from a CTLA-4 inhibitor, such as ipilimumab (Yervoy). Other combinations that may be used in combination with the antibodies of the present invention are described above.

[0026] In another aspect, the present invention provides a method for treating a patient with a tumor expressing BCMA, wherein the method comprises administering to a subject a bispecific antigen binding molecule as described above or herein or a pharmaceutical composition comprising the same 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 Cemipirimab (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 tumors expressing BCMA.

[0027] In another aspect, the present invention provides a bispecific antigen binding molecule discussed above or herein or a pharmaceutical composition discussed above or herein for use in treating a disease or condition associated with BCMA expression. In some cases, the disease or condition is cancer. In some embodiments, the cancer is multiple myeloma. In some cases, the disease or condition is Castleman's disease. In some cases, the antigen binding molecule is used in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof, optionally wherein the anti-PD-1 antibody is semimiprimonab (REGN2810).

[0028] The present invention further includes the use of the bispecific antigen binding molecules described above or discussed herein in the preparation of a medicament for treating a disease or condition associated with BCMA expression. In some cases, the disease or condition is cancer. In some embodiments, the cancer is multiple myeloma. The present invention also includes bispecific antigen binding molecules (e.g., bispecific antibodies) for treating BCMA+ cancers (e.g., multiple myeloma) in a subject.

[0029] In another aspect, the present invention provides a method for 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 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: 92, 94, 96, 84, 86 and 88, respectively, wherein the bispecific antibody is administered to the subject weekly at a dose of at least 1 mg.In some cases, the bispecific antibody is administered to a subject at a dose of at least 1 mg per week or two weeks; at least 1.5 mg per week or two weeks; at least 2.0 mg per week or two weeks; at least 2.5 mg per week or two weeks; at least 3.0 mg per week or two weeks; at least 3.5 mg per week or two weeks; at least 4 mg per week or two weeks; at least 5 mg per week or two weeks; at least 6 mg per week or two weeks; at least 7 mg per week or two weeks; at least 10 mg per week or two weeks. 8 mg; at least 9 mg per week or every two weeks; at least 10 mg per week or every two weeks; at least 15 mg per week or every two weeks; at least 20 mg per week or every two weeks; at least 25 mg per week or every two weeks; at least 30 mg per week or every two weeks; at least 35 mg per week or every two weeks; at least 40 mg per week or every two weeks; at least 45 mg per week or every two weeks; at least 50 mg per week or every two weeks; at least 55 mg per week or every two weeks; at least 60 mg per week or every two weeks; at least 70 mg per week or every two weeks; at least 80 mg per week or every two weeks; at least 90 mg per week or every two weeks; at least 100 mg per week or every two weeks; at least 110 mg per week or every two weeks; at least 120 mg per week or every two weeks; at least 130 mg per week or every two weeks; at least 140 mg per week or every two weeks; at least 150 mg per week or every two weeks At least 65 mg every two weeks; at least 70 mg every week or every two weeks; at least 75 mg every week or every two weeks; at least 80 mg every week or every two weeks; at least 85 mg every week or every two weeks; at least 90 mg every week or every two weeks; at least 95 mg every week or every two weeks; at least 100 mg every week or every two weeks; at least 150 mg every week or every two weeks; at least 200 mg every week or every two weeks; at least 250 mg every week or every two weeks; at least 300 mg every week or every two weeks; at least 400 mg every week or every two weeks , at least 350 mg; at least 400 mg per week or every two weeks; at least 450 mg per week or every two weeks; at least 500 mg per week or every two weeks; at least 550 mg per week or every two weeks; at least 600 mg per week or every two weeks; at least 650 mg per week or every two weeks; at least 700 mg per week or every two weeks; at least 750 mg per week or every two weeks; at least 800 mg per week or every two weeks; at least 850 mg per week or every two weeks; or at least 900 mg per week or every 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 for 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 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, which comprise 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, which comprise the amino acid sequences of SEQ ID NOs: 100, 102, 104, 84, 86 and 88, respectively, wherein the bispecific antibody is administered to the subject weekly at a dose of at least 1 mg.In some cases, the bispecific antibody is administered to a subject at a dose of at least 1 mg per week or two weeks; at least 1.5 mg per week or two weeks; at least 2.0 mg per week or two weeks; at least 2.5 mg per week or two weeks; at least 3.0 mg per week or two weeks; at least 3.5 mg per week or two weeks; at least 4 mg per week or two weeks; at least 5 mg per week or two weeks; at least 6 mg per week or two weeks; at least 7 mg per week or two weeks; at least 10 mg per week or two weeks. 8 mg; at least 9 mg per week or every two weeks; at least 10 mg per week or every two weeks; at least 15 mg per week or every two weeks; at least 20 mg per week or every two weeks; at least 25 mg per week or every two weeks; at least 30 mg per week or every two weeks; at least 35 mg per week or every two weeks; at least 40 mg per week or every two weeks; at least 45 mg per week or every two weeks; at least 50 mg per week or every two weeks; at least 55 mg per week or every two weeks; at least 60 mg per week or every two weeks; at least 70 mg per week or every two weeks; at least 80 mg per week or every two weeks; at least 90 mg per week or every two weeks; at least 100 mg per week or every two weeks; at least 110 mg per week or every two weeks; at least 120 mg per week or every two weeks; at least 130 mg per week or every two weeks; at least 140 mg per week or every two weeks; at least 150 mg per week or every two weeks At least 65 mg every two weeks; at least 70 mg every week or every two weeks; at least 75 mg every week or every two weeks; at least 80 mg every week or every two weeks; at least 85 mg every week or every two weeks; at least 90 mg every week or every two weeks; at least 95 mg every week or every two weeks; at least 100 mg every week or every two weeks; at least 150 mg every week or every two weeks; at least 200 mg every week or every two weeks; at least 250 mg every week or every two weeks; at least 300 mg every week or every two weeks; at least 400 mg every week or every two weeks , at least 350 mg; at least 400 mg per week or every two weeks; at least 450 mg per week or every two weeks; at least 500 mg per week or every two weeks; at least 550 mg per week or every two weeks; at least 600 mg per week or every two weeks; at least 650 mg per week or every two weeks; at least 700 mg per week or every two weeks; at least 750 mg per week or every two weeks; at least 800 mg per week or every two weeks; at least 850 mg per week or every two weeks; or at least 900 mg per week or every 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 discussed above or herein, the bispecific anti-BCMA x anti-CD3 antibody can be administered in a dosing regimen that includes a split initial dose. In some embodiments, the bispecific anti-BCMA x anti-CD3 antibody is administered to the subject weekly at a dose of at least 1 mg. In some embodiments, the dose of the bispecific anti-BCMA x anti-CD3 antibody is administered to the subject weekly at a dose of 3 mg to 900 mg. In some cases, the bispecific antibody is administered to the subject at the following doses: at least 1 mg per week or every two weeks; at least 1.5 mg per week or every two weeks; at least 2.0 mg per week or every two weeks; at least 2.5 mg per week or every two weeks; at least 3.0 mg per week or every two weeks; at least 3.5 mg per week or every two weeks; at least 4 mg per week or every two weeks; at least 5 mg per week or every two weeks; at least 6 mg per week or every two weeks; at least 7 mg per week or every two weeks; at least 10 mg per week or every two weeks; at least 15 mg per week or every two weeks; at least 16 mg per week or every two weeks; at least 17 mg per week or every two weeks; at least 18 mg per week or every two weeks; at least 19 mg per week or every two weeks; at least 20 mg per week or every two weeks; at least 21 mg per week or every two weeks; at least 22 mg per week or every two weeks; at least 24 mg per week or every two weeks; at least 25 mg per week or every two weeks; at least 26 mg per week or every two weeks; at least 27 mg per week or every two weeks; at least 28 mg per week or every two weeks 8 mg; at least 9 mg per week or every two weeks; at least 10 mg per week or every two weeks; at least 15 mg per week or every two weeks; at least 20 mg per week or every two weeks; at least 25 mg per week or every two weeks; at least 30 mg per week or every two weeks; at least 35 mg per week or every two weeks; at least 40 mg per week or every two weeks; at least 45 mg per week or every two weeks; at least 50 mg per week or every two weeks; at least 55 mg per week or every two weeks; at least 60 mg per week or every two weeks; at least 70 mg per week or every two weeks; at least 80 mg per week or every two weeks; at least 90 mg per week or every two weeks; at least 100 mg per week or every two weeks; at least 110 mg per week or every two weeks; at least 120 mg per week or every two weeks; at least 130 mg per week or every two weeks; at least 140 mg per week or every two weeks; at least 150 mg per week or every two weeks At least 65 mg every two weeks; at least 70 mg every week or every two weeks; at least 75 mg every week or every two weeks; at least 80 mg every week or every two weeks; at least 85 mg every week or every two weeks; at least 90 mg every week or every two weeks; at least 95 mg every week or every two weeks; at least 100 mg every week or every two weeks; at least 150 mg every week or every two weeks; at least 200 mg every week or every two weeks; at least 250 mg every week or every two weeks; at least 300 mg every week or every two weeks; at least 400 mg every week or every two weeks , at least 350 mg; at least 400 mg per week or every two weeks; at least 450 mg per week or every two weeks; at least 500 mg per week or every two weeks; at least 550 mg per week or every two weeks; at least 600 mg per week or every two weeks; at least 650 mg per week or every two weeks; at least 700 mg per week or every two weeks; at least 750 mg per week or every two weeks; at least 800 mg per week or every two weeks; at least 850 mg per week or every two weeks; or at least 900 mg per week or every two weeks.

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

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

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

[0037] In any method discussed above or herein, the subject may suffer from 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) previous systemic treatments (including any one or more of the previous treatments discussed above or herein).

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

[0039] In any of the methods discussed above or herein, the subject may be suffering from an extramedullary plasmacytoma.

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

[0041] In another aspect, the present invention provides a dosing regimen for use in a method for treating multiple myeloma in a subject in need thereof, wherein the dosing regimen comprises administering to the subject a bispecific antibody at an initial dose during a first week of the dosing regimen, at a second dose during a second week of the dosing regimen, and at a third dose during a third week of the dosing regimen, wherein the third dose is equal to or greater than the second dose and the second dose is greater than the initial dose, and wherein the bispecific antibody comprises: (a) a first heavy chain and a 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 a 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: 68, 70, 72, 84, 86 and 88, respectively. NO: 92, 94, 96, 84, 86 and 88, or (b) a first heavy chain and a 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 a 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 complementary determining regions (CDRs) and three light chain CDRs, which comprise the amino acid sequences of SEQ ID NO: 68, 70, 72, 84, 86 and 88, respectively, and the second antigen binding domain comprises three heavy chain CDRs and three light chain CDRs, which comprise the amino acid sequences of SEQ ID NO: 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 a 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 a 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: 92, 94, 96, 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: 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 a 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 a 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, which respectively comprise the amino acid sequences of SEQ ID NOs: 68, 70, 72, 84, 86, and 88, and the second antigen binding domain comprises three heavy chain CDRs and three light chain CDRs, which respectively comprise the amino acid sequences of SEQ ID NOs: 100, 102, 104, 84, 86, and 88. 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.

[0044] In any various embodiments of the dosing regimen, the initial dose is 1mg to 5mg. In any various embodiments of the dosing regimen, the second dose is 3mg to 400mg. In any various embodiments of the dosing regimen, the third dose is 3mg to 800mg. In some embodiments, the initial dose is 5mg, the second dose is 25mg, and the third dose is 50mg to 800mg. In some cases, during the weekly administration of the dosing regimen, the dosing regimen includes applying the third dose weekly for at least 12 weeks (for example, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or more). In some cases, the dosing regimen further includes applying the third dose once every two weeks during the biweekly administration cycle of the dosing regimen after the weekly administration cycle of the dosing regimen. In some cases, the dosing regimen also includes applying the third dose once every three weeks or once every four weeks. In various embodiments, the dosage can be: at least 1 mg per week or every two weeks; at least 1.5 mg per week or every two weeks; at least 2.0 mg per week or every two weeks; at least 2.5 mg per week or every two weeks; at least 3.0 mg per week or every two weeks; at least 3.5 mg per week or every two weeks; at least 4 mg per week or every two weeks; at least 5 mg per week or every two weeks; at least 6 mg per week or every two weeks; at least 7 mg per week or every two weeks; at least 8 mg per week or every two weeks; at least 9 mg per week or every two weeks; at least 10 mg per week or every two weeks; at least 15 mg per week or every two weeks; at least 20 mg per week or every two weeks; at least 25 mg per week or every two weeks; at least 30 mg per week or every two weeks; at least 35 mg per week or every two weeks; at least 40 mg per week or every two weeks; at least 45 mg per week or every two weeks; at least 50 mg per week or every two weeks; at least 55 mg per week or every two weeks; at least 60 mg per week or every two weeks; at least 65 mg; at least 70 mg per week or every two weeks; at least 75 mg per week or every two weeks; at least 80 mg per week or every two weeks; at least 85 mg per week or every two weeks; at least 90 mg per week or every two weeks; at least 95 mg per week or every two weeks; at least 100 mg per week or every two weeks; at least 150 mg per week or every two weeks; at least 200 mg per week or every two weeks; at least 250 mg per week or every two weeks; at least 300 mg per week or every two weeks; at least 400 mg per week or every two weeks; at least 500 mg per week or every two weeks; at least 600 mg per week or every two weeks; at least 700 mg per week or every two weeks; at least 800 mg per week or every two weeks; at least 900 mg per week or every two weeks; at least 95 mg per week or every two weeks; at least 100 mg per week or every two weeks; at least 150 mg per week or every two weeks; at least 200 mg per week or every two weeks; at least 250 mg per week or every two weeks; at least 300 mg per week or every two weeks; at least 300 mg per week or every two weeks at least 350 mg per week or every two weeks; at least 400 mg per week or every two weeks; at least 450 mg per week or every two weeks; at least 500 mg per week or every two weeks; at least 550 mg per week or every two weeks; at least 600 mg per week or every two weeks; at least 650 mg per week or every two weeks; at least 700 mg per week or every two weeks; at least 750 mg per week or every two weeks; at least 800 mg per week or every two weeks; at least 850 mg per week or every two weeks; or at least 900 mg per 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 drug. In some cases, the anti-CD38 antibody is daratumumab or isatuximab. In some cases, the proteasome inhibitor is bortezomib, carfilzomib, or ixazomib. In some cases, the immunomodulatory drug is lenalidomide or pomalidomide.

[0046] In any of the various embodiments of the dosing regimens, 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 prior therapy. In some cases, the subject is quadruple or pentarefractory to prior therapy.

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

[0049] Other embodiments will become apparent upon reading the ensuing detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 and Figure 2 The anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459 respectively show preventive dose-dependent tumor inhibition in vivo against BCMA-expressing NCI-H929 human multiple myeloma tumor cells. NCI-H929 cells express high levels of BCMA.

[0051] Figure 3 and Figure 4 Therapeutic dose-dependent tumor inhibition of the anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459, respectively, against established BCMA-expressing NCI-H929 human multiple myeloma tumor cells in vivo is shown. NCI-H929 cells express high levels of BCMA.

[0052] Figure 5 and Figure 6The anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459 respectively show preventive dose-dependent tumor inhibition in vivo against BCMA-expressing MOLP-8 human multiple myeloma tumor cells. MOLP-8 cells express moderate levels of BCMA.

[0053] Figure 7 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 relative to controls is shown. OPM-2 cells express low levels of BCMA. DETAILED DESCRIPTION

[0054] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be restrictive, because the scope of the present invention is limited only by the appended claims.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the invention belongs. As used herein, when used to refer to a specific listed value, the term "about" means that the numerical value may differ from the listed value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0056] 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 in this specification are incorporated herein by reference in their entirety.

[0057] definition

[0058] The expression "CD3" used herein refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR), and it is composed of homodimers or heterodimers formed by the association of two of the following four receptor chains: CD3-ε, CD3-δ, CD3-ζ and CD3-γ. Human CD3-ε includes the amino acid sequence as shown in SEQ ID NO: 116; human CD3-δ includes the amino acid sequence as shown in SEQ ID NO: 117; human CD3-ζ includes the amino acid sequence as shown in SEQ ID NO: 118; and human CD3-γ includes the amino acid sequence as shown in SEQ ID NO: 119. All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the corresponding protein, polypeptide or protein fragment, unless it is explicitly stated that it is from a non-human species. Therefore, the expression "CD3" means human CD3, unless specified as being from a non-human species, such as "mouse CD3", "monkey CD3" and the like.

[0059] As used herein, "antibodies that bind to CD3" or "anti-CD3 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., ε, δ, γ, or ζ), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers). The antibodies and antigen-binding fragments of the present invention may bind to soluble CD3 and / or CD3 expressed on the cell surface. Soluble CD3 includes native CD3 protein and recombinant CD3 protein variants such as monomeric and dimeric CD3 constructs that lack a transmembrane domain or are otherwise unrelated to the cell membrane.

[0060] As used herein, the expression "CD3 expressed on the cell surface" refers to one or more CD3 proteins that are expressed on the cell surface in vitro or in vivo so that at least a portion of the CD3 protein is exposed to the extracellular side of the cell membrane and can be approached by the antigen-binding portion of the antibody. "CD3 expressed on the cell surface" includes CD3 proteins contained in the context of a functional T cell receptor in the membrane of the cell. The expression "CD3 expressed on the cell surface" includes CD3 proteins (e.g., γ / ε, δ / ε and ζ / ζ CD3 dimers) expressed as a part of a homodimer or heterodimer on the cell surface. The expression "CD3 expressed on the cell surface" also includes CD3 chains (e.g., CD3-ε, CD3-δ or CD3-γ) that are expressed on the cell surface by themselves without other CD3 chain types. "CD3 expressed on the cell surface" may include or consist of CD3 proteins expressed on the surface of cells that normally express CD3 proteins. Alternatively, "cell surface expressed CD3" may comprise or consist of CD3 protein expressed on the surface of cells that normally do not express human CD3 on their surface but have been artificially engineered to express CD3 on their surface.

[0061] As used herein, the expression "BCMA" refers to B cell maturation antigen. BCMA (also known as TNFRSF17 and CD269) is a cell surface protein expressed on malignant plasma cells and plays a central role in regulating B cell maturation and differentiation into immunoglobulin-producing plasma cells. The amino acid sequence of human BCMA is shown in SEQ ID NO: 115, and can also be found in GenBank Accession No. NP_001183.2.

[0062] As used herein, “antibodies that bind to BCMA” or “anti-BCMA antibodies” include antibodies and antigen-binding fragments thereof that specifically recognize BCMA.

[0063] The term "antigen binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0064] As used herein, the term "antibody" means any antigen binding molecule or molecular complex that includes at least one complementarity determining region (CDR) that specifically binds or interacts with a specific antigen (e.g., BCMA or CD3). The term "antibody" includes immunoglobulin molecules that include four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules composed of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or V H ) and the heavy chain constant region. The heavy chain constant region consists of three domains CH 1. C H 2 and C H 3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or V L ) and the light chain constant region. The light chain constant region consists of a domain (C L 1) You can set V H Area and V L The V region is further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V L It is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-BCMA antibody or anti-CD3 antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be natural or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0065] As used herein, the term "antibody" also includes the antigen binding fragment of a complete antibody molecule. As used herein, the term "antigen binding portion thereof", "antigen binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein, which specifically binds to an antigen to form a complex. The antigen binding fragment of an antibody can be derived from a complete antibody molecule, for example, using any suitable standard technique, such as proteolytic digestion or involving manipulation and expression of DNA encoding variable and optionally constant domains of an antibody. Such DNA is known and / or is easy to obtain from, for example, commercial sources, DNA libraries (comprising, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated by chemical methods or by molecular biological techniques, for example, one or more variable domains and / or constant domains are arranged into a suitable configuration, or codons are introduced, cysteine ​​residues are produced, modified, added or deleted amino acids, etc.

[0066] Non-limiting examples of antigen-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 that mimic the hypervariable region of an antibody (e.g., isolated complementary determining regions (CDRs), such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as 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 shark variant IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0067] The antigen-binding fragment of an antibody generally includes at least one variable domain. The variable domain can be of any size or amino acid composition and will generally include at least one CDR adjacent to or within one or more framework sequences. L Domain-associated V H In the antigen-binding fragment of the structural domain, V H Domain and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and contain V H -V H 、V H -V L or V L Alternatively, the antigen-binding fragment of the antibody may contain a monomer V H or V L Structural domain.

[0068] In certain embodiments, the antigen-binding fragment of an antibody may contain 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 within the 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) VH -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 L . In any configuration of variable domains and constant domains (including any of the exemplary configurations listed above), the variable domains and constant domains may be directly connected to each other or may be connected by a complete or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible connection between adjacent variable domains and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment of the antibody of the present invention may include homodimers or heterodimers (or other multimers) of any of the variable domain and constant domain configurations listed above, which are non-covalently associated with each other and / or with one or more monomer V H or V L The domains are covalently associated (eg, through one or more disulfide bonds).

[0069] As with complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically include at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format (including the exemplary bispecific antibody formats disclosed herein) can be applied to the context of antigen-binding fragments of the antibodies of the present invention using conventional techniques available in the art.

[0070] The antibodies of the present invention can act by complement dependent cytotoxicity (CDC) or antibody dependent cell-mediated cytotoxicity (ADCC). "Complement dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of the present invention in the presence of complement. "Antibody dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) expressing Fc receptors (FcR) recognize the binding antibodies on target cells, and thereby cause the lysis of target cells. CDC and ADCC can be measured using assays well known in the art and available. (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 ability of an antibody to fix complement and mediate cell dependent cytotoxicity. Thus, the isotype of an antibody can be selected based on whether the antibody is desired to mediate cytotoxicity.

[0071] In certain embodiments of the present invention, the anti-BCMA monospecific antibody or anti-BCMA x anti-CD3 bispecific antibody of the present invention is a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable regions and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in the CDR region, particularly in the CDR3 region. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto human framework region sequences.

