Bispecific anti-BCMAx anti-CD3 antibodies and uses thereof

Abstract

B cell maturation antigen (BCMA) is expressed on malignant plasma cells. The present invention provides novel bispecific antibodies (bsAbs) that bind both BCMA and CD3 and activate T cells via CD3 complexes 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. The bispecific antigen binding molecules of the present invention are suitable for treating diseases and disorders in which an upregulated or induced immune response targeting BCMA is desired and / or therapeutically beneficial. For example, the bispecific antibodies of the present invention are suitable for treating various cancers, including multiple myeloma.

Description

[0001] Reference sequence list

[0002] This application incorporates by reference a sequence listing filed in computer readable form as File 10452WO01-Sequence, created on July 8, 2019 and containing 63,211 bytes. Technical Field

[0003] The present invention relates to antibodies and antigen-binding fragments thereof specific for B cell maturation antigen (BCMA) and methods of using the same. The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies) that bind to 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 an extracellular domain rich in cysteine. BCMA, together with closely related proteins, promotes B cell survival at different stages of development. BCMA is expressed only in cells of the B cell lineage, especially in the interfollicular regions of germinal centers and on plasmablasts and differentiated plasma cells. BCMA is selectively induced during plasma cell differentiation and is required for optimal survival of long-term surviving plasma cells in the bone marrow. In multiple myeloma, BCMA is widely expressed at higher 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 Waldenstrom's macroglobulinemia ( macroglobulinemia, Burkitt lymphoma, and diffuse large B-cell lymphoma. Tai et al., Immunotherapy, 7(11):1187-1199, 2015.

[0005] CD3 is a homodimeric or heterodimeric antigen expressed on T cells associated with the T cell receptor complex (TCR) and required for T cell activation. Functional CD3 is formed by a dimeric combination of two of four different chains: ε, ζ, δ and γ. The CD3 dimerization 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 engagement of TCR by peptide-loaded MHC molecules. 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] Antigen binding molecules targeting BCMA (including bispecific antigen binding molecules that bind BCMA and CD3) will be useful in therapeutic settings where specific targeting and T cell-mediated killing of cells expressing BCMA is desired. Summary of the invention

[0007] In one aspect, the 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 a target tumor cell, as 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 to human CD3, as measured by an in vitro FACS binding assay EC 50 Less than about 10 -6 M.

[0008] In some cases, the bispecific antigen binding molecules activate T cells in vitro, EC 50 Less than about 10 -9 M. In some cases, the bispecific antigen binding molecules mediate T cell killing of tumor cell lines expressing BCMA in vitro, EC 50 Less than about 10 -9 M. In some cases, the bispecific antigen binding molecules mediate autologous T cell killing of primary myeloma cells expressing BCMA in vitro, 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 another B cell disorder, and the B cell disorder is characterized in part by having a B cell expressing BCMA. In some cases, the bispecific antigen binding molecule suppresses the proliferation of tumor cells expressing BCMA with a dosage of about 0.04mg / kg to about 4.0mg / kg. In some cases, the dosage is 0.04mg / kg, 0.4mg / kg or 4mg / kg. In certain embodiments, the dosage is given to patients in need at least twice a week for at least seven doses. In some cases, the bispecific antigen binding molecule suppresses the proliferation of BCMA+ tumor cells, and the tumor cells are selected from the group consisting of myeloma cells, lymphoma cells and leukemia cells. In some cases, the bispecific antigen binding molecule suppresses the proliferation of BCMA+ tumor cells, and the tumor cells are selected from the group consisting of H929 cells, MOLP-8 cells and OPM cells.

[0010] In some cases, the bispecific antigen binding molecule cross-reacts with cynomolgus 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 (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.

[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 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.

[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: 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.

[0015] In another aspect, the invention provides an isolated bispecific antigen binding molecule comprising: (a) a first antigen binding domain that specifically binds to human BCMA and comprises CDRs 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 CDRs 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 instances, the first antigen binding domain comprises CDRs from 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 first antigen binding domain comprises a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-76-78-80, 4-6-8-84-86-88, 20-22-24-84-86-88, 36-38-40-84-86-88, 52-54-56-84-86-88 and 68-70-72-84-86-88, respectively. In some instances, 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 instances, 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.

[0016] In another aspect, the invention provides an isolated bispecific antigen binding molecule that competes for binding to BCMA or binds to the same epitope on BCMA as a reference antibody, wherein the reference antibody comprises a first antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO:66 / 82 and a second antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO:90 / 82 or SEQ ID NO:98 / 82.

[0017] In another aspect, the invention provides an isolated bispecific antigen binding molecule that competes for binding to human CD3 or binds to the same epitope on human CD3 as a reference antibody, wherein the reference antibody comprises a first antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO:66 / 82 and a second antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO:90 / 82 or SEQ ID NO:98 / 82.

[0018] Any bispecific antigen binding molecule discussed above or herein may 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.

[0019] In another aspect, the present invention provides a pharmaceutical composition comprising a bispecific antigen binding molecule (eg, a bispecific antibody) as discussed above or herein and a pharmaceutically acceptable carrier or diluent.

[0020] 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.

[0021] In another aspect, the present invention provides an expression vector comprising the nucleic acid molecule discussed above.

[0022] In another aspect, the present invention provides a host cell comprising the expression vector discussed above.

[0023] On the other hand, the present invention provides a method for inhibiting the growth of an individual's plasma cell tumor, comprising administering to an individual a pharmaceutical composition as described above or herein through separation of bispecific antigen binding molecules or comprising bispecific antigen binding molecules. In some cases, the plasma cell tumor is multiple myeloma. In some cases, the method further comprises administering a second therapeutic agent or treatment regimen. In certain embodiments, the second therapeutic agent includes an anti-tumor agent (e.g., a chemotherapeutic agent, including melphalan, vincristine (vincristine) (Oncovin), cyclophosphamide (cyclophosphamide) (Cytoxan), etoposide (etoposide) (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), bendamustine (bendamustine) (Treanda) or any other known chemotherapeutic agent effective in treating an individual's plasma cell tumor). In some embodiments, the second therapeutic agent includes a steroid. In some embodiments, the second therapeutic agent includes a targeted therapy, including thalidomide, enalidomide, and bortezomib, which is a therapy approved for the treatment of newly diagnosed patients. Lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab, and daratumumab are examples of second therapeutic agents that are effective in treating 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 may be an immunomodulator. In certain embodiments, the second therapeutic agent may be a proteasome inhibitor, including bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro). In certain embodiments, the second therapeutic agent may be a histone deacetylase inhibitor, such as panobinostat (Farydak). In certain embodiments, the second therapeutic agent may be a monoclonal antibody, an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a checkpoint inhibitor, or a combination thereof.

[0024] On the other hand, the present invention provides a method for treating a patient with multiple myeloma or with another B-cell malignancy expressing BCMA, wherein the method comprises administering to the individual a separated bispecific antigen binding molecule as discussed above or herein or a pharmaceutical composition comprising a bispecific antigen binding molecule. In some cases, the B-cell malignancy expressing BCMA is selected from the group consisting of 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. In some cases, the method further comprises administering a second therapeutic agent. In some embodiments, the second therapeutic agent includes an anti-tumor agent (chemotherapeutic agent), a DNA alkylating agent, an immunomodulator, a proteasome inhibitor, a histone deacetylase inhibitor, radiotherapy, stem cell transplantation, a monoclonal antibody that interacts with an antigen expressed on the surface of a tumor cell, a monoclonal antibody other than those described herein (which can interact with a different antigen on the surface of a plasma cell), a bispecific antibody (which has an arm that binds an antigen on the surface of a tumor cell and another arm that binds an antigen on a T cell), an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a checkpoint inhibitor (e.g., 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 cemiplimab (REGN2810). In certain embodiments, the checkpoint inhibitor may be selected from PD-L1 inhibitors, such as atezolizumab (Tecentriq), avelumab (Bavencio), or Durvalumab (Imfinzi). In certain embodiments, the checkpoint inhibitor may be selected from CTLA-4 inhibitors, such as ipilimumab (Yervoy). Other combinations that may be used in combination with the antibodies of the invention are described above.

[0025] In another aspect, the present invention provides a method for treating a tumor that expresses BCMA, wherein the method comprises administering to an individual a combination of an isolated bispecific antigen binding molecule as discussed above or herein or a pharmaceutical composition comprising the same and an anti-PD-1 antibody or antigen binding fragment thereof. In some cases, the anti-PD-1 antibody or antigen binding fragment is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is cemiplimab (REGN2810). In various embodiments, the combination of an anti-BCMA x anti-CD3 bispecific antigen binding molecule (e.g., a bispecific antibody) and an anti-PD-1 antibody or antigen binding fragment (e.g., an anti-PD-1 antibody) produces a synergistic therapeutic effect in treating tumors that express BCMA.

[0026] In another aspect, the present invention provides a bispecific antigen binding molecule as discussed above or herein or a pharmaceutical composition as discussed above or herein for use in treating a disease or condition associated with the expression of BCMA. 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 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 semimab (REGN2810).

[0027] The present invention further comprises the use of the bispecific antigen binding molecules discussed above or herein for the manufacture of a medicament for treating a disease or condition associated with the expression of BCMA. In some cases, the disease or condition is cancer. In some embodiments, the cancer is multiple myeloma.

[0028] In various embodiments, any features or components of the embodiments discussed above or herein can be combined, and the combination is included in the scope of the present invention. Any specific value discussed above or herein can be combined with another related value discussed above or herein to describe a range with an upper and lower value representing a range, and the range is included in the scope of the present invention.

[0029] Other embodiments will become apparent from a review of the embodiments that follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 and Figure 2 In vivo preventive dose-dependent tumor inhibition of BCMA-expressing NCI-H929 human multiple myeloma tumor cells using anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459, respectively, is demonstrated. NCI-H929 cells express high levels of BCMA.

[0031] Figure 3 and Figure 4 In vivo treatment of established BCMA-expressing NCI-H929 human multiple myeloma tumor cells with anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459, respectively, demonstrates dose-dependent tumor inhibition. NCI-H929 cells express high levels of BCMA.

[0032] Figure 5 and Figure 6 In vivo preventive dose-dependent tumor inhibition of BCMA-expressing MOLP-8 human multiple myeloma tumor cells using anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459, respectively. MOLP-8 cells express moderate levels of BCMA.

[0033] Figure 7 Demonstrates in vivo therapeutic reduction of established tumor burden in BCMA-expressing OPM-2 human multiple myeloma tumor cells by anti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459 relative to controls. OPM-2 cells express low levels of BCMA. DETAILED DESCRIPTION

[0034] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, and thus methods and conditions may vary. It should also be understood that the terminology used herein is only used for the purpose of describing specific embodiments, and is not intended to be limiting, because the scope of the present invention will be limited only by the appended claims.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. As used herein, when used with respect to a particular stated value, the term "about" means that the value may vary within a range of no more than 1% of the stated value. 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.).

[0036] 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.

[0037] definition

[0038] As used herein, the expression "CD3" 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 CD3-γ includes the amino acid sequence as shown in SEQ ID NO: 119. Unless explicitly specified as being from a non-human species, 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. Therefore, unless specified as being from a non-human species, such as "mouse CD3", "monkey CD3", etc., the expression "CD3" means human CD3.

[0039] 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., epsilon, delta, gamma, or zeta) and antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The antibodies and antigen-binding fragments of the present invention may bind to soluble CD3 and / or CD3 expressed on the cell surface. Soluble CD3 includes natural CD3 proteins as well as recombinant CD3 protein variants, such as, for example, monomeric and dimeric CD3 constructs that lack a transmembrane domain or are otherwise unrelated to a cell membrane.

[0040] As used herein, the expression "CD3 expressed on the cell surface" means one or more CD3 proteins expressed on the surface of a cell in vitro or in vivo, such that at least a portion of the CD3 protein is exposed to the extracellular side of the cell membrane and accessible to the antigen-binding portion of the antibody. "CD3 expressed on the cell surface" includes CD3 proteins contained within the context of a functional T cell receptor in the cell membrane. The expression "CD3 expressed on the cell surface" includes CD3 proteins expressed as parts of homodimers or heterodimers (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers) 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) expressed by themselves (without other CD3 chain types) on the cell surface. "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 a CD3 protein expressed on the surface of a cell that does not normally express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

[0041] 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 plasma cells that produce immunoglobulins. 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.

[0042] As used herein, “antibodies that bind to BCMA” or “anti-BCMA antibodies” include antibodies and antigen-binding fragments thereof that specifically recognize BCMA.

[0043] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0044] As used herein, the term "antibody" means any antigen binding molecule or molecular complex that includes at least one complementary determining region (CDR) that specifically binds to a specific antigen (e.g., BCMA or CD3) or interacts with a specific antigen. The term "antibody" includes immunoglobulin molecules including four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules consisting 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, C H 1. CH 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 includes a domain (C L 1). V H and V L The V regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: 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 consistent with the human germline sequence or may be naturally or artificially modified. The amino acid consensus sequence can be defined based on the juxtaposition analysis of two or more CDRs.