[0072] In some embodiments, the antibody of the present invention can be a recombinant human antibody. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or separated by a recombinant manner, such as antibodies expressed by recombinant expression vectors transfected into host cells (described further below), antibodies separated from recombinant, combined human antibody libraries (described further below), antibodies separated from transgenic animals (e.g., mice) for human immunoglobulin genes (see, e.g., Taylor et al. (1992) "Nucl. Acids Res. " 20:6287-6295), or antibodies prepared, expressed, produced or separated by any other means, the means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when using animals of transgenic human Ig sequences, undergo in vivo somatic mutagenesis), and therefore the V H Area and V L The amino acid sequence of the region is as follows: H Sequence and V L Sequences are related to but may not naturally occur in vivo in the human antibody germline repertoire.

[0073] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule includes a stable four-chain construct of about 150-160 kDa, in which the dimer is held together by an interchain heavy chain disulfide bond. In the second form, the dimer is not connected by an interchain disulfide bond and forms a molecule of about 75-80 kDa, which is composed of covalently coupled light and heavy chains (half antibodies). Even after affinity purification, these forms are extremely difficult to separate.

[0074] The frequency of the second form in various intact IgG isotypes is based on, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to the level typically observed with the human IgG1 hinge. The present invention encompasses substitutions in the hinge, C H Zone 2 or C H Region 3: Antibodies with one or more mutations, which may be desirable, for example, in production to increase the yield of a desired antibody form.

[0075] The antibody of the present invention can be an isolated antibody. As used herein, "isolated antibody" means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody" for the purposes of the present invention. Isolated antibodies also include in situ antibodies in recombinant cells. An isolated antibody is an antibody that has been subjected to at least one purification or separation step. According to certain embodiments, the isolated antibody may be substantially free of other cellular material and / or chemicals.

[0076] The present invention also includes a one-armed antibody that binds BCMA. As used herein, a "one-armed antibody" means an antigen binding molecule comprising a single antibody heavy chain and a single antibody light chain. The one-armed antibody of the present invention may include any HCVR / LCVR or CDR amino acid sequence listed in Table 1.

[0077] The anti-BCMA or anti-BCMA x anti-CD3 antibodies disclosed herein may include one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any amino acid sequence disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to one or more corresponding residues of the germline sequence from which the antibodies were derived, or to one or more corresponding residues of another human germline sequence, or to conservative amino acid substitutions of one or more corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one of ordinary skill in the art can easily generate many antibodies and antigen-binding fragments including one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and / or CDR residues within the VH domain and / or VL domain are mutated back to the residues found in the original germline sequence from which the antibodies were derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the residues found after mutation within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the residues found after mutation in CDR1, CDR2 or CDR3. In other embodiments, one or more of one or more framework and / or CDR residues are mutated to one or more corresponding residues of different germline sequences (i.e., germline sequences different from the germline sequences of the antibody originally derived). In addition, the antibodies of the present invention may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, some individual residues are mutated to the corresponding residues of a specific germline sequence, while some other residues different from the original germline sequence may be maintained or mutated to the corresponding residues of different germline sequences. Once obtained, one or more desired properties of antibodies and antigen-binding fragments containing one or more germline mutations can be easily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or exciting biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

[0078] The present invention also encompasses anti-BCMA or anti-BCMA x anti-CD3 antibodies comprising variants of any HCVR, LCVR and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention encompasses anti-BCMA or anti-BCMA x anti-CD3 antibodies having HCVR, LCVR and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any of the HCVR, LCVR and / or CDR amino acid sequences as shown in Tables 1 and 3 herein, or the anti-CD3 antibodies disclosed in WO 2014 / 047231 or WO 2017 / 053856, each of which is incorporated herein by reference.

[0079] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule called a paratope. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may comprise a portion of a sugar, phosphoryl, or sulfonyl group on the antigen.

[0080] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof means a nucleotide sequence identity of at least about 95%, more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, as measured by any well-known sequence identity algorithm such as FASTA, BLAST or Gap, as discussed below. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having an amino acid sequence that is identical or substantially similar to a polypeptide encoded by the reference nucleic acid molecule.

[0081] When applied to these polypeptides, the term "substantially similar" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity when optimally aligned using the default gap weights as by the program GAP or BESTFIT. Preferably, the residue positions that are not identical differ by conservative amino acid substitutions. "Conservative amino acid substitutions" are amino acid substitutions in which an amino acid residue is substituted by another amino acid residue having a similar side chain (R group) with chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. In the case where two or more amino acid sequences differ from each other due to conservative substitutions, the sequence identity percentage or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups having chemically similar side chains include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains are 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, incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0082] Sequence similarity of polypeptides also referred to as sequence identity is usually measured using sequence analysis software. Protein analysis software uses a measure of the similarity assigned to various substitutions, deletions and other modifications (comprising conservative amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine closely related polypeptides, such as sequence homology or sequence identity between homologous polypeptides from different organism species or between wild-type proteins and their mutant proteins. See, for example, 6.1 version GCG. FASTA using default or recommended parameters can also be used to compare polypeptide sequences, which is a program in 6.1 version GCG. FASTA (e.g., FASTA2 and FASTA3) provides alignment and sequence identity percentage of the best overlapping region between the query sequence and the search sequence (Pearson (2000), supra). When the sequence of the present invention is compared with a database containing a large number of sequences from different organisms, another preferred algorithm is a computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, e.g., 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.

[0083] Germline mutations

[0084] The anti-CD3 antibodies disclosed herein include one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy chain variable domain as compared to the corresponding germline sequence from which the antibody was derived.

[0085] The present invention also encompasses antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to one or more corresponding residues of the derived antibody, or to one or more corresponding residues of another human germline sequence, or to a conservative amino acid substitution of one or more corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"), and have weak or no detectable binding to the CD3 antigen.

[0086] In addition, the antibodies of the invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residues of a particular germline sequence, while certain other residues that differ from the original germline sequence may be maintained or mutated to the corresponding residues of a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations may be easily tested for one or more desired properties, such as improved binding specificity, weak or reduced binding affinity, improved or enhanced pharmacokinetic properties, reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner given the guidance of the present disclosure are encompassed within the present invention.

[0087] The present invention also comprises an antigen binding molecule comprising an antigen binding domain having a HCVR and / or CDR amino acid sequence substantially identical to any HCVR and / or CDR amino acid sequence disclosed herein, while maintaining or improving the desired weak affinity to the CD3 antigen. When referring to an amino acid sequence, the term "substantially identical" or "substantially identical" means that when the two amino acid sequences are optimally aligned, such as by using the program GAP or BESTFIT with the default gap weight, the two amino acid sequences share at least 95% sequence identity, even more preferably, at least 98% or 99% sequence identity. Preferably, the non-identical residue positions differ due to conservative amino acid substitutions. In the case where two or more amino acid sequences differ from each other due to conservative substitutions, the sequence identity percentage or degree of similarity may be adjusted upward to correct the conservative nature of the substitution. The method for making this adjustment is well known to those skilled in the art. See, for example, Pearson, (1994), Methods in Molecular Biology, 24: 307-331.

[0088] Sequence similarity of polypeptides also referred to as sequence identity is usually measured using sequence analysis software. Protein analysis software uses a measure of the similarity assigned to various substitutions, deletions and other modifications (comprising conservative amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine closely related polypeptides, such as sequence homology or sequence identity between homologous polypeptides from different organism species or between wild-type proteins and their mutant proteins. See, for example, 6.1 version GCG. FASTA using default or recommended parameters can also be used to compare polypeptide sequences, which is a program in 6.1 version GCG. FASTA (e.g., FASTA2 and FASTA3) provides alignment and sequence identity percentage of the best overlapping region between the query sequence and the search sequence (Pearson (2000), supra). When comparing the sequence of the present invention with a database containing a large number of sequences from different organisms, another preferred algorithm is a computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, e.g., Altschul et al., (1990), J. Molecular Biology, 215:403-410 and Altschul et al., (1997), Nucleic Acids Research, 25:3389-402.

[0089] Binding properties of antibodies

[0090] As used herein, the term "binding" in the context of binding of an antibody, immunoglobulin, antibody binding fragment or Fc-containing protein to, for example, a predetermined antigen, such as a cell surface protein or a fragment thereof, generally refers to an interaction or association between a minimum of two entities or molecular structures, such as an antibody-antigen interaction.

[0091] For example, when measured by, for example, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using an antigen as a ligand and an antibody, Ig, antibody binding fragment or Fc-containing protein as the analyte (or anti-ligand), the binding affinity is typically about 10 -7 M or lower K D Value, such as about 10 -8 M or lower, such as about 10 -9Or lower. Cell-based binding strategies such as fluorescence activated cell sorting (FACS) binding assays are also routinely used, and FACS data have a strong correlation with other methods, such as radioligand competition binding and SPR (Benedict, CA, "Journal of Immunology (J Immunol Methods)" 1997, 201 (2): 223-31; Geuijen, CA et al. "Journal of Immunology" 2005, 302 (1-2): 68-77).

[0092] Therefore, the antibody or antigen-binding protein of the present invention binds to a predetermined antigen or cell surface molecule (receptor) having a K D The affinity corresponding to the K value is at least ten times lower than the affinity of the antigen or cell surface molecule binding to a non-specific antigen (e.g., BSA, casein). According to the present invention, an affinity corresponding to the K value equal to or less than ten times that of the non-specific antigen is at least ten times lower than the affinity of the antigen or cell surface molecule binding to the non-specific antigen (e.g., BSA, casein). D The affinity of the antibody corresponding to the value can be considered as undetectable binding, but such an antibody can be paired with a second antigen-binding arm to generate a bispecific antibody of the invention.

[0093] The term "K D "(M)" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction or the dissociation equilibrium constant of the binding of an antibody or antibody binding fragment to an antigen. K D There is an inverse relationship between K and binding affinity, so K D The smaller the value, the higher the affinity, ie the stronger it is. Therefore, the term "higher affinity" or "stronger affinity" relates to a higher ability to form an interaction and therefore to a smaller K D value, and conversely, the term "lower affinity" or "weaker affinity" relates to a lower ability to form an interaction, and thus K D In some cases, a particular molecule (e.g., an antibody) has a higher binding affinity (or K) for a partner molecule (e.g., antigen X) with which it interacts than the binding affinity of the molecule (e.g., antibody) for another interacting partner molecule (e.g., antigen Y). D ) can be expressed as D value (lower or weaker affinity) divided by the smaller K D (higher or stronger affinity), for example, a binding ratio determined by a 5-fold or 10-fold greater binding affinity, as appropriate.

[0094] The term “k d "(seconds-1 or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction or the dissociation rate constant of an antibody or antibody binding fragment. This value is also called k off value.

[0095] The term “k a "(M-1×sec-1 or 1 / M) refers to the association rate constant for a specific antibody-antigen interaction or the association rate constant of an antibody or antibody binding fragment.

[0096] The term "K A ” (M-1 or 1 / M) refers to the association equilibrium constant of a specific antibody-antigen interaction or the association equilibrium constant of an antibody or antibody binding fragment. By dividing k a Divide by k d The association equilibrium constant was obtained.

[0097] The term "EC50" or "EC 50 ” refers to the half-maximal effective concentration, which comprises the concentration of the antibody that induces a response intermediate between the baseline and maximum after a specified exposure time. EC 50 Essentially represents the concentration of the antibody where 50% of its maximal effect is observed. In certain embodiments, the EC50 value is equal to the concentration of an antibody of the invention that binds to cells expressing CD3 or a tumor-associated antigen (e.g., BCMA) at half the maximum as determined by, for example, a FACS binding assay. Thus, decreased or weaker binding is observed with an increased EC50. 50 Or half of the maximum effective concentration.

[0098] In one embodiment, decreased binding can be defined as an increased EC 50 The concentration of antibody that is able to bind half the maximal number of target cells.

[0099] 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 observed with a decreased EC 50 Or the effective concentration value of half the maximum value.

[0100] Bispecific Antigen Binding Molecules

[0101] The antibodies of the present invention may be monospecific, bispecific or multispecific. A multispecific antibody may be specific for different epitopes of a target polypeptide, or may contain an antigen binding domain that is specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-BCMA monospecific antibody or anti-BCMA x anti-CD3 bispecific antibody of the present invention may be linked or co-expressed with another functional molecule, for example, another peptide or protein. For example, an antibody or a fragment thereof may be functionally linked to one or more other molecular entities such as another antibody or antibody fragment (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to produce a bispecific or antibody fragment having a second or additional binding specificity.

[0102] As used herein, the expression "anti-CD3 antibody" or "anti-BCMA antibody" is intended to include monospecific anti-CD3 or anti-BCMA antibodies and bispecific antibodies comprising a CD3 binding arm and a BCMA binding arm. Thus, the present invention includes bispecific antibodies in which one arm of the immunoglobulin binds human CD3 and the other arm of the immunoglobulin is specific for human BCMA. The CD3 binding arm may include any HCVR / LCVR or CDR amino acid sequence as listed in Table 3 herein, or an anti-CD3 antibody disclosed in WO2014 / 047231 or WO 2017 / 053856.

[0103] 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 binds or weakly associates with human and cynomolgus (monkey) CD3, but the binding interaction cannot be detected by in vitro assays known in the art. The BCMA binding arm may include any HCVR / LCVR or CDR amino acid sequence as listed in Table 1 herein.

[0104] 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-CD3xBCMA" or "CD3xBCMA" bispecific molecules, or other similar terms (e.g., anti-BCMA / anti-CD3).

[0105] As used herein, the term "BCMA" refers to human BCMA protein, unless specified as being from a non-human species (e.g., "mouse BCMA," "monkey BCMA," etc.). Human BCMA protein has the amino acid sequence shown in SEQ ID NO: 115.

[0106] The above-described bispecific antigen binding molecules that specifically bind to CD3 and BCMA may include an anti-CD3 antigen binding molecule that binds to BCMA with weak binding affinity, such as exhibiting a K of greater than about 40 nM as measured by an in vitro affinity binding assay. D Binds to CD3.

[0107] As used herein, the expression "antigen binding molecule" means a protein, polypeptide or molecular complex comprising or consisting of at least one complementarity determining region (CDR), which specifically binds to a specific antigen alone or in combination with one or more additional CDRs and / or framework regions (FRs). In certain embodiments, the antigen binding molecule is an antibody or an antibody fragment, as those terms are defined elsewhere herein.

[0108] As used herein, the expression "bispecific antigen binding molecule" means a protein, polypeptide or molecular complex comprising at least a first antigen binding domain and a second antigen binding domain. Each antigen binding domain within a bispecific antigen binding molecule includes at least one CDR, which specifically binds to a specific antigen alone or in combination with one or more additional CDRs and / or FRs. In the context of the present invention, the first antigen binding domain specifically binds to a first antigen (e.g., BCMA), and the second antigen binding domain specifically binds to a second different antigen (e.g., CD3).

[0109] In certain exemplary embodiments of the present invention, bispecific antigen binding molecules are bispecific antibodies.Each antigen binding domain of a bispecific antibody includes a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR).In the context of a bispecific antigen binding molecule (e.g., bispecific antibody) including a first antigen binding domain and a second antigen binding domain, the CDR of the first antigen binding domain can be represented by the prefix "D1", and the CDR of the second antigen binding domain can be represented by the prefix "D2".Therefore, the CDR of the first antigen binding domain can be referred to as D1-HCDR1, D1-HCDR2 and D1-HCDR3 in this article; and the CDR of the second antigen binding domain can be referred to as D2-HCDR1, D2-HCDR2 and D2-HCDR3 in this article.

[0110] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule comprises a first antigen-binding domain comprising: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising an amino acid sequence of SEQ ID NO: 66; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising an amino acid sequence of SEQ ID NO: 82. In some cases, the isolated bispecific antigen-binding molecule comprises a HCDR1 comprising an amino acid sequence of SEQ ID NO: 68, a HCDR2 comprising an amino acid sequence of SEQ ID NO: 70, and a HCDR3 comprising an amino acid sequence of SEQ ID NO: 72. In some cases, the isolated bispecific antigen-binding molecule comprises a LCDR1 comprising an amino acid sequence of SEQ ID NO: 84, a LCDR2 comprising an amino acid sequence of SEQ ID NO: 86, and a LCDR3 comprising an amino acid sequence of SEQ ID NO: 88. In some cases, the first antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO:66 and a LCVR comprising the amino acid sequence of SEQ ID NO:82.

[0111] In certain exemplary embodiments, the isolated bispecific antigen binding molecule comprises a second antigen binding domain comprising: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising an amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 98; and (b) three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising an amino acid sequence of SEQ ID NO: 82. In some cases, the second antigen binding domain comprises: (a) HCDR1 comprising an amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 100; (b) HCDR2 comprising an amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 102; and (c) HCDR3 comprising an amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 104. In some cases, the second antigen binding domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 84, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a 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 NO: 92, 94, 96, respectively; and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NO: 84, 86, 88, respectively; or (b) HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NO: 100, 102, 104, respectively; and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NO: 84, 86, 88, respectively. In some cases, the second antigen binding domain comprises: (a) a HCVR comprising the amino acid sequence of SEQ ID NO:90, and a LCVR comprising the amino acid sequence of SEQ ID NO:82; or (b) a HCVR comprising the amino acid sequence of SEQ ID NO:98, and a LCVR comprising the amino acid sequence of SEQ ID NO:82.

[0112] In certain exemplary embodiments, the isolated bispecific antigen binding molecule comprises: (a) a first antigen binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 68, 70, and 72, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively; and (b) a second antigen binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 92, 94, and 96, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid 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 a HCVR comprising the amino acid sequence of SEQ ID NO:66 and a LCVR comprising the amino acid sequence of SEQ ID NO:82; and (b) a second antigen binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:90 and a LCVR comprising the amino acid sequence of SEQ ID NO:82.

[0113] In certain exemplary embodiments, the isolated bispecific antigen binding molecule comprises: (a) a first antigen binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 68, 70, and 72, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively; and (b) a second antigen binding domain comprising HCDR1, HCDR2, and HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 100, 102, and 104, respectively, and LCDR1, LCDR2, and LCDR3 domains comprising the amino acid 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 a HCVR comprising the amino acid sequence of SEQ ID NO:66 and a LCVR comprising the amino acid sequence of SEQ ID NO:82; and (b) a second antigen binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:98 and a LCVR comprising the amino acid sequence of SEQ ID NO:82.

[0114] 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 a 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 a 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 a HCVR / LCVR amino acid sequence pair selected from 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 a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain selected from 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 a 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 CDRs of a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 90 / 82 and 98 / 82.

[0115] In certain exemplary embodiments, the isolated bispecific antigen-binding molecule competes for binding to BCMA or binds to the same epitope on BCMA as that bound by a reference antibody, wherein the reference antibody comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 66 / 82.

[0116] HCVR / LCVR pair, wherein the second antigen-binding domain comprises a HCVR / LCVR pair having an amino acid sequence of SEQ ID NO: 90 / 82 or SEQ ID NOs: 98 / 82.

[0117] 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 bound by a reference antibody, wherein the reference antibody comprises a first antigen binding domain comprising a HCVR / LCVR pair having an amino acid sequence of SEQ ID NO: 66 / 82 and a second antigen binding domain comprising a HCVR / LCVR pair having an amino acid sequence of SEQ ID NO: 90 / 82 or SEQ ID NOs: 98 / 82.

[0118] The bispecific antigen binding molecules discussed above or herein can be bispecific antibodies. In some cases, the bispecific antibodies include a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is an isotype IgG1. In some cases, the human IgG heavy chain constant region is an isotype IgG4. In various embodiments, the bispecific antibodies include a chimeric hinge that reduces Fcγ receptor binding relative to a wild-type hinge of the same isotype.

[0119] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly connected to each other to form a bispecific antigen binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be connected to a separate multimerization domain. The association of a multimerization domain with another multimerization domain promotes the association between the two antigen-binding domains, thereby forming a bispecific antigen binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide or amino acid with the ability to associate with a second multimerization domain having the same or similar structure or composition. For example, the multimerization domain can be an immunoglobulin C or a polypeptide. H A non-limiting example of a multimeric component is the Fc portion of an immunoglobulin (including the C H 2-C H 3 domains), e.g., an IgG selected from the group consisting of isotypes IgG1, IgG2, IgG3, and IgG4, and an Fc domain of any isotype within each isotype group.

[0120] The bispecific antigen binding molecules of the present invention generally include two multimerization domains, for example, two Fc domains, each of which is independently a part of a separate antibody heavy chain. The first multimerization domain and the second multimerization domain can be the same IgG isotype as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first multimerization domain and the second multimerization domain can be different IgG isotypes such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0121] 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 peptide containing short cysteine. Other multimerization domains include peptides or polypeptides including or consisting of a leucine zipper, a spiral loop motif, or a coiled coil motif.

[0122] Any bispecific antibody format or technology can be used to prepare the bispecific antigen binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked to one or more other molecular entities such as another antibody or antibody fragment having a second antigen binding specificity (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to produce a bispecific antigen binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include but are not limited to the following: for example, scFv-based or double antibody bispecific formats, IgG-scFv fusion, dual variable domains (DVD)-Ig, quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs into holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG and Mab 2 Bispecific formats (see, e.g., Klein et al., 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the aforementioned formats).

[0123] In the context of the bispecific antigen binding molecules of the present invention, a multimerization domain, e.g., an Fc domain, may include one or more amino acid changes (e.g., insertions, deletions, or substitutions) as compared to a naturally occurring Fc domain. For example, the present invention comprises bispecific antigen binding molecules comprising one or more modifications in the Fc domain that result in a modified Fc domain with a modified binding interaction (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen binding molecule comprises a C H Zone 2 or C H 3 region, wherein the modification increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome with a pH range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); modifications at positions 250 and 428 (e.g., L or F); modifications 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 modifications 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 modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, 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); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0124] The present invention also includes a bispecific antigen binding molecule comprising a first C H 3 domain and the second Ig C H 3 domains, of which the first C H 3 domain and the second Ig C H The first Ig C domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces binding of the bispecific antibody to protein A as compared to a bispecific antibody lacking the amino acid difference. H 3 domains bind protein A, and the second Ig C HThe 3 domain contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (by IMGT exon numbering; H435R by EU numbering). H 3 may further include a Y96F modification (by IMGT; Y436F by EU). See, e.g., U.S. Pat. No. 8,586,713. H Further modifications found within 3 include: in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU); in the case of IgG2 antibodies, N44S, K52N and V82I (IMGT; N384S, K392N and V422I by EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU).

[0125] In certain embodiments, the Fc domain may be chimeric, with a combination of Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may include Fc sequences derived from human IgG1, human IgG2, or human IgG4. H C in Zone 2 H 2 sequence and a portion or all of a C sequence derived from human IgG1, human IgG2 or human IgG4 H3 sequences. The chimeric Fc domain may also contain a chimeric hinge region. For example, a chimeric hinge may include an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region, which is combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any antigen-binding molecule shown herein includes, from N-terminus to C-terminus: [IgG4 CH1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any antigen-binding molecule listed herein includes, from N-terminus to C-terminus: [IgG1CH1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that can be included in any antigen binding molecule of the invention are described in U.S. Publication No. 2014 / 0243504, published on August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains and variants thereof having these general structural arrangements can have altered Fc receptor binding, which in turn affects Fc effector function.

[0126] Sequence variants

[0127] As compared with the corresponding germline sequences derived from a single antigen binding domain, the antibodies and bispecific antigen binding molecules of the present invention may include 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. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained from, for example, public antibody sequence databases. The antigen binding molecules of the present invention may include antigen binding domains derived from any exemplary amino acid sequence disclosed herein, wherein one or more amino acid mutations in one or more frameworks and / or CDR regions are mutated to one or more corresponding residues of the germline sequence of the derived antibody, or mutated to one or more corresponding residues of another human germline sequence, or mutated to conservative amino acid substitutions of one or more corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, a person of ordinary skill in the art can easily produce many antibodies and antigen binding fragments including one or more individual germline mutations or combinations thereof. In certain embodiments, V H Domain and / or V LAll mutations of the framework and / or CDR residues in the domain are back to the residues found in the original germline sequence of the initial derived antigen-binding domain. In other embodiments, only some residues are mutated back to the original germline sequence, for example, only the residues after the mutation found in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4, or only the residues after the mutation found in CDR1, CDR2 or CDR3. In other embodiments, one or more mutations of one or more CDR residues in the framework are one or more corresponding residues of different germline sequences (that is, germline sequences different from the germline sequences of the initial derived antigen-binding domains). In addition, the antigen-binding domains of the present invention can contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, wherein some individual residues are mutated to the corresponding residues of a specific germline sequence, and some other residues different from the original germline sequence can be maintained or mutated to the corresponding residues of different germline sequences. Once obtained, antigen binding domains 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 antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen binding molecules comprising antigen binding domains obtained in this general manner are encompassed by the present invention.

[0128] pH-dependent binding

[0129] The present invention includes anti-BCMA antibodies and anti-BCMA x anti-CD3 bispecific antigen binding molecules with pH-dependent binding characteristics. For example, compared with neutral pH, the anti-BCMA antibodies of the present invention may exhibit reduced binding to BCMA at acidic pH. Alternatively, compared with neutral pH, the anti-BCMA antibodies of the present invention may exhibit enhanced binding to BCMA at acidic pH. The expression "acidic pH" includes a pH value of less than about 6.2, for example, about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. As used herein, the expression "neutral pH" means a pH of about 7.0 to about 7.4. The expression "neutral pH" encompasses pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

[0130] In some cases, "reduced binding at acidic pH as compared to neutral pH" means that the K of the antibody bound to the antigen at acidic pH is D The K value of an antibody binding to its antigen at neutral pH DFor example, if an antibody or antigen-binding fragment thereof exhibits an acidic / neutral K of about 3.0 or greater, D ratio, then for the purposes of the present invention, an antibody or antigen-binding fragment thereof may be considered to exhibit "reduced binding to BCMA at acidic pH as compared to neutral pH". In certain exemplary embodiments, the acidic / neutral K of an antibody or antigen-binding fragment of the invention is D The ratio may 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.

[0131] Antibodies with pH-dependent binding characteristics can be obtained by, for example, screening for a population of antibodies that have reduced (or enhanced) binding to a specific antigen at an acidic pH as compared to a neutral pH. In addition, modification of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent properties. For example, by replacing one or more amino acids of an antigen-binding domain (e.g., within a CDR) with histidine residues, an antibody can be obtained that has reduced binding to an antigen at an acidic pH relative to a neutral pH.

[0132] Antibodies including Fc variants

[0133] According to certain embodiments of the present invention, anti-BCMA antibodies and anti-BCMA x anti-CD3 bispecific antigen binding molecules are provided, which include an Fc domain, wherein the Fc domain includes one or more mutations that enhance or reduce the binding of the antibody to the FcRn receptor at acidic pH, such as compared to neutral pH. For example, the present invention includes C in the Fc domain. H Zone 2 or C HAn antibody comprising mutations in region 3, wherein one or more mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome at a pH range of about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., modification at position 250 (e.g., E or Q); modification at positions 250 and 428 (e.g., L or F); modification 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 modification 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 modification at positions 250 and / or 428; or modification at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, modification includes 428L (e.g., M428L) and 434S (e.g., N434S) modification; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modification; 433K (e.g., H433K) and 434 (e.g., 434Y) modification; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modification; 250Q and 428L modification (e.g., T250Q and M428L); and 307 and / or 308 modification (e.g., 308F or 308P). All positions are indicated with EU numbering.

[0134] For example, the present invention comprises anti-BCMA antibodies and anti-BCMA x anti-CD3 bispecific antigen binding molecules comprising an Fc domain comprising one or more mutation pairs or groups 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 within the antibody variable domains disclosed herein are within the scope of the present invention.

[0135] Biological properties of antibodies and bispecific antigen-binding molecules

[0136] The present invention includes binding to proteins with high affinity (e.g., nanomolar or subnanomolar K D Value) Antibodies and antigen-binding fragments thereof that bind to human BCMA.

[0137] According to certain embodiments, the invention includes antibodies and antigen-binding fragments of antibodies that have a K of less than about 5 nM.D Binding to human BCMA (e.g., at 25°C) as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 4 herein. In certain embodiments, an antibody or antigen-binding fragment of the invention binds to human BCMA with 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 800 pM, less than 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. D Binding to BCMA as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 4 herein, or a substantially similar assay. The invention includes bispecific antigen binding molecules, e.g., with a K of less than about 25 pM. D Binds to human BCMA with a K of less than about 170 pM D Bispecific antibodies that bind monkey BCMA as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 4 herein, or a substantially similar assay.

[0138] The present invention also includes antibodies and antigen-binding fragments thereof that bind to BCMA with a dissociation half-life (t1 / 2) of greater than about 10 minutes or greater than about 125 minutes, as measured by surface plasmon resonance at 25°C, e.g., using an assay format as defined in Example 4 herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the invention bind to BCMA with a t1 / 2 of greater than about 3 minutes, greater than about 4 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, or greater than about 120 minutes, as measured by surface plasmon resonance at 25°C, e.g., using an assay format as defined in Example 4 herein, or a substantially similar assay. The invention includes bispecific antigen binding molecules, e.g., bispecific antibodies, that bind BCMA for greater than about 10 minutes as measured by surface plasmon resonance at 25°C, e.g., using an assay format as defined in Example 4 herein, or a substantially similar assay.

[0139] The invention also includes antibodies and antigen-binding fragments thereof that specifically bind to a human cell line expressing endogenous BCMA (e.g., NCI-H929, MOLP-8, or OMP-2) as determined by a FACS binding assay (as described in Example 6) or a substantially similar assay.

[0140] 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) inhibition of tumor growth in immunocompromised mice bearing human multiple myeloma xenografts; (b) inhibition of tumor growth of established tumors in immunocompromised mice bearing human multiple myeloma xenografts (see, e.g., Examples 10-15), and (c) inhibition of tumor growth of syngeneic melanoma and colon cancer cells engineered to express human BCMA in immunocompetent mice expressing human CD3.

[0141] 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 medium or low affinity, depending on the desired therapeutic context and specific targeting properties. In some cases, low affinity includes antibodies with a K of greater than 300 nM, greater than 500 nM, or greater than 1 μM. D or EC 50 (e.g., measured in surface plasmon resonance analysis) antibodies that bind to CD3. The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD3 with unmeasurable affinity. For example, in the case of a bispecific antigen-binding molecule, in which one arm binds CD3 and the other arm binds a target antigen (e.g., BCMA), the target antigen-binding arm may be required to bind to the target antigen with high affinity, while the anti-CD3 arm only binds to CD3 with moderate or low affinity or no affinity. In this way, preferential targeting of antigen-binding molecules to cells expressing the target antigen can be achieved while avoiding general / non-targeted CD3 binding and the adverse side effects associated therewith.

[0142] The present invention includes bispecific antigen binding molecules (e.g., bispecific antibodies) that can bind to human CD3 and human BCMA simultaneously. As measured in a suitable in vitro binding assay, the binding arm that interacts with cells expressing CD3 may have weak to undetectable binding. The extent to which bispecific antigen binding molecules bind to cells expressing CD3 and / or BCMA can be assessed by fluorescence activated cell sorting (FACS), as shown in Examples 5 and 6 herein.

[0143] For example, the invention includes antibodies, antigen-binding fragments thereof, and bispecific antibodies that specifically bind to human T cell lines that express CD3 but not BCMA (e.g., Jurkat) and / or cells that express BCMA.

[0144] The present invention encompasses antibodies, antigen-binding fragments and bispecific antibodies thereof that bind to human CD3 with weak (ie, low) or even undetectable affinity.

[0145] The present invention encompasses antibodies, antigen-binding fragments and bispecific antibodies thereof that bind to monkey (ie, cynomolgus monkey) CD3 with weak (ie, low) or even undetectable affinity.

[0146] The present invention includes antibodies, antigen-binding fragments thereof, and bispecific antibodies that bind to human CD3 and induce T cell activation.

[0147] The present invention includes anti-BCMA x anti-CD3 bispecific antigen binding molecules that are capable of depleting or reducing cells expressing tumor antigens in a subject (see, e.g., Examples 8-16 or substantially similar assays). For example, according to certain embodiments, an anti-BCMA x anti-CD3 bispecific antigen binding molecule is provided, wherein a single administration or multiple administrations of 0.04 mg / kg, 0.4 mg / kg, or 4 mg / kg of the bispecific antigen binding molecule to a subject results in a decrease in the number of cells expressing BCMA in the subject (e.g., suppressing or inhibiting tumor growth in the subject).

[0148] Epitope mapping and related technologies

[0149] The epitope on CD3 and / or BCMA to which the antigen binding molecules of the present invention bind can be composed of 3 or more single continuous sequences (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of the amino acids of CD3 or BCMA proteins. Alternatively, the epitope can be composed of multiple non-continuous amino acids (or amino acid sequences) of CD3 or BCMA. The antibodies of the present invention can interact with the amino acids contained in a single CD3 chain (e.g., CD3-ε, CD3-δ or CD3-γ), or can interact with the 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 called a paratope. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on an antigen and can have different biological effects. An epitope can be conformational or linear. Conformational epitopes are produced by spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may comprise a portion of a sugar, phosphoryl or sulfonyl group on the antigen.

[0150] Various techniques known to those of ordinary skill 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, conventional cross-blocking assays (such as described in Antibody ( Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor Laboratory, New York), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods in Molecular Biology, 248: 443-463) and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be used (Tomer, 2000, Protein Science, 9: 487-496). Another method that can be used to identify amino acids in a polypeptide that interact with the antigen binding domain of an antibody is hydrogen / deuterium exchange detected by mass spectrometry. In general, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest and then combining the antibody with the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except the residues protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal deuterium-labeled residues corresponding to specific amino acids that interact with the antibody. See, for example, Ehring (1999) Analytical Biochemistry, 267 (2): 252-259; Engen and Smith (2001) Anal. Chem., 73: 256A-265A. X-ray crystallography of antigen / antibody complexes can also be used for epitope mapping.

[0151] The present invention further comprises 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 listed in Table 1 herein). Likewise, the present invention also comprises anti-BCMA antibodies that compete for binding to BCMA with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences listed in Table 1 herein).

[0152] The present invention also includes bispecific antigen binding molecules comprising a second antigen binding domain that specifically binds to human CD3 and / or cynomolgus macaque CD3 with low or undetectable binding affinity, and a second antigen binding domain that specifically binds to human BCMA, wherein the second antigen binding domain binds to the same epitope on CD3 as any of the specific exemplary CD3-specific antigen binding domains described herein, and / or wherein 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.

[0153] Likewise, the present invention also encompasses a bispecific antigen binding molecule comprising a first antigen binding domain that specifically binds to human BCMA and a second antigen binding domain that specifically binds to human CD3, wherein the first antigen binding domain competes for binding to BCMA with any of the specific exemplary BCMA-specific antigen binding domains described herein, and / or wherein the second antigen binding domain competes for binding to CD3 with any of the specific exemplary CD3-specific antigen binding domains described herein.

[0154] By using conventional methods known in the art, it is easy to determine whether a specific antigen binding molecule (e.g., antibody) or its antigen binding domain is bound to the same epitope or competitive binding with the reference antigen binding molecule of the present invention. For example, in order to determine whether the test antibody is bound to the same epitope as the reference bispecific antigen binding molecule of the present invention on BCMA (or CD3), the reference bispecific molecule is first allowed to bind to the BCMA protein (or CD3 protein). Next, the ability of the test antibody to bind to the BCMA (or CD3) molecule is evaluated. If the test antibody can bind to BCMA (or CD3) after saturation binding with the reference bispecific antigen binding molecule, it can be concluded that the test antibody binds to an epitope different from the reference bispecific antigen binding molecule of BCMA (or CD3). On the other hand, if the test antibody cannot bind to the BCMA (or CD3) molecule after saturation binding with the reference bispecific antigen binding molecule, the test antibody can bind to the same epitope as the epitope of the reference bispecific antigen binding molecule of BCMA (or CD3) of the present invention. Then other routine experiments (e.g., peptide mutations and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope with the reference bispecific antigen binding molecule or whether steric blocking (or other phenomena) causes the observed lack of binding. Such experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assays available in the art. According to certain embodiments of the present invention, if, for example, 1 times, 5 times, 10 times, 20 times, or 100 times of an antigen-binding protein excessively inhibits another protein (as measured in a competitive binding method) by at least 50%, but preferably 75%, 90% or even 99% of the binding, then the two antigen-binding proteins are bound to the same (or overlapping) epitope (see, e.g., Junghans et al., Cancer Res., 1990:50:1495-1502). Alternatively, if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen binding protein reduce or eliminate binding of the other antigen, then the two antigen binding proteins are considered to bind to the same epitope. If only a subset of amino acid mutations that reduce or eliminate binding of one antigen binding protein reduce or eliminate binding of the other antigen, then the two antigen binding proteins are considered to have "overlapping epitopes."

[0155] To determine whether an antibody or its antigen binding domain competes for binding to a reference antigen binding molecule, the above binding method is performed in two orientations: in the first orientation, the reference antigen binding molecule is bound to the BCMA protein (or CD3 protein) under saturation conditions, and then the binding of the test antibody to the BCMA (or CD3) molecule is evaluated. In the second orientation, the test antibody is bound to the BCMA (or CD3) molecule under saturation conditions, and then the binding of the reference antigen binding molecule to the BCMA (or CD3) molecule is evaluated. If only the first (saturated) antigen binding molecule is able to bind to the BCMA (or CD3) molecule in both orientations, it can be concluded that the test antibody and the reference antigen binding molecule compete for binding to BCMA (or CD3). As will be appreciated by those of ordinary skill in the art, an antibody that competes for binding with a reference antigen binding molecule may not necessarily bind to the same epitope as the reference antibody, but may spatially block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0156] Preparation of antigen binding domains and construction of bispecific molecules

[0157] Antigen binding domains specific to specific antigens can be prepared by any antibody generation technology known in the art. Once obtained, two different antigen binding domains specific to two different antigens (e.g., CD3 and BCMA) can be appropriately arranged relative to each other to produce the bispecific antigen binding molecules of the present invention using conventional methods. (Discussion of exemplary bispecific antibody forms 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 chain and light chain) of the multispecific antigen binding molecules of the present invention are derived from chimeric antibodies, humanized antibodies, or fully human antibodies. Methods for preparing such antibodies are well known in the art. For example, VELOCIMMUNE can be used TM One or more heavy chains and / or light chains of the bispecific antigen-binding molecules of the present invention are prepared using VELOCIMMUNE TM Using the PCR technology (or any other human antibody generation technology), high affinity chimeric antibodies are initially isolated for a specific antigen (e.g., CD3 or BCMA) having a human variable region and a mouse constant region. The antibodies are characterized and selected to obtain the desired properties including affinity, selectivity, epitope, etc. The mouse constant region is replaced with the desired human constant region to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen binding molecules of the invention.

[0158] Genetically engineered animals can be used to prepare human bispecific antigen binding molecules. For example, genetically modified mice that cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, wherein the mouse only expresses one or two human light chain variable domains encoded by human immunoglobulin sequences, and the human immunoglobulin sequences are operably connected to the mouse κ constant gene at the endogenous mouse κ locus. This genetically modified mouse can be used to produce a fully human bispecific antigen binding molecule including two different heavy chains, the heavy chain is associated with the same light chain, and the light chain includes a variable domain derived from one of two different human light chain variable region gene segments. (See, for example, US 2011 / 0195454). Fully human refers to an antibody or its antigen-binding fragment or immunoglobulin domain comprising an amino acid sequence encoded by DNA, the DNA derived from a human sequence within the entire length of each polypeptide of an antibody or its antigen-binding fragment or immunoglobulin domain. In some cases, fully human sequences are derived from endogenous proteins of people. In other cases, fully human proteins or protein sequences include chimeric sequences, wherein each component sequence is derived from a human sequence. While not being limited by any one theory, chimeric proteins or chimeric sequences are generally designed to minimize the generation of immunogenic epitopes at the junction of the component sequences, for example compared to any wild-type human immunoglobulin region or domain.

[0159] Bioequivalent

[0160] The present invention encompasses antigen binding molecules having amino acid sequences that differ from the exemplary molecules disclosed herein but retain the ability to bind to CD3 and / or BCMA. Such variant molecules may include one or more additions, deletions or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity that is substantially the same as that of the bispecific antigen binding molecule.

[0161] The present invention includes antigen binding molecules that are bioequivalent to any exemplary antigen binding molecules listed herein. If, for example, two antigen binding proteins or antibodies are drug equivalents or drug substitutes that absorption rate and absorption extent do not show significant differences when using (single dose or multiple doses) with identical molar doses under similar experimental conditions, then it is considered that the two antigen binding proteins or antibodies are bioequivalent. If some antigen binding proteins are equivalent in absorption extent but their absorption rate is not equivalent, then it is considered to be equivalent or drug substitute, and can be considered to be bioequivalent, because this intentional and the difference in absorption rate reflected on the label is, for example, not necessary for obtaining effective body drug concentration during long-term use, and is considered to be medically insignificant for the specific drug product studied.

[0162] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in safety, purity, and potency.

[0163] In one embodiment, two antigen binding proteins are bioequivalent if patients can switch between the reference product and the biological product one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity or decreased effectiveness, compared to continued treatment without switching.

[0164] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms of action for one or more conditions of use (provided these mechanisms are known).

[0165] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests in humans or other mammals, where the concentration of the antibody or its metabolites is measured over time in blood, plasma, serum or other biological fluids; (b) in vitro tests that are correlated with and reasonably predictive of bioavailability data in humans; (c) in vivo tests in humans or other mammals, where appropriate acute pharmacological effects of the antibody (or its target) are measured over time; and (d) in well-controlled clinical trials to establish the safety, efficacy or bioavailability or bioequivalence of the antigen-binding protein.

[0166] Bioequivalent variants of the exemplary bispecific antigen binding molecules listed herein can be constructed, for example, by making various substitutions of residues or sequences or deleting terminal or internal residues or sequences that are not necessary for biological activity. For example, cysteine ​​residues that are not necessary for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bonds during renaturation. In other cases, bioequivalent antigen binding proteins can include variants of the exemplary bispecific antigen binding molecules listed herein that include amino acid changes that alter the glycosylation characteristics of the molecule, for example, mutations that eliminate or remove glycosylation.

[0167] Species selectivity and cross-species reactivity

[0168] 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.

[0169] According to certain exemplary embodiments of the present invention, an antigen binding molecule is provided that binds to human CD3 and / or human BCMA, and optionally may bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cattle, horse, camel, cynomolgus, marmoset, rhesus monkey or chimpanzee CD3 and / or BCMA or not bind thereto. For example, in a specific exemplary embodiment of the present invention, a bispecific antigen binding molecule comprising a first antigen binding domain that binds to human BCMA and cynomolgus BCMA and a second antigen binding domain that specifically binds to human CD3 is provided, or a bispecific antigen binding molecule comprising a first antigen binding domain that binds to human BCMA and cynomolgus BCMA and a second antigen binding domain that specifically binds to human CD3 is provided.

[0170] Therapeutic formulations and administration

[0171] The present invention provides pharmaceutical compositions comprising antigen binding molecules of the present invention. The pharmaceutical compositions of the present invention are formulated together with suitable carriers, excipients and other agents that provide improved transfer, delivery, tolerance, etc. Many suitable preparations can be found in formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These preparations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipids (cations or anions) containing vesicles (such as LIPOFECTIN TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption creams, oil-in-water and water-in-oil emulsions, emulsions of polyethylene glycol (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) "Journal of Pharmaceutical Science and Technology ( ...