[0045] As used herein, the term "antibody" also includes antigen-binding fragments of complete antibody molecules. As used herein, the term "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody and similar terms include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. Antigen-binding fragments of an antibody can be derived from a complete antibody molecule, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optional constant domains. The DNA is known and / or easily purchased or synthesized from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries). DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, one or more variable domains and / or constant domains are configured to be suitable for configuration, or codons are introduced, cysteine ​​residues are generated, amino acids are modified, added or deleted, etc.

[0046] 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) composed 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, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0047] The antigen-binding fragment of an antibody will generally include at least one variable domain. The variable domain may be of any size or amino acid composition and will generally include at least one CDR adjacent to or in frame with one or more framework sequences. L Domain associated V H In the antigen-binding fragment of the domain, V H and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region can be dimeric and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a monomer V H or V L domain.

[0048] In certain embodiments, an 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 an antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-CH 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 through a complete or partial hinge region or connecting region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which produce 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 variable domain and constant domain configuration listed above, wherein the variable domain and constant domain configuration are connected to each other and / or to one or more monomer V H or V L The domains are non-covalently associated (e.g., via a disulfide bond (S)).

[0049] As with complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will generally 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 adapted for use in the context of antigen-binding fragments of the antibodies of the invention using conventional techniques available in the art.

[0050] The antibodies of the present invention may act via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of the present invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcRs) recognize bound antibodies on target cells and thereby cause the lysis of target cells. CDC and ADCC can be measured using assays well known and available in the art. (See, e.g., U.S. Patents Nos. 5,500,362 and 5,821,337, and Clynes et al., (1998) Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.) (USA) 95: 652-656). The constant region of an antibody is crucial in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antibody can be selected based on whether it is desired that the antibody mediate cytotoxicity.

[0051] 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 induced by random or site-specific mutations in vitro or introduced by somatic mutations in vivo), for example, in CDRs and especially CDR3. 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 to human framework sequences.

[0052] In some embodiments, the antibodies of the present invention may be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, formed or separated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (further described below), antibodies separated from recombinant, combinatorial human antibody libraries (further described below), antibodies separated from transgenic animals (e.g., mice) of human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucleic Acids Res. 20: 6287-6295); or antibodies prepared, expressed, formed or separated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. The recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, the recombinant human antibodies are induced by in vitro mutations (or in vivo somatic mutations when animal transgenics of human Ig sequences are used), and thus the V H and V L The amino acid sequence of the region is derived from and is consistent with the human germline V H and V L Sequences that are related but may not naturally occur within the human antibody germline repertoire in vivo.

[0053] 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 to 160 kDa, in which the dimer is held together by interchain heavy chain disulfide bonds. In the second form, the dimer is not connected via interchain disulfide bonds and forms a molecule of about 75 to 80 kDa consisting of covalently coupled light and heavy chains (half antibodies). These forms have been extremely difficult to separate, even after affinity purification.

[0054] The frequency of the second form in various intact IgG isotypes is due to, 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 levels typically observed with the human IgG1 hinge. The present invention encompasses substitutions in the hinge, C H 2 or C H Antibodies with one or more mutations in region 3, which may be desirable (eg, in production) to increase yield of the desired antibody form.

[0055] The antibodies of the present invention may be separated antibodies. As used herein, "separated antibodies" means antibodies that have 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, for the purposes of the present invention is a "separated antibody". Separated antibodies also include antibodies in situ within recombinant cells. Separated antibodies are antibodies that have been subjected to at least one purification or separation step. According to certain embodiments, separated antibodies may be substantially free of other cellular materials and / or chemicals.

[0056] The present invention also includes a one-armed antibody that binds to BCMA. As used herein, "one-armed antibody" means an antigen binding molecule comprising a single antibody heavy chain and a single antibody light chain. The one-armed antibody of the present invention may include any HCVR / LCVR or CDR amino acid sequence as shown in Table 1.

[0057] 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 compared to the corresponding germline sequences from which the antibodies were derived. The mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences purchased from, for example, a public antibody sequence database. 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 the corresponding residues of the germline sequence from which the antibody was derived or to the corresponding residues of another human germline sequence or to the conservative amino acid substitutions of the corresponding germline residues (the sequence changes are collectively referred to herein as "germline mutations"). A person of ordinary skill in the art can easily generate a number of antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V LIn some embodiments, all framework and / or CDR residues in the domain are all mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only the mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated into corresponding residues of different germline sequences (i.e., germline sequences different from the germline sequences from which the antibody was 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, such as where certain individual residues are mutated into corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence remain unchanged or mutate into corresponding residues of different germline sequences. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present invention.

[0058] 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, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any HCVR, LCVR and / or CDR amino acid sequences 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.

[0059] 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 may have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and may have different biological effects. Epitopes may be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may comprise a carbohydrate, phosphoryl, or sulfonyl moiety on the antigen.

[0060] The term "substantially identical" or "substantially identical" when referring to a nucleic acid or a fragment thereof indicates that when optimally aligned with appropriate nucleotide insertions or deletions of another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm for sequence identity (such as FASTA, BLAST or Gap) as discussed below. In some cases, a nucleic acid molecule that is substantially identical 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.

[0061] As applied to polypeptides, the term "substantially similar" or "substantially similar" means that two peptide sequences have at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity, when optimally aligned using default gap weights, such as by the programs GAP or BESTFIT. Preferably, the difference in the inconsistent residue positions is a conservative amino acid substitution. "Conservative amino acid substitution" is a substitution in which one amino acid residue is substituted by another amino acid residue with a side chain (R group) having similar 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 percentage of sequence identity or similarity can be adjusted upward to correct for the conservative nature of the substitution. The manner in which this adjustment is performed is 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 side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0062] The sequence similarity (also referred to as sequence identity) of polypeptides is usually measured using sequence analysis software. Protein analysis software uses similarity measurements assigned to various substitutions, deletions and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used under default parameters to determine the sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from different species of organisms) or between wild-type proteins and their mutant proteins. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA (a program in GCG version 6.1) using default or recommended parameters. FASTA (such as FASTA2 and FASTA3) provides the comparison and sequence identity percentage of the best overlap region between the query sequence and the search sequence (Pearson (2000), see above). 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. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0063] Germline mutations

[0064] The 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 chain variable domain compared to the corresponding germline sequence from which the antibody was derived.