[0172] The dosage of the antigen binding molecules applied to the patient can vary according to the patient's age and body type, target disease, illness, route of administration, etc. The preferred dosage is usually calculated according to body weight or body surface area. When the bispecific antigen binding molecules of the present invention are used for the treatment of adult patients, it may be advantageous to administer the bispecific antigen binding molecules of the present invention intravenously, usually with a single dose of about 0.01 mg / kg to about 20 mg / kg body weight, more preferably, about 0.02 mg / kg to about 7 mg / kg body weight, about 0.03 mg / kg to about 5 mg / kg body weight or about 0.05 mg / kg to about 3 mg / kg body weight. Depending on the severity of the disease, the frequency and duration of treatment can be adjusted. The effective dose and schedule of the bispecific antigen binding molecules can be determined empirically; for example, patient progress can be monitored by regular assessment, and the dosage can be adjusted accordingly. In addition, the interspecific scaling of the dosage can be performed using methods well known in the art (e.g., Mordenti et al., 1991, "Pharmaceut. Res." 8: 1351).

[0173] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, 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 composition can be administered by any convenient route, e.g., by infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other bioactive agents. Administration can be systemic or local.

[0174] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are readily applicable to deliver the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once the entire pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device is preloaded with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0175] Many reusable pen and auto-injector delivery devices are useful for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM PEN (Eli Lilly and Co., Indianapolis, Indiana), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPENJUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TM PEN (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR TM Pen (Sanofi), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM PEN (Abbott Labs, Abbott Technology Park, IL), to name a few.

[0176] In some cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC: Biomedical Engineering Review (CRC Crit. Ref. Biomed. Eng.), 14: 201). In another embodiment, a polymer material can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida (CRC Pres., Boca Raton, Florida). In yet another embodiment, a controlled release system can be placed near the target of the composition, thus requiring only a small portion of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review of Langer, 1990, Science, 249: 1527-1533.

[0177] Injectable preparations can include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, instillations, etc. These injectable preparations can be prepared by known methods. For example, injectable preparations can be prepared by, for example, dissolving, suspending or emulsifying the above-mentioned antibody or its salt in a sterile aqueous medium or a conventional oily medium for injection. As aqueous media for injection, for example, there are physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with suitable solubilizers such as alcohol (for example, ethanol), polyols (for example, propylene glycol, polyethylene glycol), nonionic surfactants [for example, polysorbate 80, HCO-50 (polyoxyethylene (50 moles) adducts of hydrogenated castor oil)], etc. As oily media, for example, sesame oil, soybean oil, etc. can be used, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The injection prepared in this way is preferably filled in a suitable ampoule.

[0178] Advantageously, the pharmaceutical composition for oral or parenteral use is prepared into a unit dosage form suitable for matching a certain dose of active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the above-mentioned antibody contained is usually about 5 mg to about 500 mg per unit dosage form; especially in the form of injection, preferably the content of the above-mentioned antibody is about 5 mg to about 100 mg, and for other dosage forms, about 10 mg to about 250 mg.

[0179] Therapeutic uses of antigen binding molecules

[0180] The present invention includes a method comprising administering a therapeutic composition to a subject in need, the therapeutic composition comprising an anti-BCMA antibody or its antigen-binding fragment or a bispecific antigen binding molecule that specifically binds CD3 and BCMA. The therapeutic composition may include any antibody or bispecific antigen binding molecule disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression "subject in need" means a subject who exhibits one or more symptoms or signs of cancer (e.g., a subject who exhibits a tumor or suffers from any of the cancers mentioned herein below) or otherwise benefits from the inhibition or reduction of BCMA activity or the depletion of BCMA+ cells (e.g., multiple myeloma cells) or non-human animals.

[0181] The antibodies and bispecific antigen binding molecules of the present invention (and therapeutic compositions comprising the same) are particularly useful for treating any disease or condition in which stimulation, activation and / or targeting of an immune response would be 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 improve any disease or condition associated with or mediated by BCMA expression or activity or proliferation of BCMA+ cells. The mechanism of action for achieving the therapeutic method of the present invention includes killing cells expressing BCMA in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis or by a combination of two or more of these mechanisms. Cells expressing BCMA that can be inhibited or killed using the bispecific antigen binding molecules of the present invention include, for example, multiple myeloma cells.

[0182] 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 suitable for treating patients suffering from multiple myeloma. According to other related embodiments of the present invention, a method is provided, which comprises administering an anti-BCMA antibody or anti-BCMA x anti-CD3 bispecific antigen binding molecule as disclosed herein to a patient suffering from multiple myeloma. Analysis / diagnostic methods known in the art, such as tumor scanning, can be used to determine whether a patient carries multiple myeloma or other B cell line cancers.

[0183] The present invention also encompasses methods for treating residual cancer in a subject.As used herein, the term "residual cancer" means the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[0184] According to certain aspects, the invention provides a method for treating a disease or condition associated with BCMA expression (e.g., multiple myeloma), comprising administering to the subject one or more of the anti-BCMA or bispecific antigen binding molecules described elsewhere herein after determining that the subject has multiple myeloma. For example, the invention comprises a method for treating myeloma comprising administering to the patient an anti-BCMA antibody or 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.

[0185] Combination therapies and formulations

[0186] The present invention provides a method including administering a pharmaceutical composition, the pharmaceutical composition including any exemplary antibody and bispecific antigen binding molecule described herein and a combination of one or more additional therapeutic agents. The exemplary additional therapeutic agent that can be combined with the antigen binding molecules of the present invention or administered in combination therewith includes, for example, an anti-tumor agent (e.g., a chemotherapeutic agent, including melphalan (melphalan), vincristine (Oncovin (Oncovin)), cyclophosphamide (Cytoxan (Cytoxan)), etoposide (VP-16), doxorubicin (Adriamycin (Adriamycin)), liposomal doxorubicin (Doxil (Doxil)), obendamustine (obendamustine, bendamustine (Treanda)) or any other chemotherapeutic agent known to be effective for treating plasma cell tumors of a subject). In some embodiments, the second therapeutic agent includes a steroid. In some embodiments, the second therapeutic agent includes targeted therapy, which includes thalidomide, lenalidomide and bortezomib, which are approved for the treatment of newly diagnosed patients. For example, lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab and daratumumab are examples of second therapeutic agents for effective treatment of relapsed myeloma. In certain embodiments, the second therapeutic agent is a regimen including radiotherapy or stem cell transplantation. In certain embodiments, the second therapeutic agent can be an immunomodulator. In certain embodiments, the second therapeutic agent can be a proteasome inhibitor, including bortezomib (Velcade), carfilzomib / Kyprolis, ixazomib / Ninlaro. In certain embodiments, the second therapeutic agent can be a histone deacetylase inhibitor, such as panobinostat (panobinostat / Farydak). In certain embodiments, the second therapeutic agent can be a monoclonal antibody, an antibody drug conjugate, a bispecific antibody conjugated with an anti-tumor agent, a checkpoint inhibitor or a combination thereof. Other agents that can be beneficially combined with the antigen binding molecules of the present invention include cytokine inhibitors, and the cytokine inhibitors include small molecule cytokine inhibitors and with cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18 or their respective receptor-bound antibodies.The pharmaceutical compositions of the invention (e.g., pharmaceutical compositions comprising anti-BCMA x anti-CD3 bispecific antigen binding molecules as disclosed herein) can also be administered as part of a treatment regimen comprising one or more therapeutic combinations selected from the following: a monoclonal antibody different from the monoclonal antibodies described herein, which can interact with a different antigen on the surface of plasma cells; a bispecific antibody, one arm of which binds to an antigen on the surface of a tumor cell and the other arm binds to an antigen on a T cell; an antibody drug conjugate; a bispecific antibody conjugated to an anti-tumor agent; a checkpoint inhibitor, such as a checkpoint inhibitor targeting PD-1 or CTLA-4; or a combination thereof. In certain embodiments, the checkpoint inhibitor can be selected from a PD-1 inhibitor, such as pembrolizumab (Keytruda), nivolumab (Opdivo), or cemipril monoclonal antibody (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 may be used in conjunction with the antibodies of the present invention are described above.

[0187] The present invention also comprises a therapeutic combination comprising any antigen binding molecule mentioned herein and an inhibitor of one or more 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 above cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, siRNA, a peptibody, a nanobody or an antibody fragment (e.g., a Fab fragment; a F(ab')2 fragment; a Fd fragment; a Fv fragment; a scFv; a dAb fragment; or other engineered molecules such as a double antibody, a three-antibody, a four-antibody, a microantibody and a minimum recognition unit). The antigen binding molecules of the present invention can also be administered in combination with antiviral agents, antibiotics, analgesics, corticosteroids and / or NSAIDs and / or co-formulated. The antigen binding molecules of the invention may also be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy.

[0188] The additional therapeutically active component may be administered prior to, simultaneously with, or shortly after administration of the antigen binding molecules of the invention; (for purposes of this disclosure, such administration regimens are considered administration of the antigen binding molecules "in combination" with the additional therapeutically active component).

[0189] The invention encompasses pharmaceutical compositions in which the antigen binding molecules of the invention are co-formulated with one or more additional therapeutically active ingredients as described elsewhere herein.

[0190] Administration regimen

[0191] According to certain embodiments of the present invention, multiple doses of antigen binding molecules (e.g., anti-BCMA antibodies or bispecific antigen binding molecules that specifically bind BCMA and CD3) can be administered to a subject within a defined time course. The method according to this aspect of the present invention includes sequentially administering multiple doses of the antigen binding molecules of the present invention to a subject. As used herein, "sequential administration" means that the antigen binding molecules of each dose are administered to a subject at different time points, for example, at different dates separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods, the methods including sequentially administering a single initial dose of antigen binding molecules to a patient, then administering one or more second doses of antigen binding molecules, and optionally subsequently administering one or more third doses of antigen binding molecules.

[0192] The terms "initial dose", "secondary dose" and "tertiary dose" refer to the time series of the antigen binding molecules of the present invention. Therefore, "initial dose" is the dosage (also referred to as "baseline dose") applied at the beginning of the treatment regimen; "secondary dose" is the dosage applied after the initial dose; "tertiary dose" is the dosage applied after the second dose. Initial dose, second dose and third dose can all contain the same amount of antigen binding molecules, but may generally be different from each other in terms of frequency of application. However, in certain embodiments, the amount of antigen binding molecules contained in initial dose, second dose and / or third dose during treatment is different from each other (for example, appropriately adjusted or reduced). In certain embodiments, two or more (for example, 2, 3, 4 or 5) dosages are applied as "loading doses" at the beginning of the treatment regimen, followed by subsequent doses (for example, "maintenance doses") applied on a less frequent basis. In any embodiment, initial dose (for example, first weekly dose) can be divided into two dosages, applied on different days (for example, consecutive days) of no more than three days. In any embodiment, the first nominal dose (i.e., the second dose) can be divided into two doses, administered on different days (e.g., consecutive days) not exceeding three days. For example, if the initial dose or the second dose is 6 mg, the dose can be divided into two 3 mg doses, for example, administered on consecutive days or on different days separated by not exceeding three days. In various embodiments, the dose (e.g., the dose administered weekly, as a single dose or as two divided parts of a dose) is or is at least 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg g, 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, 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, 180 mg, 185mg, 190mg, 195mg, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270m g, 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, 415mg, 420mg, 425mg, 430mg, 435mg, 440mg, 445mg, 450mg, 455mg, 460mg, 465mg, 470mg, 475mg, 480mg, 485mg, 490mg, 495mg, 500mg, 510mg, 520mg, 530mg, 540mg, 550mg, 560mg, 570mg, 580mg, 5 90mr, 600mg, 610mg, 620mg, 630mg, 640mg, 650mg, 660mg, 670mg, 680mg, 690mg, 700mg, 710mg, 720mg, 730mg, 740mg, 750mg, 760mg, 77 5g, 8g, 8.5g, 9g, 9.5g, 10g or more. Any of these amounts may be used to define the initial,The range of the second or third dose is included in the scope of the present disclosure. In some embodiments, all doses are administered as a single dose (e.g., a single infusion), including doses administered within the first and second weeks of the dosing regimen. For example, an initial dose of 1 mg to 5 mg may be administered as a single dose in the first week, a second dose of 3 mg to 400 mg may be administered as a single dose in the second week, and a third dose of 50 mg to 800 mg may be administered as a single dose in the third week, followed by administration during the weekly dosing portion of the dosing regimen. In another example, an initial dose of 5 mg may be administered as a single dose in the first week, a second dose of 25 mg may be administered as a single dose in the second week, and a third dose of 50 mg to 800 mg may be administered as a single dose in the third week, followed by administration during the weekly dosing portion of the dosing regimen. In some cases, the dosing regimen may thereafter (e.g., after 12 to 16 weeks) include administration every two weeks, every three weeks, once a month, etc.

[0193] In an exemplary embodiment of the invention, each second dose and / or third 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, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 / 2 or more) weeks of administration. As used herein, the phrase "previous dose" means the dosage of the antigen binding molecules administered to the patient before the next dose in the sequence in the absence of an intermediate dose in a sequence of multiple administrations.

[0194] The method according to this aspect of the invention may include administering any number of second doses and / or third doses of antigen binding molecules (e.g., anti-BCMA antibodies or bispecific antigen binding molecules that specifically bind BCMA and CD3) to the patient. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) second doses are administered to the patient. Similarly, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) third doses are administered to the patient.

[0195] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as other second doses. For example, each second dose can be administered to the patient 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose can be administered at the same frequency as other third doses. For example, each third dose can be administered to the patient 2 to 4 weeks after the previous dose. Alternatively, the frequency of administering the second dose and / or the third dose to the patient can change during the course of the treatment regimen. The frequency of administration can also be adjusted during the physician's treatment according to the needs of individual patients after clinical examination.

[0196] Diagnostic Uses of Antibodies

[0197] The anti-BCMA antibodies of the present invention can also be used to detect and / or measure BCMA or BCMA-expressing cells in a sample, for example, for diagnostic purposes. For example, anti-BCMA antibodies or fragments thereof can be used to diagnose a disorder or disease characterized by abnormal expression of BCMA (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for BCMA can include, for example, contacting a sample obtained from a patient with an anti-BCMA antibody of the present invention, wherein the anti-BCMA antibody is labeled with a detectable marker or reporter molecule. Alternatively, an unlabeled anti-BCMA antibody can be combined with a second antibody that is itself detectably labeled for diagnostic applications. The detectable marker or reporter molecule can be, for example, 3 H. 14 C. 32 P. 35 S or 125I; 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 includes 89 Zr-deferoxamine labeled 89 Zr-labeled antibodies for non-invasive identification and tracking of tumor cells in a subject (e.g., positron emission tomography (PET) imaging). (See, e.g., Tavare, R et al., Cancer Research, 2016 Jan 1;76(1):73-82; and Azad, BB et al., Oncotarget., 2016 Mar 15;7(11):12344-58). Specific exemplary assays that can be used to detect or measure BCMA in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).

[0198] Samples that can be used for BCMA diagnostic assays according to the present invention include any tissue or fluid sample that can be obtained from a patient containing a detectable amount of BCMA protein or fragments thereof under normal or pathological conditions. Typically, the level of BCMA in a specific sample obtained from a healthy patient (e.g., a patient who does not suffer from a disease or condition associated with abnormal BCMA levels or activity) will be measured to preliminarily 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 or condition (e.g., a tumor containing cells expressing BCMA).

[0199] Example

[0200] The following examples are presented to provide a complete disclosure and description of how to prepare and use the methods and compositions of the present invention to those of ordinary skill in the art, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy with respect to the numerals used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is or is close to atmospheric pressure.

[0201] Example 1: Generation of anti-BCMA antibodies

[0202] Anti-BCMA antibodies are obtained by immunizing genetically modified mice or engineered mice comprising DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions with human BCMA antigen (eg, hBCMA, SEQ ID NO: 115).

[0203] After immunization, spleen cells are collected from each mouse and either (1) fused with mouse myeloma cells to maintain their viability and form hybridoma cells and screened for BCMA specificity, or (2) B cell sorting (as described in US2007 / 0280945A1) using human BCMA fragments as sorting agents to bind and identify reactive antibodies (antigen-positive B cells).

[0204] Initially isolated chimeric antibodies against BCMA with human variable regions and mouse constant regions. Antibodies are characterized and selected for desired characteristics (including affinity, selectivity, etc.). If necessary, the mouse constant region is replaced with a desired human constant region (e.g., a wild-type or modified IgG1 or IgG4 constant region) to produce fully human anti-BCMA antibodies. Although the selected constant region can vary depending on the specific use, high affinity antigen binding and target specificity properties are present in the variable region.

[0205] Anti-BCMA antibody heavy and light chain variable region amino acid and nucleic acid sequences: 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 present invention. The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0206] Table 1: Amino Acid Sequence Identifiers

[0207]

[0208]

[0209] Table 2: Nucleotide sequence identifiers

[0210]

[0211] Example 2: Generation of anti-CD3 antibodies

[0212] Anti-CD3 antibodies were generated as described in WO 2017 / 053856, which is incorporated herein by reference. Two such anti-CD3 antibodies were selected for the production of bispecific anti-BCMA x anti-CD3 antibodies according to the present invention. Table 3 lists 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 listed in Table 4. Other anti-CD3 antibodies for the preparation of bispecific antibodies according to the present invention can be found, for example, in WO 2014 / 047231.

[0213] Table 3: Amino Acid Sequence Identifiers

[0214]

[0215] Table 4: Nucleotide sequence identifiers

[0216]

[0217] Example 3: Generation of bispecific antibodies that bind BCMA and CD3

[0218] 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-CD3xBCMA or anti-BCMA x anti-CD3 bispecific molecules". The anti-BCMA portion of the anti-BCMAx anti-CD3 bispecific molecule can be used to target tumor cells expressing BCMA (also known as CD269), and the anti-CD3 portion of the bispecific molecule can be used to activate T cells. The simultaneous binding of BCMA to CD3 on tumor cells and T cells promotes the directed killing (cell lysis) of target tumor cells by activated T cells.

[0219] Bispecific antibodies comprising an anti-BCMA specific binding domain and an anti-CD3 specific binding domain are constructed using standard methods, wherein the anti-BCMA antigen binding domain and the anti-CD3 antigen binding domain each comprise a different, distinct HCVR paired with a common LCVR. In an exemplary bispecific antibody, the molecule is constructed using 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 using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-BCMA antibody, and an antibody light chain known to be effectively promiscuous or paired with a variety of heavy chain arms.

[0220] Table 5: Summary of components of anti-BCMA x anti-CD3 bispecific antibodies

[0221]

[0222]

[0223] Table 6 shows the amino acid sequence identifiers for the bispecific anti-BCMA x anti-CD3 antibodies exemplified herein.

[0224] Table 6: Amino acid sequences of anti-BCMA x anti-CD3 bispecific antibodies

[0225]

[0226] The bsAb25441D bispecific antibody (REGN5458) 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 the bsAb25441D bispecific antibody (REGN5458) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 130. The second heavy chain of the bsAb25441D bispecific antibody (REGN5458) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 131. The common light chain of the bsAb25441D bispecific antibody (REGN5458) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 132.

[0227] 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 the bsAb25442D bispecific antibody (REGN5459) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 130. The second heavy chain of the bsAb25442D bispecific antibody (REGN5459) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 131. The common light chain of the bsAb25442D bispecific antibody (REGN5459) comprises a constant region comprising the amino acid sequence of SEQ ID NO: 132.

[0228] Example 4: Surface Plasmon Resonance-Derived Binding Affinities and Kinetic Constants of Anti-BCMA Antibodies and Anti-BCMA x Anti-CD3 Bispecific Antibodies

[0229] The equilibrium dissociation constant (K) for hBCMA.mmh (SEQ ID NO: 106) binding to purified anti-BCMA mAb and anti-BCMA x anti-CD3 bispecific mAb was determined using a real-time surface plasmon resonance biosensor using a Biacore 4000 instrument. DValues). 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-hexa-histidine tag (human BCMA-MMH; SEQ ID NO: 106) or monkey BCMA expressed with a C-terminal myc-myc-hexa-histidine tag (monkey BCMA-MMH; SEQ ID NO: 110) were prepared in HBS-ET running buffer (ranging from 90 to 1.11 nM, 3-fold dilution). For dimer affinity, different concentrations of the extracellular domain of human BCMA expressed with a C-terminal mFc tag (human BCMA-MFC; SEQ ID NO: 108) and monkey BCMA expressed with a C-terminal mFc tag (monkey BCMA-MFC; SEQ ID NO: 112) prepared in HBS-ET running buffer (ranging from 30 to 0.37 nM, 3-fold dilution), or 30 nM BCMA expressed with a C-terminal mFc tag (mouse BCMA-MFC; SEQ ID NO: 114) were prepared. The antigen sample was then injected over the surface capturing the 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 minutes while dissociation was monitored for 10 minutes in HBS-ET running buffer. All binding kinetic experiments were performed at 25°C. The kinetic association (k) was determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software a ) and dissociation (k d ) rate constant. Binding and dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows:

[0230] and

[0231] As shown in Table 7, at 25°C, all anti-BCMA antibodies of the present invention bound to human BCMA-MMH with K D The values ​​ranged from 1.06 nM to 3.56 nM. As shown in Table 8, at 25°C, all anti-BCMA antibodies of the present invention bound to human BCMA-MFC with K DThe values ​​ranged from 22.3 pM to 103 pM. As shown in Table 9, at 25°C, the two anti-BCMA antibodies of the present invention bound to monkey BCMA-MMH, with K D The values ​​ranged from 38.8 nM to 49.92 nM. As shown in Table 10, at 25°C, the four anti-BCMA antibodies of the present invention bound to monkey BCMA-MFC, with K D The values ​​ranged from 148 pM to 14.7 nM. As shown in Table 11, at 25°C, the four anti-BCMA antibodies of the present invention bound to mouse BCMA-MFC, with K D The values ​​ranged from 677 pM to 18.8 nM.

[0232] Table 7: Binding kinetic parameters of anti-BCMA monoclonal antibodies binding to human BCMA-MMH at 25°C

[0233]

[0234] Table 8: Binding kinetic parameters of anti-BCMA monoclonal antibodies binding to human BCMA-MFC at 25°C

[0235]

[0236] Table 9: Binding kinetic parameters of anti-BCMA monoclonal antibodies binding to monkey BCMA-MMH at 25°C

[0237]

[0238] Table 10: Binding kinetic parameters of anti-BCMA monoclonal antibodies binding to monkey BCMA-MFC at 25°C

[0239]

[0240] Table 11: Binding kinetic parameters of anti-BCMA monoclonal antibodies binding to mouse BCMA-MFC at 25°C

[0241]

[0242]

[0243] Example 5: FACS binding of anti-BCMA x anti-CD3 bispecific antibodies to cells expressing human and cynomolgus CD3

[0244] Flow cytometry analysis was used to determine the binding of BCMAxCD3 bispecific antibodies to human and cynomolgus CD3 (Jurkat cells, mfCD3 engineered Jurkat cells, primary human CD8+ and cynomolgus CD8+ T cells). In brief, 1e05 cells / well were incubated on ice for 30 minutes with serially diluted BCMAxCD3 and control antibodies in the presence of FACS wash solution and block (PBS+1% filtered FBS+5% mouse serum). After incubation, the cells were washed twice with cold FACS wash solution (PBS+1% filtered FBS) and the bound antibodies were detected by incubating on ice with Alexa647-conjugated anti-human secondary antibodies for another 30 minutes. Wells without antibodies or only containing secondary antibodies were used as controls. In order to detect monkey and human T cells, a mixture of human and cynomolgus cross-reactive antibodies for 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 flow cytometry on a BD FACS Canto II. Cells were gated by FSC-H and FSC-A to select singlet events, followed by side and forward scatter to select real-time events. For monkey T cells, an additional gate was performed on CD8+ / CD16- cells.

[0245] EC50 values ​​for FACS binding were calculated using 4-parameter nonlinear regression analysis in Prism software.

[0246] Jurkat cells are a T-cell lymphoblastoid cell line expressing human CD3. REGN5458 binds to human CD3 on Jurkat cells and primary human CD8+ T cells with a median EC50 of 1.50x10 -8 M and 3.20x10 -8 M. REGN5459 binds weakly to human CD3, with a median EC50 of 5.58x10 -7 M, and the median EC50 for primary human CD8+ T cells was 4.71x10 -6 Using CRISPR / Cas9 technology, the Jurkat cell line was engineered to express cynomolgus monkey CD3ε and CD3δ chains in place of the human versions. The median EC50 for REGN5458 binding to the mfCD3 engineered Jurkat cell line was 1.51x10 -8 M, the median EC50 for binding to primary cynomolgus monkey CD8+ T cells was 4.66x10 -8 M. REGN5459 does not bind to mfCD3-expressing cells.

[0247] For the negative isotype control antibody (referred to as mAbl5260), no binding was observed on any of the cell lines.

[0248] Table 12: Binding to cells expressing CD3: Median EC50

[0249]

[0250] Example 6: FACS binding assay to evaluate cell surface antigen binding ability

[0251] 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, Cat# S-004-C) and seeded at 500,000 cells per well in 96-well V-bottom plates in staining buffer (PBS without calcium and magnesium (Irving 9240) + 2% FBS (ATCC 30-2020). Cells were stained with two-fold serial dilutions of Alexa 647-conjugated anti-BCMA x CD3 antibody (mAb25442D-A647) or Alexa 647-conjugated isotype control with the same CD3 binding arm paired with an irrelevant tumor targeting arm (Isotype-A647) for 30 minutes at 4°C. Cells were washed twice with staining buffer and incubated with LIVE / DEAD according to the manufacturer's instructions. TM Fixable Green Dead Cell Stain Kit (Invitrogen, L34970) was used for labeling to distinguish live cells from dead cells. The cells were then washed and fixed at 4°C for 25 minutes using a 50% BD Cytofix (BD, Cat#554655) solution diluted in PBS. The samples were run on an Accuri C6 flow cytometer (BD Biosciences) and analyzed in Flowjo 10.2 (Tree Star). After gating live cells and single cells, the mean fluorescence intensity (MFI) was determined, and the MFI values ​​were plotted on a 10-point response curve using a four-parameter logistic equation in Graphpad Prism to calculate the EC 50 The zero condition for each dose response curve was also included in the analysis as a continuation of the two-fold serial dilution and is represented as the lowest dose. The signal-to-noise ratio (S / N) was determined by taking the ratio of the mAb25442D-A647 MFI to the Isotype-A647 MFI. (Table 13). The mAb25442D-A647 S / N ranged from 2 to 470, with an EC 50Values ​​ranged from 27 to 83 nM. No detectable binding was observed on HEK293 cells.

[0252] Table 13: Binding to cells

[0253]

[0254]

[0255] ND = Not determined due to non-S-shaped curve

[0256] Example 7: Activation of T cells by bispecific anti-BCMA x anti-CD3 antibodies in the presence of BCMA-expressing cells

[0257] The activity of the anti-BCMA x anti-CD3 bispecific antibody was evaluated in a Jurkat / NFATLuc reporter bioassay using several cell lines with different BCMA surface expression levels. Jurkat cells were engineered to express a NFAT luciferase reporter (Jurkat / NFATLuc.3C7), and 50,000 Jurkat reporter cells were combined with 50,000 BCMA-positive (Daudi, MM1-S, NCI-H929, OPM-2, RPMI-8226, MOLP-8, or Raji) or BCMA-negative (HEK293) cells in a Thermo Nunclon delta 96-well white microplate (Thermo Scientific, Cat#136102) in 50 ul of assay medium (RPMI medium with 10% FBS and 1% P / S / G). Three-fold serial dilutions of BCMA x CD3 bispecific antibodies (mAb25441D or mAb25442D or bivalent anti-BCMA antibody (mAb21581) were immediately added to 50uL of assay buffer. The plates were gently agitated and incubated in a 37°C, 5% CO2 incubator for 4-6 hours. NFAT-luciferase activity was determined using Promega One-Glo (Cat# E6130) and a Perkin Elmer Envision plate reader. RLU was plotted on a 12-point response curve using a four-parameter logistic equation in GraphPadPrism to calculate the EC 50 Values. The no antibody treatment condition for each dose response curve was also included in the analysis as a continuation of the three-fold serial dilution and is represented as the lowest dose. The signal-to-noise ratio (S:N) was determined by the ratio of the highest RLU to the lowest RLU on the curve.

[0258] mAb25441D activated Jurkat / NFATLuc cells in the presence of BCMA expressing cells with EC50 ranging from 0.61nM to 2.1nM and S:N ranging from 8 to 123. mAb25442D activated Jurkat / NFATLuc cells in the presence of BCMA expressing cells with EC50 ranging from 2.6nM to 11nM and S:N ranging from 7 to 120. The BCMA x CD3 bispecific mAb25441D with a higher affinity CD3 binding arm was consistently more effective than mAb25442D with a lower affinity CD3 binding arm; while the S:N of the two bispecifics were similar. In the presence of HEK293 cells, neither antibody activated Jurkat / NFATLuc cells, and the control bispecific antibody did not significantly increase the Jurkat reporter gene activity of any cell line tested. The results are shown in Tables 14A and 14B below.

[0259] Table 14A: Activation of T cells

[0260]

[0261] Table 14B: Activation of T cells

[0262]

[0263] Example 8: FACS-based cytotoxicity assay to evaluate T cell-mediated killing of BCMA-expressing multiple myeloma cells in the presence of anti-BCMA x anti-CD3 bispecific antibodies

[0264] 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 and following the manufacturer's instructions (Bangs Laboratories).

[0265] Briefly, multiple myeloma (MM) cell lines (H929, MM1S, U266, MOLP8, and RPMI8226) and Quantum Simply Cellular beads were incubated at 4°C for 30 minutes while titrating APC-conjugated anti-hBCMA-19F2 antibodies. 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. Template (Bangs Labs), saturating levels of ABC for anti-BCMA 19F2 for each cell line were interpolated from a standard curve generated from the channel intensity of the bead population at saturation.

[0266] Killing of BCMA-expressing target cells by resting human or cynomolgus monkey T cells was determined by flow cytometry. Separately, human or cynomolgus monkey peripheral blood mononuclear cells (PBMCs) were cultured at 1×10 6 Cells / mL were plated in supplemented RPMI (human) or X-Vivo (cynomolgus monkey) medium and incubated overnight at 37°C to enrich lymphocytes by depleting adherent macrophages, dendritic cells, and some monocytes. The next day, target cells expressing BCMA were labeled with 1uM Violet CellTrace and incubated at 37°C with PBMCs (effector / target cell 4:1 ratio) depleted of adherent cells and serially diluted BCMAxCD3 bispecific antibodies or control antibodies. After 48-72 hours, cells were removed from the cell culture plates, stained with mixed phenotypic antibodies and live / dead cell viability dyes, and analyzed by FACS. To quantify the number of live target cells present in the wells, 20μl CountBright absolute counting beads were added to the wells before acquisition. To assess the specificity of killing, cells were gated on the population labeled with Violet Cell Tracker. The survival percentage of target cells was calculated as follows: Target survival = (R1 / R2)*100, where R1 = the absolute number of live target cells in the presence of effector cells and antibody, R2 = the number of live target cells alone (cultured without effector cells or test antibody).

[0267] Human CD8+ T cells were gated as CD45+ / CD14- / CD4- / CD8+. Cynomolgus CD8+ T cells were gated as CD45+ / CD20- / CD14- / CD4- / CD8+ T cell activation was reported as the percentage of CD25+ or CD69+ T cells to total CD8+ T cells.

[0268] EC50 values ​​for target cell survival and T cell activation were calculated using 4-parameter nonlinear regression analysis in Prism software.

[0269] The anti-BCMA x anti-CD3 bispecific antibody was tested for its ability to activate resting human and cynomolgus macaque T cells to kill a panel of BCMA-expressing cells with different surface BCMA levels. Using resting human T cells as effector cells, REGN5458 mediated killing of 5 different BCMA cell lines with EC 50 The value range is 7.07x10 -10 M to 3.45x10 -11 M. REGN5459 showed killing of the same five cell lines with EC50 values ​​ranging from 1.66x10 -9 M to 1.06x10 -10 M. EC of T cell activation measured by upregulation of CD25 on CD8+ T cells 50Similar to killing EC 50 Modest T cell activation was observed in the presence of the 1-arm CD3 isotype control mAb 17664D, but only against the U266 cell line. No cytotoxicity was observed for the isotype controls tested.

[0270] BCMAxCD3-mediated killing of cynomolgus monkey T cells was only tested on the MM cell line H929. EC cytotoxicity mediated by REGN5458 and REGN5459 50 2.34x10 -11 and 6.92x10 -11 No cytotoxicity or T cell activation was observed for the isotype control antibody mAb15260 with human or cynomolgus monkey effector cells. The results are shown in Tables 15A, 15B and 16 below.

[0271] Table 15A: Median EC 50 , human effector cells

[0272]

[0273] Table 15B: Median EC 50 , human effector cells

[0274]

[0275] Table 16: Median EC 50 , Cynomolgus monkey effector cells

[0276]

[0277] Example 9: FACS cytotoxicity assay of autologous T cell-mediated killing of primary multiple myeloma blasts in the presence of anti-BCMA x anti-CD3 bispecific antibodies

[0278] To monitor specific killing of multiple myeloma cells by flow cytometry, bone marrow mononuclear cells (BMMCs) from multiple myeloma patients were plated on human stromal cells (HS5) and incubated overnight at 37°C. Separately, matched patient peripheral blood mononuclear cells (PBMCs) were thawed and cultured at 1×10 6Cells / mL were cultured overnight at 37°C to enrich lymphocytes by consuming adherent cells. The next day, BMMCs were co-incubated with naive PBMCs depleted of adherent cells on stromal cells (HS5) and serial 10-fold dilutions of BCMAxCD3 bispecific or 1-arm CD3 isotype controls (starting concentration 66.7 nM) at 37°C. Cells were removed from the cell culture plates on days 3, 4, or 7 and analyzed by FACS. To assess the specificity of killing, multiple myeloma cells were gated as single, live, CD90 negative (to exclude stromal cells), CD2 negative, and CD56 positive. With the exception of MM455, the CD45 content of multiple myeloma cells in most samples was low. The reported percentage of live target cells was used to calculate the adjusted survival rate as follows: adjusted survival rate = (R1 / R2) * 100, where R1 =% live target cells in the presence of antibody, and R2 =% live target cells in the absence of test antibodies.

[0279] T cells were gated as CD2 positive, CD56 negative, and CD4 or CD8 positive. T cell activation was reported as the percentage of CD25+CD4 or CD8 T cells to total CD4 or CD8 T cells.

[0280] The ability of BCMAxCD3 bispecific antibodies to redirect autologous donor PBMCs to kill primary multiple myeloma blasts was tested. The maximum BCMAxCD3-mediated cytotoxicity on primary MM blasts ranged from 52-96% with an EC50 range of 9.89x10 for REGN5458. -11 M to 3.67x10 -9 M, 4.96x10 for REGN5459 -10 M to 7.94x10 -8 M. T cell activation was measured by assessing upregulation of CD25 on CD8+ T cells. The EC50 range for T cell activation was 3.23x10 -9 Up to 1.69x10 -10 For the 1-arm CD3 (no target binding) isotype control, moderate cytotoxicity and T cell activation were observed. The results are shown in Tables 17A and 17B below.

[0281] Table 17A: MM% lysis

[0282]

[0283] Table 17B: MM lysis EC50 and T cell activation

[0284]

[0285] Example 10: Anti-BCMA x anti-CD3 bispecific antibody prevents the growth of BCMA-expressing tumors (NCI-H929) in vivo in a xenogeneic tumor model

[0286] To determine the in vivo efficacy of the BCMAxCD3 bispecific antibody (Ab), xenograft studies were performed. scid Iq tm1Wjl / SzJ(NSG) mice were implanted subcutaneously with 10x10 6 0.5x10 NCI-H929 multiple myeloma cells expressing BCMA and 0.5x10 isolated from normal donors 6 A mixture of human peripheral blood mononuclear cells (PBMCs). Mice (n=7 per group) were immediately administered PBS vehicle control, irrelevant anti-FelD1 bivalent isotype control Ab (REGN2759), CD3 binding control bispecific Ab (mAb17664D), BCMAxCD3 (G; REGN5458) bispecific Ab, or BCMAxCD3 (G20; REGN5459) bispecific Ab at a dose of 4 mg / kg. Mice were administered Ab twice weekly for a total of three weeks and tumor growth was assessed over 40 days. Although BCMA + Tumors grew similarly in vehicle-, isotype control-, and CD3-binding control-treated mice, but both BCMAxCD3 Abs tested blocked tumor growth in vivo.

[0287] Syngeneic tumor implantation and measurement: NSG mice were subcutaneously implanted with 10x10 6 0.5x10 NCI-H929 multiple myeloma cells expressing BCMA and 0.5x10 derived from normal donors 6 Mice (n=7 per group) were immediately administered PBS vehicle control, irrelevant anti-FelD1 bivalent isotype control Ab (REGN2759), CD3 binding control bispecific Ab (mAb17664D), BCMAxCD3 (G; REGN5458) bispecific Ab, or BCMAxCD3 (G20; REGN5459) bispecific Ab at a dose of 4 mg / kg. Mice were administered Ab twice a week for a total of three weeks. Tumor growth was measured twice a week with a caliper for the duration of the experiment. Mice were sacrificed 40 days after tumor implantation.

[0288] Calculation of Tumor Growth and Inhibition: To determine tumor volume by external caliper gauges, the maximum longitudinal diameter (length, in mm) and the maximum transverse diameter (width, in mm) were measured. Tumor volume based on caliper gauge measurements was calculated by the following formula: Volume (mm 3 )=(length×width 2 ) / 2.

[0289] In xenograft tumor models, the BCMAxCD3 bispecific Ab blocked BCMA in vivo + Growth of NCI-H929 tumors. The results are shown in Table 18 below.

[0290] Table 18: Average tumor volume at different time points

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301] Example 11: Anti-BCMA x anti-CD3 bispecific antibody prevents the growth of BCMA-expressing tumors (NCI-H929) in a dose-dependent manner in a xenogeneic in vivo tumor model

[0302] To determine the in vivo efficacy of the anti-BCMA x anti-CD3 bispecific antibody (Ab), xenograft studies were performed. scid Iq tm1Wjl / SzJ(NSG) mice were implanted subcutaneously with 10x10 6 0.5x10 NCI-H929 human multiple myeloma cells expressing BCMA and 0.5x10 isolated from normal healthy donors 6A mixture of 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, BCMAxCD3 (G; REGN5458) bispecific Ab at doses of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg, or BCMAxCD3 (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 60 days. Although BCMA + NCI-H929 tumors grew similarly in vehicle- and CD3-binding control-treated mice, but both anti-BCMA x anti-CD3 Abs tested blocked tumor growth in vivo in a dose-dependent manner.

[0303] Implantation and measurement of xenograft tumors: NSG mice were subcutaneously implanted with 10x10 6 0.5x10 NCI-H929 multiple myeloma cells expressing BCMA and 0.5x10 derived from normal healthy donors 6 The 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), BCMAxCD3 (G; REGN5458) bispecific Ab, or BCMAxCD3 (G20; REGN5459) bispecific Ab. mAb17664D and REGN4460 were dosed at 4 mg / kg, while REGN5458 and REGN5459 were administered at doses of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. The mice were administered Ab twice weekly for a total of seven times. Tumor growth was measured twice weekly with a caliper for the duration of the experiment.

[0304] Calculation of xenograft tumor growth and inhibition: To determine tumor volume by external caliper gauges, the maximum longitudinal diameter (length, in mm) and the maximum transverse diameter (width, in mm) were measured. Tumor volume based on caliper measurements was calculated by the following formula: Volume (mm 3 )=(length×width 2 ) / 2.

[0305] In this xenogeneic in vivo tumor model, the BCMAxCD3 bispecific Ab blocked BCMA in a dose-dependent manner. + The results are shown in Table 19 below and are Figure 1 and Figure 2 In the description.

[0306] Table 19: Average tumor volume at different time points

[0307] Antibody treatment <![CDATA[Average tumor volume on day 4 (mm 3 ) ± SEM]]> PBS (vehicle control) 60.1±7.9 mAb17664D (CD3 binding control-G) – 4 mg / kg 42.5±4.7 REGN4460 (CD3 binding control-G20) – 4 mg / kg 52.0±5.9 REGN5458 (BCMAxCD3-G) – 4 mg / kg 18.0±1.2 REGN5458(BCMAxCD3-G)–0.4mg / kg 31.9±2.0 REGN5458(BCMAxCD3-G)–0.04mg / kg 32.0±2.9 REGN5459(BCMAxCD3-G20)–4mg / kg 21.8±3.4 REGN5459(BCMAxCD3-G20)–0.4mg / kg 19.6±4.4 REGN5459(BCMAxCD3-G20)–0.04mg / kg 33.0±4.4

[0308] Antibody treatment <![CDATA[Average tumor volume on day 7 (mm 3 ) ± SEM]]> PBS (vehicle control) 138.2±25.1 mAb17664D (CD3 binding control-G) – 4 mg / kg 108.6±17.8 REGN4460 (CD3 binding control-G20) – 4 mg / kg 132.4±21.1 REGN5458 (BCMAxCD3-G) – 4 mg / kg 1.3±1.3 REGN5458(BCMAxCD3-G)–0.4mg / kg 11.3±3.0 REGN5458(BCMAxCD3-G)–0.04mg / kg 30.8±5.5 REGN5459(BCMAxCD3-G20)–4mg / kg 8.0±4.3 REGN5459(BCMAxCD3-G20)–0.4mg / kg 7.3±3.6 REGN5459(BCMAxCD3-G20)–0.04mg / kg 8.4±4.0

[0309]

[0310]

[0311] Antibody treatment <![CDATA[Average tumor volume on day 15 (mm 3 ) ± SEM]]> PBS (vehicle control) 921.4±147.5 mAb17664D (CD3 binding control-G) – 4 mg / kg 874.8±86.6 REGN4460 (CD3 binding control-G20) – 4 mg / kg 1190.7±91.2 REGN5458 (BCMAxCD3-G) – 4 mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.4mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.04mg / kg 133.4±50.9 REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg 7.9±7.9

[0312] Antibody treatment <![CDATA[Average tumor volume on day 19 (mm 3 ) ± SEM]]> PBS (vehicle control) 1785.3±282.2 mAb17664D (CD3 binding control-G) – 4 mg / kg 1833.4±186.6 REGN4460 (CD3 binding control-G20) – 4 mg / kg 2336.5±188.3 REGN5458 (BCMAxCD3-G) – 4 mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.4mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.04mg / kg 413.7±162.7 REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg 23.1±23.1

[0313] Antibody treatment <![CDATA[Average tumor volume on day 22 (mm 3 ) ± SEM]]> PBS (vehicle control) 2601.5±414.5 mAb17664D (CD3 binding control-G) – 4 mg / kg 2878.5±257.6 REGN4460 (CD3 binding control-G20) – 4 mg / kg 3374.3±267.2 REGN5458 (BCMAxCD3-G) – 4 mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.4mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.04mg / kg 669.4±248.5 REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg 69.5±69.5

[0314] Antibody treatment <![CDATA[Average tumor volume on day 26 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.4mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.04mg / kg 1167.0±431.7 REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg 156.7±156.7

[0315]

[0316]

[0317] Antibody treatment <![CDATA[Average tumor volume on day 34 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.4mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.04mg / kg Euthanized animals REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg Euthanized animals

[0318] Antibody treatment <![CDATA[Average tumor volume on day 39 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.4mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.04mg / kg Euthanized animals REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg Euthanized animals

[0319]

[0320]

[0321] Antibody treatment <![CDATA[Average tumor volume on day 46 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.4mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.04mg / kg Euthanized animals REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg Euthanized animals

[0322] Antibody treatment <![CDATA[Average tumor volume on day 55 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.4mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.04mg / kg Euthanized animals REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg Euthanized animals

[0323]

[0324]

[0325] Example 12: Anti-BCMA x anti-CD3 bispecific antibody reduces the volume and prevents the growth of established BCMA-expressing tumors (NCI-H929) in a dose-dependent manner in a xenogeneic in vivo tumor model

[0326] To determine the in vivo efficacy of the anti-BCMA x anti-CD3 bispecific antibody (Ab), xenograft studies were performed. scid Iq tm1Wjl / SzJ(NSG) mice were implanted subcutaneously with 10x10 6 0.5x10 NCI-H929 human multiple myeloma cells expressing BCMA and 0.5x10 isolated from normal healthy donors 6 Tumors were allowed to grow and establish for 5 days until they were approximately 70 mm in size. 3 . On day 5, mice (n=7-8 per group) were administered PBS vehicle control, CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 4 mg / kg, CD3-binding control bispecific Ab (G20; REGN4460) at a dose of 4 mg / kg, BCMAxCD3 (G; REGN5458) bispecific Ab at doses of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg, or BCMAxCD3 (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 55 days. Although BCMA + NCI-H929 tumors grew similarly in vehicle- and CD3-binding control-treated mice, but both BCMAxCD3 Abs tested shrank established tumors and prevented tumor growth in vivo in a dose-dependent manner.