[0065] The present invention also encompasses 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 regions and / or CDR regions are mutated to the corresponding residues in the germline sequence from which the antibody is derived, or to the corresponding residues in another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"), and have weak or undetectable binding to the CD3 antigen.

[0066] 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 are different from the original germline sequence remain unchanged or mutate to the corresponding residues of a different germline sequence. After being obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be 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 under the guidance of the present invention are encompassed by the present invention.

[0067] The present invention also comprises an antigen binding molecule comprising an antigen binding domain having a HCVR and / or CDR amino acid sequence substantially consistent with any HCVR and / or CDR amino acid sequence disclosed herein, while maintaining or improving the desired weak affinity with the CD3 antigen. When referring to an amino acid sequence, the term "substantially consistent" or "substantially consistent" means that two amino acid sequences have at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity when optimally aligned (such as by the program GAP or BESTFIT using the default gap weights). Preferably, the difference in the inconsistent residue positions is a conservative amino acid substitution. In the case where two or more amino acid sequences differ from each other due to conservative substitutions, the percentage of sequence identity or similarity can be adjusted upward to correct for the conservative nature of the substitution. The manner in which this adjustment is performed is well known to those skilled in the art. See, for example, Pearson (1994) Methods in Molecular Biology 24: 307-331.

[0068] The sequence similarity (also referred to as sequence identity) of polypeptides is usually measured using sequence analysis software. Protein analysis software uses similarity measurements assigned to various substitutions, deletions and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used under default parameters to determine the sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from different species of organisms) or between wild-type proteins and their mutant proteins. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA (a program in GCG version 6.1) using default or recommended parameters. FASTA (such as FASTA2 and FASTA3) provides the comparison and sequence identity percentage of the best overlap region between the query sequence and the search sequence (Pearson (2000), see above). 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 Res. 25:3389-402.

[0069] Binding properties of antibodies

[0070] As used herein, the term "binding" in the context of an antibody, immunoglobulin, antibody binding fragment or Fc-containing protein binding to, for example, a predetermined antigen (such as a cell surface protein or a fragment thereof) generally refers to the mutual interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0071] 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 an analyte (or anti-ligand), the binding affinity typically corresponds to about 10 -7 M or smaller, such as about 10 -8 M or smaller, such as about 10 -9 M or smaller K D Cell-based binding strategies such as fluorescence activated cell sorting (FACS) binding analysis are also routinely used, and FACS data correlate well with other methods such as radioligand competition binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0072] Thus, an antibody or antigen binding protein of the invention binds to a predetermined antigen or cell surface molecule (receptor) with a K corresponding to an affinity at least ten-fold lower than its binding to a non-specific antigen (e.g., BSA, casein). D According to the present invention, this corresponds to a K value equal to or lower than ten times lower than that of the nonspecific antigen. D Antibodies with an affinity of less than or equal to 100 μg / ml can be considered to have no detectable binding, yet such antibodies can be paired with a second antigen-binding arm to generate the bispecific antibodies of the invention.

[0073] The term "K D "(M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction, or the dissociation equilibrium constant for an antibody or antibody binding fragment binding 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. Therefore, the term "higher affinity" or "stronger affinity" relates to a higher ability to form an interaction and therefore 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 a larger K D In some cases, a higher binding affinity of a particular molecule (e.g., an antibody) to its interaction partner molecule (e.g., antigen X) than the binding affinity of the molecule to another interaction partner molecule (e.g., antigen Y) can be expressed as a binding rate, which is expressed by dividing the greater K D value (lower or weaker affinity) divided by the smaller K D The binding affinity of a protein may be determined by (higher or stronger affinity), for example, 5-fold or 10-fold greater binding affinity as the case may be.

[0074] The term “k d ”(sec -1 , or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction or the dissociation rate constant of an antibody or antibody binding fragment. The value is also referred to as k off value.

[0075] The term “k a ”(M -1 ×sec -1 The on-rate constant (or 1 / M) refers to the on-rate constant for a particular antibody-antigen interaction, or the on-rate constant for an antibody or antibody binding fragment.

[0076] The term "K A ”(M -1 k or 1 / M) refers to the association equilibrium constant for a particular antibody-antigen interaction or the association equilibrium constant for an antibody or antibody binding fragment. a Divide by kd The association equilibrium constant was obtained.

[0077] The term "EC50" or "EC 50 ” refers to the half-maximal effective concentration, which comprises the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC 50 The concentration of antibody at which 50% of the maximal effect of the antibody is observed is substantially represented. 50 The EC value is equal to the concentration of the antibody of the invention that produces half-maximal binding to cells expressing CD3 or tumor-associated antigens (e.g., BCMA) as determined by, for example, FACS binding analysis. 50 Reduced or weaker binding was observed at or below half-maximal effective concentration values.

[0078] In one embodiment, decreased binding can be defined as an increased EC capable of binding to half maximal number of target cells. 50 Antibody concentration.

[0079] 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. 50 Enhanced cytotoxic activity (e.g., T cell-mediated tumor cell killing) is observed at or below half-maximal effective concentration values.

[0080] Bispecific Antigen Binding Molecules

[0081] The antibodies of the present invention may be monospecific, bispecific or multispecific. Multispecific antibodies may be specific for different epitopes of a target polypeptide or may contain antigen binding domains that are 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 (e.g., another peptide or protein). For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bispecific or multispecific antibody with a second or other binding specificity.

[0082] The expression "anti-CD3 antibody" or "anti-BCMA antibody" used herein 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 shown in Table 3 herein or an anti-CD3 antibody disclosed in WO 2014 / 047231 or WO 2017 / 053856.

[0083] In certain embodiments, the CD3 binding arm binds human CD3 and induces human T cell activation. In certain embodiments, the CD3 binding arm weakly binds human CD3 and induces human T cell activation. In other embodiments, in the case of a bispecific or multispecific antibody, the CD3 binding arm weakly binds human CD3 and induces cell killing expressing a tumor associated antigen. In other embodiments, the CD3 binding arm weakly binds or associates with human and cynomolgus macaque (monkey) CD3, however, the binding interaction is not detectable by in vitro assays known in the art. The BCMA binding arm may include any HCVR / LCVR or CDR amino acid sequence as shown in Table 1 herein.

[0084] According to certain exemplary embodiments, the present invention comprises a bispecific antigen binding molecule that specifically binds to CD3 and BCMA. Such molecules may be referred to herein as, for example, "anti-BCMA x anti-CD3" or "anti-CD3 / anti-BCMA" or "anti-CD3 xBCMA" or "CD3 x BCMA" bispecific molecules or other similar terms (e.g., anti-BCMA / anti-CD3).