[0327] Implantation and measurement of xenograft tumors: NSG mice were subcutaneously implanted with 10x10 6 0.5x10 NCI-H929 multiple myeloma cells expressing BCMA and 0.5x10 derived from normal healthy donors 6 Tumors were allowed to grow and establish for 5 days until they were approximately 70 mm in size. 3 . On day 5, mice (n=7-8 per group) were then administered PBS vehicle control, CD3 binding control bispecific Ab (G; mAb17664D), CD3 binding control bispecific Ab (G20; REGN4460), BCMAxCD3 (G; REGN5458) bispecific Ab, or BCMAxCD3 (G20; REGN5459) bispecific Ab. mAb17664D and REGN4460 were dosed at 4 mg / kg, while REGN5458 and REGN5459 were administered at doses of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. Mice were administered Ab twice weekly for a total of seven times. Tumor growth was measured twice weekly with a caliper for the duration of the experiment.

[0328] Calculation of xenograft tumor growth and inhibition: To determine tumor volume by external caliper gauges, the maximum longitudinal diameter (length, in mm) and the maximum transverse diameter (width, in mm) were measured. Tumor volume based on caliper measurements was calculated by the following formula: Volume (mm 3 )=(length×width 2 ) / 2.

[0329] In this xenogeneic in vivo tumor model, the anti-BCMA x anti-CD3 bispecific antibody reduced established BCMA in a dose-dependent manner. + The results are shown in Table 20 below and are Figure 3 and Figure 4 In the description.

[0330] Table 20: Average tumor volume at different time points

[0331] Antibody treatment <![CDATA[Average tumor volume on day 5 (mm 3 ) ± SEM]]> PBS (vehicle control) 61.5±6.4 mAb17664D (CD3 binding control-G) – 4 mg / kg 63.7±5.4 REGN4460 (CD3 binding control-G20) – 4 mg / kg 62.6±3.6 REGN5458 (BCMAxCD3-G) – 4 mg / kg 71.9±10.3 REGN5458(BCMAxCD3-G)–0.4mg / kg 69.3±7.3 REGN5458(BCMAxCD3-G)–0.04mg / kg 58.1±5.6 REGN5459(BCMAxCD3-G20)–4mg / kg 61.8±5.2 REGN5459(BCMAxCD3-G20)–0.4mg / kg 69.5±4.1 REGN5459(BCMAxCD3-G20)–0.04mg / kg 74.9±6.4

[0332] Antibody treatment <![CDATA[Average tumor volume on day 8 (mm 3 ) ± SEM]]> PBS (vehicle control) 124.3±17.3 mAb17664D (CD3 binding control-G) – 4 mg / kg 145.3±22.0 REGN4460 (CD3 binding control-G20) – 4 mg / kg 170.7±15.5 REGN5458 (BCMAxCD3-G) – 4 mg / kg 64.7±16.4 REGN5458(BCMAxCD3-G)–0.4mg / kg 120.3±16.3 REGN5458(BCMAxCD3-G)–0.04mg / kg 130.3±16.7 REGN5459(BCMAxCD3-G20)–4mg / kg 45.8±9.8 REGN5459(BCMAxCD3-G20)–0.4mg / kg 171.9±23.2 REGN5459(BCMAxCD3-G20)–0.04mg / kg 152.3±20.0

[0333]

[0334]

[0335] Antibody treatment <![CDATA[Average tumor volume on day 15 (mm 3 ) ± SEM]]> PBS (vehicle control) 1150.4±105.7 mAb17664D (CD3 binding control-G) – 4 mg / kg 1041.4±101.3 REGN4460 (CD3 binding control-G20) – 4 mg / kg 1298.4±71.0 REGN5458 (BCMAxCD3-G) – 4 mg / kg 25.6±19.2 REGN5458(BCMAxCD3-G)–0.4mg / kg 476.2±133.5 REGN5458(BCMAxCD3-G)–0.04mg / kg 1031.2±164.3 REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 327.2±135.6 REGN5459(BCMAxCD3-G20)–0.04mg / kg 1094.2±78.9

[0336]

[0337]

[0338] Antibody treatment <![CDATA[Average tumor volume on day 22 (mm 3 ) ± SEM]]> PBS (vehicle control) 3717.9±214.5 mAb17664D (CD3 binding control-G) – 4 mg / kg 3688.9±272.0 REGN4460 (CD3 binding control-G20) – 4 mg / kg 4492.2±344.0 REGN5458 (BCMAxCD3-G) – 4 mg / kg 78.3±60.8 REGN5458(BCMAxCD3-G)–0.4mg / kg 2068.5±465.0 REGN5458(BCMAxCD3-G)–0.04mg / kg 3745.7±541.2 REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 815.4±387.1 REGN5459(BCMAxCD3-G20)–0.04mg / kg 3285.9±227.3

[0339] Antibody treatment <![CDATA[Average tumor volume on day 27 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 252.3±185.1 REGN5458(BCMAxCD3-G)–0.4mg / kg 3463.9±1025.0 REGN5458(BCMAxCD3-G)–0.04mg / kg 1589.1±0 REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 1849.9±903.1 REGN5459(BCMAxCD3-G20)–0.04mg / kg Euthanized animals

[0340]

[0341]

[0342] Antibody treatment <![CDATA[Average tumor volume on day 35 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 633.5±473.5 REGN5458(BCMAxCD3-G)–0.4mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.04mg / kg Euthanized animals REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 996.8±771.0 REGN5459(BCMAxCD3-G20)–0.04mg / kg Euthanized animals

[0343] Antibody treatment <![CDATA[Average tumor volume on day 40 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 369.5±369.5 REGN5458(BCMAxCD3-G)–0.4mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.04mg / kg Euthanized animals REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 375.6±375.6 REGN5459(BCMAxCD3-G20)–0.04mg / kg Euthanized animals

[0344]

[0345]

[0346] Example 13: Anti-BCMA x anti-CD3 bispecific antibody prevents the growth of BCMA-expressing tumors (MOLP-8) in a dose-dependent manner in a xenogeneic in vivo tumor model

[0347] To determine the in vivo efficacy of the anti-BCMA x anti-CD3 bispecific antibody (Ab), xenograft studies were performed. scid Iq tm1Wjl / SzJ(NSG) mice were implanted subcutaneously with 5x10 6 1x10 MOLP-8 human multiple myeloma cells expressing BCMA and 1x10 isolated from normal healthy donors 6 A mixture of 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, BCMAxCD3 (G; REGN5458) bispecific Ab at doses of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg, or BCMAxCD3 (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 56 days. Although BCMA + MOLP-8 tumors grew similarly in vehicle- and CD3-binding control-treated mice, but both BCMAxCD3Abs tested blocked tumor growth in vivo in a dose-dependent manner.

[0348] Implantation and measurement of xenograft tumors: NSG mice were subcutaneously implanted with 5x10 6 1x10 MOLP-8 multiple myeloma cells expressing BCMA and 1x10 derived from normal healthy donors 6 The 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), BCMAxCD3 (G; REGN5458) bispecific Ab, or BCMAxCD3 (G20; REGN5459) bispecific Ab. mAb17664D and REGN4460 were dosed at 4 mg / kg, while REGN5458 and REGN5459 were administered at doses of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. The mice were administered Ab twice weekly for a total of seven times. Tumor growth was measured twice weekly with a caliper for the duration of the experiment.

[0349] Calculation of xenograft tumor growth and inhibition: To determine tumor volume by external caliper gauges, the maximum longitudinal diameter (length, in mm) and the maximum transverse diameter (width, in mm) were measured. Tumor volume based on caliper measurements was calculated by the following formula: Volume (mm 3 )=(length×width 2 ) / 2.

[0350] In this xenograft in vivo tumor model, the anti-BCMA x anti-CD3 bispecific antibody blocked BCMA in a dose-dependent manner. + The volume growth of MOLP-8 tumors. The results are shown in Table 21 below and are Figure 5 and Figure 6 In the description.

[0351] Table 21: Average tumor volume at different time points

[0352] Antibody treatment <![CDATA[Average tumor volume on day 3 (mm 3 ) ± SEM]]> PBS (vehicle control) 10.3±3.0 mAb17664D (CD3 binding control-G) – 4 mg / kg 11.6±2.0 REGN4460 (CD3 binding control-G20) – 4 mg / kg 14.1±3.9 REGN5458 (BCMAxCD3-G) – 4 mg / kg 12.5±1.3 REGN5458(BCMAxCD3-G)–0.4mg / kg 13.5±1.5 REGN5458(BCMAxCD3-G)–0.04mg / kg 9.3±2.4 REGN5459(BCMAxCD3-G20)–4mg / kg 12.9±1.3 REGN5459(BCMAxCD3-G20)–0.4mg / kg 14.0±1.6 REGN5459(BCMAxCD3-G20)–0.04mg / kg 11.7±2.1

[0353] Antibody treatment <![CDATA[Average tumor volume on day 7 (mm 3 ) ± SEM]]> PBS (vehicle control) 73.4±13.5 mAb17664D (CD3 binding control-G) – 4 mg / kg 50.0±6.6 REGN4460 (CD3 binding control-G20) – 4 mg / kg 45.7±6.1 REGN5458 (BCMAxCD3-G) – 4 mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.4mg / kg 1.0 1.0 REGN5458(BCMAxCD3-G)–0.04mg / kg 18.3±5.0 REGN5459(BCMAxCD3-G20)–4mg / kg 0.6±0.6 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg 37.0±5.7

[0354]

[0355]

[0356] Antibody treatment <![CDATA[Average tumor volume on day 14 (mm 3 ) ± SEM]]> PBS (vehicle control) 677.0±62.7 mAb17664D (CD3 binding control-G) – 4 mg / kg 530.0±44.6 REGN4460 (CD3 binding control-G20) – 4 mg / kg 549.1±59.2 REGN5458 (BCMAxCD3-G) – 4 mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.4mg / kg 0±0 REGN5458(BCMAxCD3-G)–0.04mg / kg 255.4±79.7 REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg 356.7±84.6

[0357] Antibody treatment <![CDATA[Average tumor volume on day 17 (mm 3 ) ± SEM]]> PBS (vehicle control) 1349.5±149.7 mAb17664D (CD3 binding control-G) – 4 mg / kg 935.3±71.3 REGN4460 (CD3 binding control-G20) – 4 mg / kg 1027.1±86.6 REGN5458 (BCMAxCD3-G) – 4 mg / kg 14.5±7.3 REGN5458(BCMAxCD3-G)–0.4mg / kg 1.7±1.7 REGN5458(BCMAxCD3-G)–0.04mg / kg 494.3±144.3 REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg 645.6±140.9

[0358]

[0359]

[0360] Antibody treatment <![CDATA[Average tumor volume on day 23 (mm 3 ) ± SEM]]> PBS (vehicle control) 4155.1±401.8 mAb17664D (CD3 binding control-G) – 4 mg / kg 3288.4±204.6 REGN4460 (CD3 binding control-G20) – 4 mg / kg 3592.7±224.2 REGN5458 (BCMAxCD3-G) – 4 mg / kg 193.3±117.7 REGN5458(BCMAxCD3-G)–0.4mg / kg 9.7±9.7 REGN5458(BCMAxCD3-G)–0.04mg / kg 1882.3±551.5 REGN5459(BCMAxCD3-G20)–4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg 2124.4±444.1

[0361] Antibody treatment <![CDATA[Average tumor volume on day 28 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 627.4±318.1 REGN5458(BCMAxCD3-G)–0.4mg / kg 47.4±47.4 REGN5458(BCMAxCD3-G)–0.04mg / kg 2542.5±613.3 REGN5459(BCMAxCD3-G20)–4mg / kg 1.9±1.9 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg 1939.3±840.6

[0362] Antibody treatment <![CDATA[Average tumor volume on day 31 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 1018.5±498.3 REGN5458(BCMAxCD3-G)–0.4mg / kg 104.7±92.6 REGN5458(BCMAxCD3-G)–0.04mg / kg 2906.1±532.6 REGN5459(BCMAxCD3-G20)–4mg / kg 3.8±3.0 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg 2688.7±1176.6

[0363] Antibody treatment <![CDATA[Average tumor volume on day 35 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 1342.9±629.6 REGN5458(BCMAxCD3-G)–0.4mg / kg 375.1±307.5 REGN5458(BCMAxCD3-G)–0.04mg / kg 3538.0±0.0 REGN5459(BCMAxCD3-G20)–4mg / kg 9.3±7.5 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg 612.1±0

[0364]

[0365]

[0366] Antibody treatment <![CDATA[Average tumor volume on day 49 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 1683.5±1683.5 REGN5458(BCMAxCD3-G)–0.4mg / kg No records REGN5458(BCMAxCD3-G)–0.04mg / kg Euthanized animals REGN5459(BCMAxCD3-G20)–4mg / kg No records REGN5459(BCMAxCD3-G20)–0.4mg / kg No records REGN5459(BCMAxCD3-G20)–0.04mg / kg Euthanized animals

[0367] Antibody treatment <![CDATA[Average tumor volume on day 56 (mm 3 ) ± SEM]]> PBS (vehicle control) Euthanized animals mAb17664D (CD3 binding control-G) – 4 mg / kg Euthanized animals REGN4460 (CD3 binding control-G20) – 4 mg / kg Euthanized animals REGN5458 (BCMAxCD3-G) – 4 mg / kg 3108.1±3108.1 REGN5458(BCMAxCD3-G)–0.4mg / kg 1742.4±635.2 REGN5458(BCMAxCD3-G)–0.04mg / kg Euthanized animals REGN5459(BCMAxCD3-G20)–4mg / kg 17.2±17.2 REGN5459(BCMAxCD3-G20)–0.4mg / kg 0±0 REGN5459(BCMAxCD3-G20)–0.04mg / kg Euthanized animals

[0368] Example 14: Anti-BCMA x anti-CD3 bispecific antibody delays the growth of BCMA-expressing tumors (MOLP-8) in a xenogeneic in vivo tumor model

[0369] To determine the in vivo efficacy of the anti-BCMA x anti-CD3 bispecific antibody (Ab), a xenograft study was performed. scid Iq tm1Wjl / SzJ(NSG) mice were injected intraperitoneally with 4x10 6 Human peripheral blood mononuclear cells (PBMCs). On day 0, mice were intravenously administered 2x10 6 BCMA + MOLP-8 human multiple myeloma tumor cells, which were engineered to also express firefly luciferase (MOLP-8-luciferase cells). The mice (n=5 per group) were then immediately administered a 4 mg / kg dose of a CD3-binding control bispecific Ab (G; mAb17664D) or a 4 mg / kg dose of a BCMAxCD3 (G; REGN5458) bispecific Ab. The 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, one group of mice (n=5) was given only MOLP-8-luciferase cells without PBMCs or antibodies. To measure background BLI levels, one group of mice (n=5) was untreated and did not receive tumors, PBMCs, or antibodies. Although BCMA + MOLP-8-luciferase tumors gradually grew in CD3+ control-treated mice, but treatment with the BCMAxCD3 Ab of REGN5458 delayed tumor growth in vivo.

[0370] Xenograft Implantation and Measurements: On day -11, immunodeficient NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ(NSG) mice were injected intraperitoneally with 5x10 6 On day 0, mice were intravenously administered 2x10 6 BCMA +MOLP-8-luciferase cells. Then mice (n=5 per group) were immediately administered a 4mg / kg dose of CD3-binding control bispecific Ab (G; mAb17664D) or a 4mg / kg dose of BCMAxCD3 (G; REGN5458) bispecific Ab. On the 3rd and 7th days, mice were administered these Abs twice more, for a total of three doses. Tumor growth was assessed within 48 days by measuring tumor BLI in anesthetized animals. As a positive control, a group of mice (n=5) were given only MOLP-8-luciferase cells without PBMC or antibodies. In order to measure background BLI levels, a group of mice (n=5) were untreated and did not receive tumors, PBMCs or antibodies.

[0371] Measurement of xenograft tumor growth: BLI imaging was used to measure tumor burden. Mice were injected IP with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after this injection, BLI imaging of mice was performed under isoflurane anesthesia using the Xenogen IVIS system. Image acquisition was performed at a 3D field of view, 1.5 cm subject height, and mid-group level, with automatic exposure times determined by the Living Image software. BLI signals were extracted using the Living Image software: a region of interest was drawn around each tumor mass, and photon intensity was recorded as p / s / cm2 / sr.

[0372] The anti-BCMA x anti-CD3 bispecific antibody REGN5458 delayed BCMA in this xenogeneic in vivo tumor model + Growth of MOLP-8-luciferase tumors. The results are shown in Table 22 below.

[0373] Table 22: Average tumor volume at different time points (by radiation intensity)

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386] Example 15: Anti-BCMA x anti-CD3 bispecific antibody reduces tumor (OPM-2) burden to background levels in vivo

[0387] To determine the in vivo efficacy of the anti-BCMA x anti-CD3 bispecific antibody (Ab), a xenograft study was performed. scid Iq tm1Wjl / SzJ(NSG) mice were intravenously administered 2x10 6 BCMA + OPM-2 human multiple myeloma tumor cells, which were engineered to also express firefly luciferase (OPM-2-luciferase cells). On day 10, mice were injected intraperitoneally with 4x10 6 Human peripheral blood mononuclear cells (PBMCs). On day 21, mice (n=5 per group) were administered a 0.4 mg / kg dose of a CD3-binding control bispecific Ab (G; mAb17664D), 0.4 mg / kg of BCMAxCD3 (G; REGN5458 bispecific Ab, or 0.4 mg / kg of BCMAxCD3 (G20; REGN5459) bispecific Ab. Mice were administered two more doses of these Abs on days 25 and 28, for a total of three doses. Tumor growth over 61 days was assessed by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, one group of mice (n=5) was given only OPM-2-luciferase cells without PBMCs or antibodies. To measure background BLI levels, one group of mice (n=5) was untreated and received no tumor, PBMCs, or antibodies. Although BCMA + OPM-2-luciferase tumors progressively outgrew in CD3-binding control-treated mice, but treatment with the BCMAxCD3 Abs of REGN5458 and REGN5459 reduced tumor burden to background levels in most animals.

[0388] Xenograft Implantation and Measurements: On day 0, immunodeficient NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ(NSG) mice were intravenously administered 2x10 6 BCMA +OPM-2 human multiple myeloma tumor cells, which were engineered to also express firefly luciferase (OPM-2-luciferase cells). On day 10, mice were injected intraperitoneally with 4x10 6 Human peripheral blood mononuclear cells (PBMCs). On day 21, mice (n = 5 per group) were administered a 0.4 mg / kg dose of a CD3-binding control bispecific Ab (G; mAb17664D), 0.4 mg / kg of a BCMAxCD3 (G; REGN5458 bispecific Ab, or 0.4 mg / kg of a BCMAxCD3 (G20; REGN5459) bispecific Ab. Mice were administered two more doses of these Abs on days 25 and 28, for a total of three doses. Tumor growth over 61 days was assessed by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, one group of mice (n = 5) was given only OPM-2-luciferase cells without PBMCs or antibodies. To measure background BLI levels, one group of mice (n = 5) was untreated and did not receive tumors, PBMCs, or antibodies.

[0389] Measurement of xenograft tumor growth: BLI imaging was used to measure tumor burden. Mice were injected IP with 150 mg / kg of the luciferase substrate D-luciferin suspended in PBS. Five minutes after this injection, BLI imaging of mice was performed under isoflurane anesthesia using the Xenogen IVIS system. Image acquisition was performed at a 3D field of view, 1.5 cm subject height, and mid-group level, with automatic exposure times determined by the Living Image software. BLI signals were extracted using the Living Image software: a region of interest was drawn around each tumor mass, and photon intensity was recorded as p / s / cm2 / sr.

[0390] Although BCMA + OPM-2-luciferase tumors gradually grew in mice treated with CD3 binding control, but BCMAxCD3 Ab treatment with REGN5458 and REGN5459 reduced tumor burden to background levels in most animals. The results are shown in Table 23 below and in Figure 7 Medium picture.

[0391] Table 23: Average tumor volume at different time points (by radiation intensity)

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405] Example 16: BCMAxCD3 bispecific antibody inhibits the growth of syngeneic tumors in vivo in a dose-dependent manner

[0406] 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 murine CD3deg (CD3 humanized mice) were subcutaneously implanted with 0.5x10 cells engineered to express full-length human BCMA (B16 / BCMA cells). 6 1x10 B16 melanoma cells or 1x10 engineered to express full-length human BCMA (MC38 / BCMA) 6 MC38 colon cancer cells. Mice (n=7 per group) were then immediately administered a 0.4 mg / kg dose of a CD3 binding control bispecific Ab (G; mAb17664D) or a 0.4 mg / kg or 0.04 mg / kg dose of a BCMAxCD3 (G; REGN5458) bispecific Ab. Mice were administered these antibodies twice more on days 4 and 7 for a total of three doses, and tumor growth was assessed throughout the experiment. While B16 / BCMA tumors and MC38 / BCMA tumors grew in CD3 binding control treated mice, BCMAxCD3 REGN5458 was able to inhibit the growth of both tumor lines in vivo in a dose-dependent manner.

[0407] Implantation and measurement of syngeneic tumors: C57BL / 6 mice expressing human CD3deg instead of murine CD3deg (CD3 humanized mice) were subcutaneously implanted with 0.5x10 cells engineered to express full-length human BCMA (B16 / BCMA cells). 61x10 B16F10 melanoma cells or 1x10 engineered to express full-length human BCMA (MC38 / BCMA) 6 MC38 colon cancer cells. Mice (n = 7 per group) were then immediately administered a 0.4 mg / kg dose of a CD3-binding control bispecific Ab (G; mAb17664D) or a 0.4 mg / kg or 0.04 mg / kg dose of a BCMAxCD3 (G; REGN5458) bispecific Ab. Mice were administered these antibodies twice more on days 4 and 7 for a total of three doses, and tumor growth was assessed throughout the experiment.

[0408] Calculation of Syngeneic Tumor Growth and Inhibition: To determine tumor volume by external calipers, the maximum longitudinal diameter (length, in mm) and the maximum transverse diameter (width, in mm) were measured. Tumor volume based on caliper measurements was calculated by the following formula: Volume (mm 3 )=(length×width 2 ) / 2.

[0409] While B16 / BCMA tumors and MC38 / BCMA tumors grew in CD3 binding control treated mice, BCMAxCD3 REGN5458 was able to inhibit the growth of both tumor lines in vivo in a dose-dependent manner. The results are shown in Table 24 below.

[0410] Table 24: Average tumor volume at different time points

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417] Example 17: Epitope Mapping of REGN5458 Binding to BCMA by Hydrogen-Deuterium Exchange

[0418] H / D exchange epitope mapping was performed using mass spectrometry (HDX-MS) to identify amino acid residues of BCMA (recombinant human BCMA, amino acid sequence SEQ ID NO: 115) that interact with REGN5458 (BCMA x CD3 bispecific antibody). A general description of H / D exchange is shown in, for example, Ehring (1999) Analytical Biochemistry 267(2): 252-259; and Engen and Smith (2001) Analytical Chemistry 73: 256A-265A.

[0419] 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 stage (μBinary Solvent Manager) for sample digestion and loading, a Waters Acquity M stage (μBinary Solvent Manager) for analytical gradients, and a Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

[0420] The labeling solution was prepared as PBS buffer (10 mM phosphate buffer, 140 mM NaCl and 3 mM KCl, equivalent to pH 7.4 at 25°C) in D2O at pD 7.0. For deuterium labeling, 10 μL hBCMA.hFc (REGN2746, 54.5 μM; SEQ ID NO: 120 or hBCMA.hFc) premixed with REGN5458 at a 1:2 molar ratio (Ag-Ab complex) was incubated with 90 μL D2O labeling solution at 20°C in duplicate at various time points (e.g., undeuterated control = 0 seconds; deuterium labeled, 5 minutes and 10 minutes). The deuteration reaction was quenched by adding 100 μL pre-chilled quench buffer (0.5M TCEP-HCl, 8M urea and 1% formic acid) to each sample for 5 minutes at 20°C. The quenched samples were then injected into the Waters HDX Manager for online pepsin / protease XIII digestion. The 50 μL DO labeling solution was then incubated with 10 μL DO labeling solution at 20°C for 5 minutes. Digested peptides were separated using a 13-min gradient from 10% to 32% B (mobile phase A: 0.5% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile) on a 4x50 mm, NovaBioassays. The eluted peptides were analyzed by Q Exactive HF mass spectrometry in LC-MS / MS or LC-MS mode.

[0421] The LC-MS / MS data of the undeuterated BCMA samples were searched against a database containing BCMA and its random sequences using the Byonic search engine (Protein Metrics). The search parameters (in ELN) were set to default values ​​using nonspecific enzymatic digestion and human glycosylation as common variable modifications. The list of identified peptides was then imported into HDX Workbench software (version 3.3) to calculate the deuterium uptake for each peptide detected by LC-MS from all deuterated samples. For a given peptide, the center-of-mass mass (intensity-weighted average mass) at each time point was used to calculate the deuterium uptake (D) and the percentage of deuterium uptake (%D);

[0422]

[0423] A total of 8 peptides from hBCMA.hFc were identified from hBCMA.hFc alone and hBCMA.hFc in complex with REGN5458 samples, representing 100% sequence coverage of hBCMA. The average standard deviation (SD) of all peptides was estimated to be 1.4% (detailed calculations are defined in ELN and Pascal, BD et al. (2012) Journal of the American Society for Mass Spectrometry 23 (9): 1512-1521). Therefore, any peptide that exhibited a percentage difference in D-absorption magnitude greater than 4.2% (3 times the average SD) was defined as being significantly protected. For 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).

[0424] Table 25: Selected BCMA.hFc peptides with significant protection when combined with REGN5458

[0425]

[0426] Example 18: FACS binding assay of BCMAxCD3 bispecific antibodies and other BCMA antibodies on multiple myeloma cell lines after overnight incubation with anti-BCMA antibodies

[0427] Flow cytometric 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 667nM anti-BCMA antibodies, DAPT (a γ-secretase inhibitor), or medium alone. After 18 hours, the wells were washed with cold FACS wash buffer (PBS + 1% filtered FBS) and resuspended in 667nM of the same anti-BCMA antibody in cold staining buffer (Miltenyi 130-091-221) on ice for 30 minutes. After incubation, the cells were washed twice with cold FACS wash buffer (PBS + 1% filtered FBS) and the bound antibodies were detected by incubating with the appropriate anti-human secondary antibody (anti-hIgG or anti-HIS) for another 30-45 minutes on ice. 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. The fold increase in staining was calculated by dividing the MFI of stained cells previously incubated overnight in BCMA abs or DAPT by the MFI of stained cells incubated overnight in medium alone.

[0428] BCMA is rapidly cleaved from the cell surface by the enzyme γ-secretase. Overnight incubation with γ-secretase inhibitors such as DAPT prevents BCMA cleavage, resulting in elevated cell surface BCMA levels. Tables 26-29 report the multiple increases in the median fluorescence intensity (MFI) of BCMA on cells incubated overnight in anti-BCMA antibodies or DAPT compared to cells incubated in culture medium alone. We observed that overnight incubation with DAPT increased the BCMA levels detected by anti-BCMA antibodies (BCMAxCD3 bispecific R5458, parental BCMA antibody mAb15281, and other internal BCMA antibodies) on H929, Molp8, U266, and MM.1S by 2.3-4 times, 2.4-8.6 times, 5.3-9.0 times, and 11.9 times, respectively.

[0429] Notably, we also observed that overnight incubation of MM cell lines with 66.7 or 667 nM REGN5458 or the parental bivalent anti-BCMA antibody mAb21581 similarly resulted in increased surface BCMA levels detected by FACS, suggesting that binding of the anti-BCMA antibody prevented γ-secretase cleavage of BCMA. The antibody-induced increase in surface BCMA varied between cell lines, with greater fold increases in Molp8 and MM1S cells compared to H929 or U266. This phenomenon was not limited to REGN5458 but was also observed with other in-house BCMA antibodies.

[0430] Table 26: MFI fold change for cells incubated in medium alone (NCI-H929)

[0431]

[0432] Table 27: MFI fold change for cells incubated in medium only (Molp8)

[0433]

[0434]

[0435] Table 28: MFI fold change for cells incubated in medium only (U266)

[0436]

[0437] Table 29: MFI fold change for cells incubated in medium only (MM1S)

[0438]

[0439] Example 19: Autologous T cell-mediated killing of human and cynomolgus monkey plasma cells in the presence of BCMAxCD3 bispecific antibody

[0440] Evaluation of unstimulated autologous T cells enriched for CD138 by flow cytometry + Specific killing of human or cynomolgus monkey plasma cells. Provide human or cynomolgus monkey bone marrow aspirates and blood within 24 hours of harvest. Use EasySep Human CD138 according to the manufacturer's instructions. + Positive Selection Kit, enrichment of CD138 from bone marrow by positive selection + Plasma cells. PBMCs were isolated from whole blood by density separation. PBMCs were labeled with 1 μM Vybrant CFDA-SE fluorescent tracer dye. After labeling, 1x10 4 CD138 enriched + Plasma cells were seeded at an E:T ratio of 10:1 with Vybrant CFDA-SE labeled PBMC and serial dilutions of REGN5458, CD3 binding control bsAb, or BCMA binding control mAb in round-bottom 96-well plates in complete medium for 72 hours at 37°C. At the end of the culture period, viable CD138 +Plasma cells. Percent survival was normalized to control conditions (plasma cells in the presence of PBMCs only). T cell activation was assessed by flow cytometry. Activation was reported as CD2 + / CD4 + or CD2 + / CD8 + / CD16 - Percentage of T cells. The percentage of T cell activation was normalized to the control condition (plasma cells in the presence of PBMCs only).

[0441] In vitro studies evaluated the effects of REGN5458 or negative controls (BCMA-binding control mAb or CD3-binding control bsAb) on primary human and cynomolgus macaque T cell activation and cytotoxicity of autologous plasma cells. EC values ​​for cytotoxicity and percentage of T cell activation for each donor were 50 The values ​​are summarized in Table 30.

[0442] 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, EC 50 The values ​​were 42.8 pM and 191 pM, respectively, and resulted in maximum cytotoxicity percentages of 91% and 89%, respectively. Meanwhile, in the presence of human plasma cells from donors 1 and 2, REGN5458 mediated T cell activation in a concentration-dependent manner, CD8 + T cell activated EC 50 The values ​​were 214pM and 860pM, respectively, and CD8 + The maximum percentages of T cell activation were 2% and 36%, respectively. Cytotoxicity of plasma cells in both donors and increased CD8 in donor 2 were observed only at nanomolar concentrations of CD3 binding control. + T cell activation. No effects on cytotoxicity or T cell activation were observed using the BCMA binding control at any concentration tested in either donor.

[0443] REGN5458 mediates cytotoxicity in primary cynomolgus monkey plasma cells from two donors in a concentration-dependent manner; EC for donor 1 was calculated 50 The EC of donor 2 was 1.31 nM, but the EC of donor 2 could not be determined. 50 In both donors, REGN5458 treatment resulted in increased cytotoxicity of plasma cells (maximum cytotoxicity percentages were 94% and 91% for donors 1 and 2, respectively). Meanwhile, REGN5458 mediated T cell activation in a concentration-dependent manner in the presence of cynomolgus macaque plasma cells from donors 1 and 2, respectively, CD4 + T cell activated EC 50 The values ​​were 28.1nM and 18.1nM, while CD8 + T cell activated EC50 The values ​​were 22.4 nM and 76.7 nM for donors 1 and 2, respectively. The maximum T cell activation percentages obtained were for CD4 + T cells were 9% and 16%, respectively, for CD8 + T cells were 12% and 17%.

[0444] No target cell killing was observed at any concentration of BCMA binding control 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 nanomolar concentrations of CD3 binding control.

[0445] Table 30: EC of cytotoxicity and T cell activation percentage for each donor 50 value

[0446]

[0447] a Autologous plasma cells were tested for each donor.

[0448] Example 20: Anti-BCMA x anti-CD3 bispecific antibody synergizes with anti-PD-1 antibody to enhance anti-tumor efficacy in vivo

[0449] To determine whether the BCMAxCD3 bispecific antibody (Ab) synergizes with PD-1 blockade to provide superior in vivo anti-tumor efficacy, syngeneic tumor studies were performed in mice expressing human CD3. The results showed that the combination of REGN5458 and PD-1 blockade provided superior anti-tumor efficacy compared with REGN5458 or PD-1 blockade alone.

[0450] Implantation and measurement of syngeneic tumors: C57BL / 6 mice expressing human CD3deg instead of murine CD3deg (CD3 humanized mice) were subcutaneously implanted with 1x10 6 MC38 colon cancer cells. Tumors were allowed to establish for 3 days, at which time mice (n = 6 or 7 per group) were administered a dose of 0.4 mg / kg of a CD3 binding control bispecific Ab (G; H4sH17664D) or a dose of 0.04 mg / kg or 0.24 mg / kg of a BCMAxCD3 (G; REGN5458) bispecific Ab, together with 4 mg / kg of an alternative anti-mouse PD-1 antibody (clone RPM1-14) or 4 mg / kg of an isotype control Ab (clone 2A3). Specific treatment groups are shown in Table 31 below.

[0451] Table 31: Treatment Groups

[0452]

[0453]

[0454] The mice were administered these antibodies two more times, on days 7 and 11, for a total of three doses, and tumor growth was assessed throughout the experiment.

[0455] Calculation of Syngeneic Tumor Growth and Inhibition: To determine tumor volume by external calipers, the maximum longitudinal diameter (length, in mm) and the maximum transverse diameter (width, in mm) were measured. Tumor volume based on caliper measurements was calculated by the following formula: Volume (mm 3 )=(length×width 2 ) / 2.

[0456] The results showed that the combination of REGN5458 and PD-1 blockade provided superior anti-tumor efficacy compared to REGN5458 or PD-1 blockade alone. In particular, the results showed that on day 24 (the last day for collecting data for all treatment groups), the combination of BCMAxCD3 bispecific antibody and anti-PD-1 antibody produced a statistically significant synergistic therapeutic effect in inhibiting tumor growth (Table 32, 0.04 mg / kg of BCMAxCD3 and 4 mg / kg of anti-PD-1). Using 2-way ANOVA test on day 24, p<0.0001 between (i) REGN5458 (0.04 mg / kg) + isotype and REGN5458 (0.04 mg / kg) + anti-PD-1 antibody combination (Group 3 vs. Group 4), (ii) REGN5458 (0.24 mg / kg) + isotype and REGN5458 (0.24 mg / kg) + anti-PD-1 antibody combination (Group 5 vs. Group 6), (iii) anti-PD-1 and REGN5458 (0.04 mg / kg) + anti-PD-1 antibody combination (Group 2 vs. Group 6). Using 2-way ANOVA test on day 24, p=0.0005 between anti-PD-1 and REGN5458 (0.04 mg / kg) + anti-PD-1 antibody combination (Group 2 vs. Group 4). In this experiment, increasing the dose of BCMAxCD3 bispecific antibody (0.24 mg / kg) combined with PD-1 blockade resulted in tumor inhibition comparable to lower doses of bispecific antibody plus PD-1 blockade. The demonstrated synergy with lower doses of bispecific antibody is advantageous because the use of lower doses reduces the risk of any adverse side effects. Similarly, at the end of the experiment (day 28), the combination of BCMAxCD3 bispecific antibody and anti-PD-1 antibody showed a synergistic therapeutic effect on the number of tumor-free mice at both doses of bispecific antibody (0.04 mg / kg and 0.24 mg / kg), as shown in Table 33.

[0457] Table 32: Average tumor volume at different time points

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470] Table 33: Tumor-free mice at the end of the experiment

[0471]

[0472] Example 21: Anti-BCMA x anti-CD3 bispecific antibody synergizes with anti-PD-1 antibody to enhance anti-tumor efficacy in vivo

[0473] Similar results were obtained in the second experiment, identical to those discussed above in Example 20, except the number of mice per group = 10, and the higher dose of BCMAxCD3 REGN5458 was 0.4 mg / kg. The specific treatment groups for the second experiment are shown in Table 34 below.

[0474] Table 34: Treatment Groups

[0475]

[0476]

[0477] The results showed that the combination of REGN5458 and PD-1 blockade provided superior anti-tumor efficacy compared to REGN5458 or PD-1 blockade alone. In particular, the results showed that on day 21 (the last day for collecting data for all treatment groups), the combination of BCMAxCD3 bispecific antibody and anti-PD-1 antibody produced a synergistic therapeutic effect in inhibiting tumor growth (Table 35, 0.04 mg / kg of BCMAxCD3 and 4 mg / kg of anti-PD-1). Using a 2-way ANOVA test, p<0.0001 between (i) REGN5458 (0.04 mg / kg) + isotype and REGN5458 (0.04 mg / kg) + anti-PD-1 antibody combination (Group 3 vs. Group 4), (ii) anti-PD-1 and REGN5458 (0.04 mg / kg) + anti-PD-1 antibody combination (Group 2 vs. Group 4), (iii) anti-PD-1 and REGN5458 (0.4 mg / kg) + anti-PD-1 antibody combination (Group 2 vs. Group 6) on Day 21. As discussed above in Example 20, in this experiment, increasing doses of BCMAxCD3 bispecific antibody (0.4 mg / kg) combined with PD-1 blockade resulted in tumor inhibition comparable to lower bispecific antibody doses combined with PD-1 blockade. The demonstrated synergy with lower doses of the bispecific antibody is advantageous because the use of lower doses reduces the risk of any adverse side effects. Similarly, at the end of the experiment (day 25), the combination of the BCMAxCD3 bispecific antibody and the anti-PD-1 antibody showed a synergistic therapeutic effect on the number of tumor-free mice at both doses of the bispecific antibody (0.04 mg / kg and 0.4 mg / kg), as shown in Table 36.

[0478] Table 35: Average tumor volume at different time points

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486] Table 36: Tumor-free mice at the end of the experiment

[0487]

[0488] Example 22: Method for treating multiple myeloma using anti-BCMA x anti-CD3 bispecific antibodies

[0489] A Phase 1 / 2 study evaluating the safety, tolerability, preliminary anti-tumor activity, and pharmacokinetics (PK) of REGN5458 (anti-BCMA x anti-CD3 bispecific antibody) in patients with relapsed or refractory multiple myeloma who have exhausted all treatment options, including proteasome inhibitors, immunomodulatory drugs, and anti-CD38 antibody therapy, is ongoing and has shown meaningful clinical benefit.

[0490] As part of the REGN5458 program, patients with advanced multiple myeloma who are difficult to treat are being studied, including those whose cancerous plasma cells are extramedullary (outside the bone marrow) and non-secretory (do not secrete detectable biomarkers).

[0491] In multiple myeloma clinical trials, treatment evaluation is based on a reduction in myeloma protein levels and eradication of myeloma cells. Myeloma protein response evaluation is based on a reduction in the level of monoclonal (M) protein, a biomarker found in the patient's urine and blood that is used to determine the extent of myeloma disease. A partial response (PR) is defined as a ≥50% reduction in serum / urine M protein or a ≥50% reduction in the difference between involved and uninvolved free light chain (FLC) levels, and a ≥50% reduction in soft tissue plasmacytomas. A very good partial response (VGPR) is defined as a ≥90% reduction in serum / urine M protein or a ≥90% reduction in the difference between involved and uninvolved FLC levels, a ≥90% reduction in soft tissue plasmacytomas, and detection of M protein by immunofixation but not by electrophoresis. A complete response (CR) is defined as negative M protein by immunofixation in serum and urine, disappearance of any soft tissue plasmacytomas, and <5% plasma cells in the bone marrow aspirate. A stringent complete response was defined as a complete response (as described above) plus a normal FLC ratio (a K / λ ratio ≤4:1 or ≥1:2 for patients with K and λ). Minimal residual disease (MRD), reflecting eradication of myeloma cells, was measured separately from the M protein, with MRD negativity defined as the absence of cancer plasma cells per 100,000 bone marrow cells.

[0492] Objectives: Primary and secondary endpoints will be explored.

[0493] Research Main Objectives yes:

[0494] (1) In the Phase 1 portion of the study: Evaluate safety, tolerability, and dose-limiting toxicities (DLTs) and determine the recommended Phase 2 dose regimen (RP2DR) (defined as the maximum tolerated dose regimen [MTDR] or the biologically effective dose regimen [BEDR]) of REGN5458 as a monotherapy for patients with relapsed or refractory multiple myeloma (MM) who have exhausted all treatment options that are expected to provide meaningful clinical benefit. The determination of the RP2DR will be based on a review of nonclinical and all clinical data, including data related to safety, pharmacokinetics (PK), PK / PD (pharmacokinetic / pharmacodynamic) relationships, and efficacy.

[0495] (2) In the Phase 2 portion of the study: Evaluate the preliminary anti-tumor activity of REGN5458, measured by objective response rate (ORR)

[0496] The study Secondary Objectives Yes (in both Phase 1 and Phase 2 sections):

[0497] (1) Evaluate the preliminary anti-tumor activity of REGN5458 as measured by duration of response (DOR), progression-free survival (PFS), minimal residual disease (MRD) status, and overall survival (OS);

[0498] (2) evaluate the (PK) properties of REGN5458;

[0499] (3) characterize the immunogenicity of REGN5458;

[0500] (4) In the Phase 1 portion only: evaluate the preliminary anti-tumor activity of REGN5458 as measured by ORR; and

[0501] (5) Phase 2 only: Evaluation of the safety and tolerability of REGN5458.

[0502] Study Design: The Phase 1 portion will follow a standard 4+3 dose escalation design with a DLT observation period of 28 days to evaluate the safety of REGN5458 and select the RP2DR (defined as MTDR or BEDR) of REGN5458 as monotherapy.