[0085] 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.

[0086] The aforementioned bispecific antigen binding molecules that specifically bind to CD3 and BCMA may include an anti-CD3 antigen binding molecule that binds to CD3 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 .

[0087] 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 other 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.

[0088] 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 that specifically binds to a specific antigen alone or in combination with one or more other 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).

[0089] In certain exemplary embodiments of the present invention, the bispecific antigen binding molecule is a bispecific antibody. Each antigen binding domain of a bispecific antibody includes a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the case of a bispecific antigen binding molecule (e.g., a bispecific antibody) including a first and second antigen binding domain, the CDR of the first antigen binding domain can be specified with a prefix "D1" and the CDR of the second antigen binding domain can be specified with a prefix "D2". Therefore, the CDR of the first antigen binding domain may be referred to herein as D1-HCDR1, D1-HCDR2, and D1-HCDR3; and the CDR of the second antigen binding domain may be referred to herein as D2-HCDR1, D2-HCDR2, and D2-HCDR3.

[0090] In certain exemplary embodiments, the separated bispecific antigen-binding molecule comprises a first antigen-binding domain comprising: (a) three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) contained in 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 in a light chain variable region (LCVR) comprising an amino acid sequence of SEQ ID NO: 82. In some cases, the separated 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 separated 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.

[0091] 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) 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.

[0092] 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.

[0093] In certain exemplary embodiments, the separated bispecific antigen-binding molecule comprises: (a) a first antigen-binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively; and (b) a second antigen-binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 100, 102, 104, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively. In some cases, the isolated bispecific antigen binding molecule comprises: (a) a first antigen binding domain comprising 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.

[0094] In certain exemplary embodiments, the isolated bispecific antigen binding molecule comprises: (a) a first antigen binding domain that specifically binds to human BCMA and comprises CDRs of 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 CDRs 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 instances, the first antigen binding domain comprises CDRs from 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 first antigen binding domain comprises a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-76-78-80, 4-6-8-84-86-88, 20-22-24-84-86-88, 36-38-40-84-86-88, 52-54-56-84-86-88 and 68-70-72-84-86-88, respectively. In some instances, 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 instances, 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.

[0095] In certain exemplary embodiments, the isolated bispecific antigen binding molecule competes for binding to BCMA or binds to the same epitope on BCMA as a reference antibody, wherein the reference antibody comprises a first antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO:66 / 82 and a second antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO:90 / 82 or SEQ ID NO:98 / 82.

[0096] In certain exemplary embodiments, the isolated bispecific antigen binding molecule competes for binding to human CD3 or binds to the same epitope on human CD3 as a reference antibody, wherein the reference antibody comprises a first antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO:66 / 82 and a second antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO:90 / 82 or SEQ ID NO:98 / 82.

[0097] The bispecific antigen binding molecules discussed above or herein may 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.

[0098] The first antigen binding domain and the second antigen binding domain may 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 are each connected to a separate multimerization domain. The association of one multimerization domain with another multimerization domain promotes the association between the two antigen binding domains, thereby forming a bispecific antigen binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide or amino acid that can associate with a second multimerization domain having the same or similar structure or configuration. For example, the multimerization domain may include immunoglobulin C. H A non-limiting example of a multimeric component is an Fc portion of an immunoglobulin (including a C H 2-C H 3 domains), for example, an Fc domain of IgG selected from the isotypes IgG1, IgG2, IgG3 and IgG4, and any allotype of IgG within each isotype group.

[0099] The bispecific antigen binding molecules of the present invention will generally include two multimerization domains, such as two Fc domains that are each independently part of a single antibody heavy chain. The first and second multimerization domains may have the same IgG isotype, such as, for example, IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerization domains may have different IgG isotypes, such as, for example, IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0100] In certain embodiments, the multimerization domain is an Fc fragment or amino acid sequence containing at least one cysteine ​​residue with a length of 1 to about 200 amino acids. In other embodiments, the multimerization domain is a cysteine ​​residue or a short peptide containing cysteine. Other multimerization domains comprise peptides or polypeptides that include or consist of a leucine zipper, a helical loop motif, or a coiled coil motif.

[0101] Any bispecific antibody format or technology can be used to make 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 (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities (e.g., another antibody or antibody fragment having a second antigen-binding specificity) 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, for example, scFv-based or bifunctional antibody bispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadromas, mortise-in-knob, common light chains (e.g., common light chains with mortise-in-knob, etc.), interchange Mabs, interchange Fabs, (SEED) bodies, leucine zippers, Duobodies, IgG1 / IgG2, dual-acting Fab (DAF)-IgG, and Mabs 2 Bispecific formats (for review of the aforementioned formats, see, eg, Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein).

[0102] In the case of the bispecific antigen-binding molecules of the present invention, the multimerization domain (e.g., Fc domain) may include one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to the wild-type, naturally occurring version of the Fc domain. For example, the present invention comprises bispecific antigen-binding molecules comprising one or more modifications in the Fc domain, wherein the modifications produce a modified Fc domain having a binding interaction (e.g., enhanced or reduced) between the modified Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a C H 2 or C H3 region, wherein the modification enhances the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome at a pH ranging from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T) and 256 (e.g., S / R / Q / E / D or T); or position 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or position 250 and / or 428; or position 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0103] The present invention also includes a first C H 3 domains and the second Ig C H A bispecific antigen-binding molecule with three domains, wherein the first and second Ig C H The first Ig C H 3 domain binding protein A and the second Ig C H The 3 domain contains a mutation that reduces or abolishes 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. HFurther modifications within 3 comprise in the case of an IgG1 antibody: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU); in the case of an IgG2 antibody: N44S, K52N and V82I (IMGT; N384S, K392N and V422I by EU); and in the case of an IgG4 antibody: Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU).

[0104] In certain embodiments, the Fc domain may be a chimeric, combined Fc sequence derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may include a human IgG1, human IgG2, or human IgG4C. H Part or all of Zone 2 C H 2 sequences and part or all of the C sequences derived from human IgG1, human IgG2 or human IgG4 H 3 sequences. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may include an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region, combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. Specific examples of chimeric Fc domains that may be included in any antigen-binding molecule listed herein include (from N-terminus to C-terminus): [IgG4C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules listed herein includes (from N-terminus to C-terminus): [IgG1 C H

[00136] In some embodiments, the chimeric Fc domains of the present invention may be included in any antigen-binding molecule of the present invention.

[00137] In some embodiments, the chimeric Fc domains of the present invention may be included in any antigen-binding molecule of the present invention.

[00138] In some embodiments, the chimeric Fc domains of the present invention may be included in any antigen-binding molecule of the present invention. [00139 ...1] In some embodiments, the chimeric Fc domains of the present invention may be included in any antigen-binding molecule of the present invention.

[00133] In some embodiments, the chimeric Fc domains of the present invention may be included in any

[0105] Sequence variants

[0106] Compared to the corresponding germline sequences from which individual antigen binding domains are derived, 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 regions and / or CDR regions of the heavy and light chain variable domains. The mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences purchased 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 sequences disclosed herein, wherein one or more amino acids in one or more framework regions and / or CDR regions mutate into corresponding residues from the germline sequences from which antibodies are derived or mutate into corresponding residues from another human germline sequence or mutate into conservative amino acid substitutions of corresponding germline residues (the sequence changes are collectively referred to herein as "germline mutations"). 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, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V L All framework and / or CDR residues in the domain are mutated back to the residues found in the original germline sequence from which the antigen binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated into corresponding residues of different germline sequences (i.e., germline sequences different from the germline sequences from which the antigen binding domain was originally derived). In addition, the antigen binding domain may contain any combination of two or more germline mutations in the framework and / or CDR regions, such as where certain individual residues are mutated into corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence remain unchanged or mutate into 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 one or more antigen binding domains obtained in this general manner are encompassed by the present invention.

[0107] pH-dependent binding

[0108] The present invention comprises anti-BCMA antibodies and anti-BCMA x anti-CD3 bispecific antigen binding molecules having pH-dependent binding characteristics. For example, compared to neutral pH, the anti-BCMA antibodies of the present invention may exhibit weakened binding to BCMA at acidic pH. Alternatively, compared to 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 below about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or lower. As used herein, the expression "neutral pH" means a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

[0109] In some cases, "reduced binding at acidic pH compared to neutral pH" is based on the K of the antibody binding to its antigen at acidic pH. D The K value of the antibody binding to its antigen at neutral pH D For example, for the purposes of the present invention, if an antibody or antigen-binding fragment thereof exhibits an acidic / neutral K of about 3.0 or greater, D ratio, then the antibody or antigen-binding fragment thereof may be considered to exhibit "reduced binding to BCMA at acidic pH compared to neutral pH". In certain exemplary embodiments, the acidic / neutral K of the 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.

[0110] Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening for a population of antibodies that have reduced (or enhanced) binding to a particular antibody at acidic pH compared to neutral pH. In addition, modifications of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For example, antibodies with reduced antigen binding at acidic pH relative to neutral pH can be obtained by replacing one or more amino acids of the antigen-binding domain (e.g., within the CDR) with histidine residues.

[0111] Antibodies including Fc variants

[0112] 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 (e.g., at an acidic pH value compared to a neutral pH value). For example, the present invention includes a C in the Fc domain H 2 or C H The antibody of the mutation is included in the 3rd region, wherein the mutation increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., an endosome in which the pH is in the range of about 5.5 to about 6.0). The mutation may result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of the Fc modification include, for example, position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T) and 256 (e.g., S / R / Q / E / D or T); or position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or position 250 and / or 428; or position 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0113] 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 aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are encompassed within the scope of the present invention.

[0114] Biological characteristics of antibodies and bispecific antigen-binding molecules

[0115] The present invention comprises high affinity (e.g., or subnanomolar K DValue) Antibodies and antigen-binding fragments thereof that bind to human BCMA.

[0116] According to certain embodiments, the invention comprises antibodies and antigen-binding fragments of antibodies that bind human BCMA (e.g., at 25° C.), wherein the K as measured by surface plasmon resonance (e.g., using an assay format as defined in Example 4 herein) is D In certain embodiments, the antibodies or antigen-binding fragments of the invention bind to BCMA with a K as measured by surface plasmon resonance (e.g., using an assay format as defined in Example 4 herein or a substantially similar assay). D The invention encompasses bispecific antigen binding molecules, for example, that bind to human BCMA (wherein K D is less than about 25 pM) and binds to monkey BCMA (wherein the K as measured by surface plasmon resonance (e.g. using an assay format as defined in Example 4 herein or a substantially similar assay) D is less than about 170 pM) of the bispecific antibody.

[0117] The present invention also comprises antibodies and antigen-binding fragments thereof that bind to BCMA, wherein the dissociation half-life (t1 / 2) is greater than about 10 minutes or greater than about 125 minutes as measured by surface plasmon resonance (e.g., using an assay format as defined in Example 4 herein or a substantially similar assay) at 25°C. In certain embodiments, the antibodies or antigen-binding fragments of the invention bind to BCMA, wherein the t1 / 2 is 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 100 minutes, greater than about 110 minutes, or greater than about 120 minutes as measured by surface plasmon resonance (e.g., using an assay format as defined in Example 4 herein or a substantially similar assay) at 25°C. The present invention comprises bispecific antigen binding molecules, e.g., bispecific antibodies that bind BCMA, wherein the t1 / 2 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) is greater than about 10 minutes.

[0118] The present invention also encompasses 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 set forth in Example 6, or a substantially similar assay.

[0119] The present invention also comprises an anti-BCMA x anti-CD3 bispecific antigen binding molecule that exhibits one or more characteristics selected from the group consisting of: (a) suppressing tumor growth in immunocompromised mice bearing human multiple myeloma xenografts; (b) suppressing tumor growth of established tumors in immunocompromised mice bearing human multiple myeloma xenografts (see, e.g., Examples 10-15); and (c) suppressing tumor growth of allogeneic melanoma and colon carcinoma cells engineered to express human BCMA in immunocompetent mice expressing human CD3.

[0120] The present invention comprises antibodies and antigen-binding fragments thereof that bind to human CD3 with high affinity. The present invention also comprises antibodies and antigen-binding fragments thereof that bind to human CD3 with medium or low affinity (depending on the desired therapeutic situation and specific targeting characteristics). In some cases, low affinity comprises binding with a K of greater than 300 nM, greater than 500 nM, or greater than 1 μM. D or EC 50 Antibodies that bind to CD3 (e.g., as measured in surface plasmon resonance analysis). The present invention also encompasses 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, where one arm binds CD3 and the other arm binds a target antigen (e.g., BCMA), it may be desirable for the target antigen-binding arm 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 / untargeted CD3 binding and the attendant adverse side effects associated therewith.