[0503] Once RP2DR is determined, the Phase 2 portion will begin to further evaluate preliminary anti-tumor activity, safety and tolerability, PK properties, and biomarker responses in patients receiving REGN5458 monotherapy.

[0504] Each patient will receive 16 weekly (QW) infusions of REGN5458 according to the designated dosing schedule, followed by 12 additional doses of REGN5458 every 2 weeks (Q2W).

[0505] Each patient will receive an initial dose of REGN5458 followed by a nominal dose if the initial dose is well tolerated. The initial dose of REGN5458 will be administered as divided (fractionated) infusions at the assigned dose (preferably on 2 consecutive days, but not more than 3 days apart). If the initial dose is well tolerated, the patient will receive a divided infusion at the assigned dose in week 2 (preferably on consecutive days, but not more than 3 days apart) and a higher nominal dose in week 3, after which the nominal dose will be administered as a single infusion.

[0506] The dose escalation scheme increases the nominal dose in each consecutive dose group by approximately 3 times compared to the nominal dose in the previously evaluated dose group. Similarly, it increases the initial dose in week 1 of each consecutive dose group by approximately 3 times. However, during the dose-limiting toxicity (DLT) observation period, if 1 patient in ≥2 patients in the dose group develops a DLT or ≥ Grade 2 adverse event (except for Grade ≥2 AEs that are clearly unrelated to the study drug), but the dose regimen is still determined to be tolerable, the increase in the initial dose and nominal dose will not exceed 2 times (i.e., an increase of 100%) of the respective initial dose and nominal dose of the previously evaluated dose group. For example, if 1 DLT is observed in 6 or 7 patients in DL2 (3 mg initial dose and 10 mg nominal dose), and the dose regimen is determined to be tolerable, in the next dose group (i.e., DL3), the initial dose is 6 mg and the nominal dose is 20 mg, and the subsequent dose escalation of the subsequent dose group will not be more than 2 times greater than the previous dose group.

[0507] Study Duration: The planned study duration for each patient is 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 durable clinical activity and safety (total follow-up period is approximately 60 weeks).

[0508] Study population:

[0509] Phase 1 Part : According to the 4+3 design, up to 7 DLT-evaluable patients can be enrolled in each dose group. If the dose regimen of the designated dose level (DL) is determined to be tolerable and the maximum tolerated dose (MTD) is not exceeded, additional enrollment of up to 3 patients in the respective DL (up to 10 patients in each DL) can be initiated. The actual sample size of these dose-escalation groups will depend on the number of patients observed to have documented DLTs, the number of DLs implemented, and the number of patients who withdraw.

[0510] Phase 2 Part: Approximately 10 to 14 patients will be evaluable for safety and efficacy. The analysis of these patients will be combined with that of 6 to 10 patients treated in the Phase 1 RP2DR, yielding a total of 20 patients treated with RP2DR. This study will enroll patients with MM who have exhausted all treatment options that are expected to provide meaningful clinical benefit through disease relapse, treatment-refractory disease, or intolerance or refusal of treatment. In addition, each patient must have progressed after at least 3 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 shown failure to respond to both an IMiD and a proteasome inhibitor, the patient may be eligible to participate in the study even if fewer than 3 lines of treatment have been previously given. Refractory disease is defined as no response or relapse of MM within 60 days of the last treatment.

[0511] Inclusion Criteria - Patients must meet the following criteria to be eligible for study inclusion:

[0512] 1. Aged 18 or above

[0513] 2. Eastern Cooperative Oncology Group (ECOG) performance status ≤ 1

[0514] Individual cases of patients with ECOG 2 performance status, whose ECOG status is expected to improve with effective treatment, can be discussed with the Medical Monitor for potential enrollment.

[0515] 3. Active MM was confirmed according to the International Myeloma Working Group (IMWG) diagnostic criteria.

[0516] 4. Patients must have symptomatic myeloma at the time of study entry, accompanied by myeloma-related organ damage or tissue dysfunction (such as hypercalcemia, renal insufficiency, osteolytic lesions, or anemia)

[0517] 5. The patient must have myeloma, which can be measured by assessment of the monoclonal component in serum or urine or by serum assay (FLC)

[0518] Measurable disease is defined as 1 or more of the following:

[0519] a. Serum M protein ≥ 1g / dL,

[0520] b. Urine M-protein ≥ 200 mg / 24 hours, and / or

[0521] c. FLC test, involving FLC level ≥10mg / dL, abnormal serum FLC ratio

[0522] - Patients with immunoglobulin A (IgA) myeloma but without measurable M-protein may be enrolled if quantitative IgA levels are elevated and longitudinal follow-up is available

[0523] - Patients with non-secretory MM may be considered for inclusion following discussion with the sponsor including the feasibility of a response assessment plan according to IMWG guidelines.

[0524] 6. Disease progression based on IMWG criteria

[0525] 7. MM patients who have exhausted all treatment options that are expected to provide meaningful clinical benefit, either through disease relapse, treatment-refractory disease, or intolerance or refusal of treatment, including:

[0526] a. Progression on or after at least 3 lines of therapy or intolerance to therapy (including proteasome inhibitors, IMiDs, and anti-CD38 antibodies), or

[0527] b. Progressed on or after anti-CD38 antibody and have disease that is "double refractory" to proteasome inhibitors and IMiDs, or is intolerant to treatment. Anti-CD38 antibody may have been administered alone or in combination with another drug (such as a proteasome inhibitor). Refractory disease is defined as no response or relapse within 60 days of the last treatment.

[0528] 8. Adequate hematologic function as measured by:

[0529] a. Platelet count>50x10 9 / L. To meet this platelet eligibility requirement, the patient may not have received a platelet transfusion within 7 days.

[0530] b.ANC>1.0x10 9 / L. Patients may not have received granulocyte colony stimulating factor (G-CSF) within 2 days to meet this absolute neutrophil count eligibility requirement.

[0531] c. Hemoglobin > 8.0 g / dL

[0532] 9. Adequate liver function, defined as:

[0533] a.Total bilirubin ≤1.5 x ULN

[0534] b. Transaminase (ALT, AST) ≤ 2.5 x ULN

[0535] c. Alkaline phosphatase ≤ 2.5 x ULN

[0536] - Patients with Gilbert's syndrome do not need to meet this total bilirubin requirement as long as the total bilirubin does not change from baseline values.

[0537] 10. Cockcroft-Gault serum creatinine clearance > 30 mL / min

[0538] - Patients with creatinine clearance passing Cockcroft-Gault but not meeting eligibility criteria may be considered for enrollment if the measured creatinine clearance (based on a 24-hour urine collection or other reliable method) is >30 mL / min.

[0539] 11. If previously treated with CAR T therapy or any gene therapy product, the patient must have recovered from the toxicity of that therapy

[0540] 12. Life expectancy is at least 6 months

[0541] 13. Willing and able to comply with clinic visits and study-related procedures, including serial bone marrow assessments according to the protocol schedule

[0542] - Must provide bone marrow aspirate and biopsy or other tissue infiltrated with malignant plasma cells at screening to assess BCMA levels in malignant cells, but BCMA levels do not need to be shown prior to enrollment.

[0543] 14. Provide informed consent signed by the research patient

[0544] 15. Ability to understand and complete research-related questionnaires.

[0545] Exclusion criteria: - Patients who meet any of the following criteria will be excluded from this study:

[0546] 1. Presence of plasma cell leukemia, Macroglobulinemia (lymphoplasmacytic lymphoma) or POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes)

[0547] 2. Patients with known MM brain lesions or MM meningeal involvement (suspected central nervous system (CNS) myeloma should be excluded by radiographic imaging and / or lumbar puncture as appropriate)

[0548] 3. History of neurodegenerative conditions or CNS movement disorders

[0549] 4. Cardiac ejection fraction <40% as determined by echocardiography or multi-depth acquisition scan (MUGA).

[0550] 5. Ongoing systemic steroid treatment with more than 10 mg of prednisone or anti-inflammatory equivalent per day within 72 hours of study drug initiation.

[0551] 6. Vaccination with a replication-competent vector within 28 days prior to the first study drug administration

[0552] 7. Treatment with any systemic standard or investigational anti-myeloma therapy within 5 half-lives or 28 days, whichever is shorter, prior to the first dose of study drug

[0553] 8. Prior treatment with any anti-BCMA antibody (including antibody-drug conjugates or bispecific antibodies) or BCMA-directed CAR T therapy

[0554] 9. Any infection requiring hospitalization or treatment with IV anti-infective drugs within 2 weeks of the first administration of study drug

[0555] 10. Uncontrolled infection with human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV); or other uncontrolled infection

[0556] Patients with controlled HIV infection (undetectable viral load and CD4 counts exceeding 350 cells / μL either spontaneously or on a stable antiviral regimen) are allowed.

[0557] b. Patients with controlled hepatitis B infection (positive hepatitis B surface antigen [HepBsAg+]) (serum HBV DNA polymerase chain reaction [PCR] below the detection limit and receiving hepatitis B antiviral treatment) are allowed.

[0558] c. Patients who are HCV antibody positive (HCV Ab+) and whose infection is controlled (undetectable HCV RNA by PCR, either spontaneously or in response to previous successful anti-HCV treatment) are allowed.

[0559] 11. Documented history of severe allergic reaction or acute hypersensitivity reaction caused by previous antibody treatment

[0560] - For this purpose, severe allergic reactions were defined as those meeting CTCAE v5.0 Grade 3 or 4 severity criteria (i.e., characterized by bronchospasm; or life-threatening consequences; or requiring intravenous intervention, other urgent intervention, or hospitalization for clinical sequelae) or requiring emergency room visit.

[0561] 12. History of allergy to any compound in the tetracycline antibiotic group (precautionary measures are taken due to the possible presence of trace components in the study drug materials)

[0562] 13. Known to be active against allopurinol and rasburicase Both Hypersensitivity

[0563] 14. History of allogeneic stem cell transplantation at any time, or history of autologous stem cell transplantation within 12 weeks after the start of study treatment

[0564] 15. Members of the clinical site research team or his / her immediate family, unless approved in advance by the Sponsor

[0565] 16. Women of childbearing age (WOCBP) with a positive serum β-human chorionic gonadotropin (β-hCG) pregnancy test are not eligible for this study.

[0566] 17. Patients who have been institutionalized pursuant to an order issued by a judicial or administrative authority

[0567] 18. Pregnant or breastfeeding women

[0568] 19. Women of childbearing potential*, and males** who are unwilling to use highly effective contraception before the initial dose / start of the first treatment, during the study, and for at least 6 months after the last dose.

[0569] *Highly effective contraceptive measures for women include:

[0570] a. Stable use of combined (estrogen and progestin) hormonal contraception (oral, vaginal, transdermal) or progestin-only hormonal contraception (oral, injectable, implantable) and suppression of ovulation for 2 or more menstrual cycles prior to screening

[0571] b. Intrauterine device (IUD); intrauterine hormone-releasing system (IUS)

[0572] c. Bilateral tubal ligation

[0573] d. Vasectomy partner (provided that the male vasectomy partner is the study participant’s only sexual partner and the partner has been medically evaluated as having had a successful surgery)

[0574] e. and / or sexual abstinence

[0575] Women of childbearing potential are defined as women who remain fertile from menarche until postmenopause, unless sterility is permanent. Permanent sterilization methods include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy.

[0576] Postmenopausal status is defined as the absence of menstruation for 12 months without other medical reasons. High follicle-stimulating hormone (FSH) levels in the postmenopausal range can be used to confirm postmenopausal status in women who are not using hormonal contraception or hormone replacement therapy.

[0577] However, a single FSH measurement is not sufficient to determine the onset of the postmenopausal state in the absence of 12 months of amenorrhea.

[0578] **Highly effective contraceptive measures for men include condoms or sexual abstinence, unless vasectomy has been performed (which has been medically evaluated as successful)

[0579] Sexual abstinence is considered a highly effective method only if it is defined as abstention from heterosexual intercourse during the entire risk period associated with study treatment. The reliability of sexual abstinence needs to be assessed with respect to the duration of the clinical trial and the patient's preferences and usual lifestyle.

[0580] Periodic abstinence (calendar, ovulatory, postovulatory methods), withdrawal (coitus interruptus), spermicides alone, and the lactational amenorrhea method (LAM) are unacceptable methods of contraception. Female and male condoms should not be used together.

[0581] Treatment: REGN5458 for IV infusion will be provided by the sponsor in liquid form in sterile, single-use vials. Each vial will contain REGN5458 at a concentration of 10 mg / mL.

[0582] A pharmacist or other qualified individual will be identified at each site to prepare REGN5458 for administration.

[0583] For the initial dose and the first nominal dose, treatment will be administered as 2 separate 4-hour infusions, preferably for 2 consecutive days, but no more than 3 days apart (e.g., Day 1 of Week 1 and Day 2 of Week 1). The first nominal dose administered as a single infusion will be administered within 4 hours. If this infusion is well tolerated without any level of CRS or IRR events, then the subsequent REGN5458 infusion can be reduced to 2 hours based on the clinical judgment of the investigator. If this 2-hour REGN5458 infusion is well tolerated without any level of CRS or IRR events, then the subsequent REGN5458 infusion can be reduced to 1 hour based on the clinical judgment of the investigator. Thereafter, each dose of REGN5458 can be administered within the IV infusion duration associated with the absence of CRS or IRR events.

[0584] Treatment after the patient receives the initial dose and the first nominal dose may be administered as a single infusion. The investigator may choose to divide the dose into 2 separate infusions over 2 days (preferably consecutively, but not more than 3 days apart).

[0585] The dose of REGN5458 each patient receives will be assigned based on the DL group. The dose administered at each DL will be a fixed dose and will not depend on patient weight or body surface area (BSA).

[0586] Study endpoints:

[0587] In the first stage The primary endpoints of the study were:

[0588] (1) The incidence of DLTs from the first dose to the end of the DLT observation period; and

[0589] (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.

[0590] In the Phase 2 section The primary endpoint of the study was ORR measured using the International Myeloma Working Group (IMWG) criteria up to 14 months after the last dose.

[0591] Secondary End Points Yes (in both Phase 1 and Phase 2 sections):

[0592] (1) Changes in serum REGN5458 concentration over time;

[0593] (2) the incidence of anti-drug antibodies (ADA) in response to REGN5458 treatment over time;

[0594] (3) DOR using IMWG criteria up to 14 months after the last dose;

[0595] (4) PFS measured using IMWG criteria up to 14 months after the last dose;

[0596] (5) the rate of MRD-negative status using IMWG criteria up to 14 months after the last dose;

[0597] (6) OS up to 14 months after the last dose

[0598] (7) In the Phase 1 portion only - ORR measured using IMWG criteria, up to 14 months after the last dose; and

[0599] (8) In Phase 2 only - the incidence and severity of TEAEs and AESIs during REGN5458 treatment and up to 14 months after the last dose.

[0600] Procedure and Assessment:

[0601] Screening only: Demographics, complete physical examination, height, medical and oncological history, revised International Staging System (ISS) staging (including chromosomal abnormalities and β2-microglobulin), brain magnetic resonance imaging (MRI), echocardiography or multi-gated acquisition scan (MUGA), HIV / HBV / HCV testing, prothrombin time (PT) (international normalized ratio [INR]), and aPTT / PTT.

[0602] Safety Sex: vital signs, limited physical examination, weight, electrocardiogram, ECOG status, laboratory evaluations, adverse events (AEs), concomitant medications (CMs).

[0603] effect : 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, quantitative immunoglobulins (immunoglobulin A [IgA], immunoglobulin M [IgM], immunoglobulin G [IgG], immunoglobulin D [IgD], immunoglobulin E [IgE]), evaluation for extramedullary plasmacytoma (measured by clinical examination and / or radiographic examination if relevant [biopsy is optional]), and skeletal evaluation.

[0604] Blood samples will be collected for analysis of drug concentrations and assessment of ADA in serum.

[0605] Statistics Program:

[0606] Phase 1 Part : According to the 4+3 design, up to 7 DLT-evaluable patients can be recruited in each dose group. If it is determined that the dose regimen in the specified DL is tolerable and does not exceed the MTD, additional recruitment of up to 3 patients in the corresponding DL can be initiated (a total of up to 10 patients in each DL). The actual sample size of these dose-escalation groups will depend on the number of patients with DLT observed, the number of DLs implemented, and the number of patients who withdrew.

[0607] Phase 2 Part : The sample size of 20 patients was determined based on clinical considerations to further explore the safety and preliminary anti-tumor activity of REGN5458 in patients treated at RP2DR. In Phase 1, 6 to 10 patients will receive study drug at RP2DR, and these will contribute to the total sample size of 20 patients for analysis. The remaining 10 to 14 patients who are evaluable for safety and efficacy will be included in Phase 2.

[0608] Phase 2 conditions for recruitment suspension and safety review: To further evaluate the tolerability of RP2DR, the cumulative number rate of patients experiencing unacceptable toxicity (cUT) will be estimated from both the Phase 1 and Phase 2 parts in patients treated with RP2DR. Stopping boundaries based on frequentist intervals using the lower limit of the 1-sided 80% confidence interval (CI) will be included. If the lower limit of the 1-sided 80% CI of the estimated cUT does not include 20%, recruitment in the Phase 2 part will be paused.

[0609] This assessment will be performed in the first 12 patients treated at RP2DR in both the Phase 1 and Phase 2 parts; it will be repeated in the first 16 patients treated at RP2DR (i.e., when 4 additional patients are enrolled) in both the Phase 1 and Phase 2 parts. Further enrollment in the Phase 2 part will be paused if the cUT rate excludes 20% (i.e., 4 or more patients in the first 12 patients treated at RP2DR have unacceptable toxicity, or 5 or more patients in the first 16 patients have unacceptable toxicity). Further enrollment in the Phase 2 part will also be paused if 4 patients with unacceptable toxicity are observed before 12 patients are enrolled, or 5 patients with unacceptable toxicity are observed before 16 patients are enrolled.

[0610] Preliminary results: Forty-nine patients (median age, 64 years; 31% 70 years or older) had received a median of five prior systemic therapies (range, 2-17; 33 patients (67.3%) had received prior autologous stem cell transplantation. 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 resistant to anti-CD38 antibodies, with 30.6% being at least triple-resistant, 30.6% being quadruple-resistant, and 57.1% being pentaresistant, respectively. 80% of patients were resistant to carfilzomib and 92% were resistant to pomalidomide. Patients received 3 mg to 96 mg of levofloxacin. REGN5458 was treated in groups over six dose levels. The median follow-up duration was 2.63 (range 0.5-13.4) months. The majority (63%) had a modified International Staging System (ISS) stage of II.

[0611] The most common adverse events (AEs) were cytokine release syndrome (CRS; 38.8%), anemia (36.7%), fatigue (34.7%), nausea (31%), pyrexia (31%), and back pain (27%). Grade 3 or higher AEs occurred in 43% of patients, with the most common being anemia (22.4%), neutropenia (14.3%), and lymphopenia (12.2%). The most common serious adverse events were due to infections (20.4%) and CRS (12.2%); no patient experienced grade ≥ 3 CRS, and less than 40% of patients experienced CRS. CRS occurred primarily in the first week of treatment, with 33% of patients having grade 1 and 6% having grade 2. No correlation was observed between CRS and dose level. No patient experienced grade ≥ 3 neurotoxicity.

[0612] The objective response rate (ORR) across all dose levels was 38.8% (29.2% at dose levels 1 to 3, 41.2% at dose levels 4 and 5, and 62.5% at dose level 6), with 95% of responders achieving at least a very good partial response (VGPR); 42.1% had a complete response (CR) or a stringent CR. Four of seven evaluable patients (57%) achieved minimal residual disease (MRD)-negative status, with a sensitivity of 10 -5 . Tumor responses were not affected by BCMA expression in core biopsies, as assessed by immunohistochemistry. A total of 63.2% of responders had a duration of response (DOR) ≥ 4 months, 52.6% of responders had a DOR ≥ 6 months, and 36.8% of responders had a DOR ≥ 8 months. The observed median duration of response was 6.01 months. Responses occurred early (most by week 4) and deepened over time. Among responding patients with ≥ 6 months follow-up, 83% (10 / 12) showed sustained responses 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%, compared with 45% for patients without EMP, including 20% ​​complete response or strict complete response, and 22.5% very good partial response. The ORR for patients with bone marrow plasmacytosis < 50% was 71.4%, compared with 9.1% for patients with bone marrow plasmacytosis ≥ 50%. Among patients with bone marrow plasmacytosis <50%, 35.7% achieved a stringent complete response and 35.7% achieved a very good partial response. Significant improvements in global health status / quality of life were observed at week 4 and have persisted through week 24 (to date). Tumor responses were not associated with BCMA expression as assessed by immunohistochemistry. A summary of the observed responses is shown in Table 37 below.

[0613] Table 37: Efficacy Results by Dose Level

[0614]

[0615] Thus, it is believed that the bispecific antibodies described herein can be used to treat human subjects with BCMA-expressing cancers (e.g., multiple myeloma), especially those who are refractory to previous therapy (e.g., triple-refractory, quadruple-refractory, or penta-refractory) or who have relapsed after previous treatment, by administering a dose of at least 3 mg weekly, in divided doses (e.g., during the first and second weeks) or as a single infusion (e.g., in week 3 and thereafter).

[0616] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. Use of a bispecific antibody in the preparation of a medicament for treating refractory multiple myeloma that is triple refractory to anti-CD38 antibodies, proteasome inhibitors, and immunomodulatory drugs in a subject, wherein the bispecific antibody comprises a first heavy chain consisting of the amino acid sequence of SEQ ID NO: 126, a second heavy chain consisting of the amino acid sequence of SEQ ID NO: 127, and a common light chain consisting of the amino acid sequence of SEQ ID NO:

129.

2. The use according to claim 1, 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.

3. The use according to claim 1, wherein the subject is quadruple or quintuple refractory to previous treatment.

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