[0121] The present invention comprises bispecific antigen binding molecules (e.g., bispecific antibodies) that are capable of binding to human CD3 and human BCMA simultaneously. The binding arm that interacts with cells expressing CD3 may have weak to undetectable binding, as measured in a suitable in vitro binding assay. The extent to which bispecific antigen binding molecules bind to cells expressing CD3 and / or BCMA can be assessed by fluorescence activated cell sorting (FACS), as described in Examples 5 and 6 herein.

[0122] For example, the present invention comprises antibodies, antigen-binding fragments thereof, and bispecific antibodies that specifically bind to human T cell lines expressing CD3 but not BCMA (eg, Jurkat) and / or cells expressing BCMA.

[0123] The present invention encompasses antibodies, antigen-binding fragments thereof, and bispecific antibodies that bind to human CD3 with weak (ie, low) or even undetectable affinity.

[0124] The present invention encompasses antibodies, antigen-binding fragments thereof, and bispecific antibodies that bind to monkey (ie, cynomolgus monkey) CD3 with weak (ie, low) or even undetectable affinity.

[0125] The present invention comprises antibodies, antigen-binding fragments thereof, and bispecific antibodies that bind to human CD3 and induce T cell activation.

[0126] The present invention comprises an anti-BCMA x anti-CD3 bispecific antigen binding molecule capable of depleting or reducing tumor antigen-expressing cells in a subject (see, e.g., Examples 8 to 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 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 BCMA-expressing cells in the subject (e.g., suppressing or inhibiting tumor growth in the subject).

[0127] Epitope mapping and related technologies

[0128] The epitope on CD3 and / or BCMA to which the antigen binding molecules of the present invention bind may consist of a single continuous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of CD3 or BCMA proteins. Alternatively, the epitope may consist of multiple non-continuous amino acids (or amino acid sequences) of CD3 or BCMA. The antibodies of the present invention may interact with amino acids contained in a single CD3 chain (e.g., CD3-ε, CD3-δ or CD3-γ) or may interact with amino acids on two or more different CD3 chains. As used herein, the term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule called a paratope. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on an antigen and may have different biological effects. An epitope may be conformational or linear. A conformational epitope is 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 certain instances, an epitope may comprise a carbohydrate, phosphoryl or sulfonyl moiety on an antigen.

[0129] Various techniques known to those of ordinary skill in the art can be used to determine whether the antigen binding domain of an antibody "interacts with one or more amino acids within a polypeptide or protein." Exemplary techniques include, for example, conventional cross-blocking analysis, as described Antibodies , Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY); 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 antigen chemical modification can be used (Tomer 2000 Protein Science 9: 487-496). Another method that can be used to identify amino acids in a polypeptide that interacts with the antigen binding domain of an antibody is hydrogen / deuterium exchange detected by mass spectrometry. In general, hydrogen / deuterium exchange methods involve deuterium labeling of a protein of interest, followed by binding of the antibody to the deuterium-labeled protein. Subsequently, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except residues protected by the antibody (which remain deuterium-labeled). After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing deuterium-labeled residues corresponding to the 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 crystallization of antigen / antibody complexes can also be used for epitope mapping purposes.

[0130] 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 shown 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 shown in Table 1 herein).

[0131] The present invention also encompasses 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.

[0132] 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 specific exemplary BCMA-specific antigen binding domain described herein, and / or wherein the second antigen binding domain competes for binding to CD3 with any specific exemplary CD3-specific antigen binding domain described herein.

[0133] Whether a specific antigen binding molecule (e.g., antibody) or its antigen binding domain binds to the same epitope or competes for binding as a reference antigen binding molecule of the present invention can be easily determined by using conventional methods known in the art. For example, to determine whether a test antibody binds to the same epitope on BCMA (or CD3) as a reference bispecific antigen binding molecule of the present invention, the reference bispecific molecule is first bound to the BCMA protein (or CD3 protein). Subsequently, the ability of the test antibody to bind to the BCMA (or CD3) molecule is evaluated. If the test antibody is able to bind to BCMA (or CD3) after saturation binding with the reference bispecific antigen binding molecule, it can be inferred that the test antibody binds to an epitope of BCMA (or CD3) different from the reference bispecific antigen binding molecule. On the other hand, if the test antibody is not able to bind to the BCMA (or CD3) molecule after saturation binding with the reference bispecific antigen binding molecule, the test antibody may bind to an epitope of BCMA (or CD3) identical to the epitope bound by the reference bispecific antigen binding molecule of the present invention. Other routine experiments (such as peptide mutations and binding analysis) may then be performed to confirm whether the observed insufficient binding of the test antibody is actually due to binding to the same epitope as the reference bispecific antigen binding molecule, or whether steric blocking (or another phenomenon) causes the observed insufficient binding. Such experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding analysis available in the art. According to certain embodiments of the present invention, if, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990: 50: 1495-1502), for example, 1, 5, 10, 20, or 100 times excess of one antigen-binding protein inhibits the binding of another antigen-binding protein by at least 50%, but preferably 75%, 90%, or even 99%, then the two antigen-binding proteins bind to the same (or overlapping) epitope. Alternatively, if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antigen-binding protein reduce or eliminate the binding of another antigen-binding protein, then it is considered that the two antigen-binding proteins bind to the same epitope. Two antigen binding proteins are said to have "overlapping epitopes" 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 binding protein.

[0134] To determine whether an antibody or its antigen binding domain competes with a reference antigen binding molecule for binding, the above binding method is performed in the following two directions: in the first direction, 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 direction, 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 in both directions, only the first (saturated) antigen binding molecule is able to bind to the BCMA (or CD3) molecule, it is inferred that the test antibody competes with the reference antigen binding molecule for binding to BCMA (or CD3). As will be appreciated by those of ordinary skill in the art, an antibody that competes with a reference antigen binding molecule for binding 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.

[0135] Preparation of antigen binding domains and construction of bispecific molecules

[0136] Antigen binding domains with specificity for a particular antigen can be prepared by any antibody production technology known in the art. After obtaining, two different antigen binding domains with specificity for two different antigens (e.g., CD3 and BCMA) can be appropriately arranged relative to each other using conventional methods to produce the bispecific antigen binding molecules of the present invention. (Discussions of exemplary bispecific antibody types that can be used to construct bispecific antigen binding molecules of the present invention are provided elsewhere herein). In certain embodiments, one or more of the individual components (e.g., heavy chains and light chains) of the multispecific antigen binding molecules of the present invention are derived from chimeric, humanized or fully human antibodies. Methods for preparing the antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen binding molecules of the present invention can be prepared using VELOCIMMUNE TM Technical preparation. Using VELOCIMMUNE TM Using the PCR technology (or any other human antibody production technology), high affinity chimeric antibodies for a specific antigen (e.g., CD3 or BCMA) are initially isolated, which have human variable regions and mouse constant regions. Antibodies are characterized and selected for desired characteristics (including affinity, selectivity, epitope, etc.). The mouse constant regions are replaced with the desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen binding molecules of the invention.

[0137] Genetically engineered animals can be used to make 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 of mouse κ constant genes operably connected to endogenous mouse κ locus. The genetically modified mice can be used to produce fully human bispecific antigen binding molecules including two different heavy chains, and the heavy chains are associated with consistent light chains including variable domains derived from one of two different human light chain variable region gene segments. (See, for example, US 2011 / 0195454). Fully human refers to antibodies or their antigen-binding fragments or immunoglobulin domains including amino acid sequences encoded by DNA, and the DNA is derived from human sequences on 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 proteins that are endogenous to humans. In other cases, fully human proteins or protein sequences include chimeric sequences in which 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 in the junction of the component sequences, for example compared to any wild-type human immunoglobulin region or domain.

[0138] Bioequivalent

[0139] The present invention encompasses antigen binding molecules having amino acid sequences that differ from those of the exemplary molecules disclosed herein but retain the ability to bind to CD3 and / or BCMA. The 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 equivalent to that of the described bispecific antigen binding molecules.

[0140] The present invention includes antigen binding molecules, which are bioequivalents of any exemplary antigen binding molecules listed herein. For example, if two antigen binding proteins or antibodies are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in their absorption rate and degree when given with the same molar dose in a single dose or multiple doses under similar experimental conditions, then the two antigen binding proteins or antibodies are considered to be bioequivalent. If some antigen binding proteins are equivalent in their absorption degree rather than their absorption rate, then they are considered to be equivalents or pharmaceutical substitutes, and they can be considered to be bioequivalent again, because the difference in absorption rate is intentional and reflected in the labeling, for reaching effective main drug concentration in terms of, for example, long-term use, and are considered to be medically insignificant for the specific drug studied.

[0141] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in safety, purity, and potency.

[0142] In one embodiment, two antigen binding proteins are bioequivalent if the patient can switch one or more times between the reference product and the biological product without an increased risk of expected side effects, including clinically significant changes in immunogenicity or diminished effectiveness, compared to continued therapy without such switching.

[0143] In one embodiment, two antigen binding proteins are bioequivalent if they both act by a common mechanism or mechanism of action for the conditions of use, to the extent that the mechanism is known.

[0144] 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 in blood, plasma, serum or other biological fluids is measured over time; (b) in vitro tests that correlate with and reasonably predict the in vivo bioavailability data in humans; (c) in vivo tests in humans or other mammals, where the appropriate acute pharmacological effects of the antibody (or its target) are measured over time; and (d) well-controlled clinical trials that establish the safety, efficacy or bioavailability or bioequivalence of the antigen-binding protein.

[0145] Bioequivalent variants of the exemplary bispecific antigen binding molecules listed herein can be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences that are not essential for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or inappropriate intramolecular disulfide bridges after renaturation. In other cases, bioequivalent antigen binding proteins may include variants of the exemplary bispecific antigen binding molecules listed herein, which variants include amino acid changes that modify the glycosylation characteristics of the molecule, such as mutations that eliminate or remove glycosylation.

[0146] Species selectivity and cross-species reactivity

[0147] According to certain embodiments of the present invention, antigen binding molecules are provided that bind to human CD3 but not CD3 from other species. Antigen binding molecules that bind to human BCMA but not 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.

[0148] 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 may or may not bind to one or more of the following, depending on the specific circumstances: 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. 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.

[0149] Therapeutic formulations and administration

[0150] The present invention provides pharmaceutical compositions comprising antigen binding molecules of the present invention. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and similar properties. A wide variety of suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (such as LIPOFECTIN TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol with various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) "J Pharm Sci Technol" 52:238-311.

[0151] The dosage of the antigen binding molecule administered to the patient may vary depending on the patient's age and stature, target disease, condition, route of administration, etc. The preferred dosage is usually calculated based on body weight or body surface area. When the bispecific antigen binding molecule of the present invention is used for the treatment of adult patients, the bispecific antigen binding molecule of the present invention is preferably administered intravenously, usually at a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dose and duration of the administration of the bispecific antigen binding molecule can be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dosage can be adjusted accordingly. In addition, the dosage can be adjusted proportionally between species using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351). 。

[0152] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, such as 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, such as by infusion or rapid injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. It can be administered systemically or topically.

[0153] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are readily applicable to the delivery of the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable sleeve containing the pharmaceutical composition. After all of the pharmaceutical composition in the sleeve has been administered and the sleeve is empty, the empty sleeve can be easily discarded and replaced with a new sleeve containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable sleeve. Specifically, the disposable pen delivery device is pre-filled with a pharmaceutical composition stored in a reservoir within the device. After the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0154] Many reusable pen and automatic injection delivery devices are used for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include but are not limited to AUTOPEN, to name a few. 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, IN); NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark); NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark); BD TM OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM OPTICLIK TM (sanofi-aventis, Frankfurt, Germany). 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-Aventis), 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 Park IL).

[0155] 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 "Critical Reference for Biomedical Engineering (CRC Crit. Ref. Biomed. Eng.)" 14:201). In another embodiment, a polymeric material can be used; see "Medical Applications of Controlled Release", Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida (CRC Pres., Boca Raton, Florida). In another embodiment, the controlled release system can be placed close to the target of the composition, so that only a portion of the systemic dose is required (see, for example, 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 (Science)" 249:1527-1533.

[0156] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections; drip infusions, etc. These injectable preparations can be prepared by well-known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injection. As aqueous media for injection, such as physiological saline, isotonic solutions containing glucose and other adjuvants, etc., they can be used in combination with appropriate solubilizers (such as alcohols (such as ethanol), polyols (such as propylene glycol, polyethylene glycol), nonionic surfactants [such as polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.). As oily media, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with solubilizers (such as benzyl benzoate, benzyl alcohol, etc.). The injection thus prepared is preferably filled in an appropriate ampoule.

[0157] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared into a dosage form in a unit dose suitable for the dosage of the active ingredient. The dosage form in the unit dose includes, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The content of the aforementioned antibody is generally about 5 to about 500 mg / dosage form / unit dose; especially in the injection dosage form, preferably the content of the aforementioned antibody is about 5 to about 100 mg and about 10 to about 250 mg for other dosage forms.

[0158] Therapeutic uses of antigen binding molecules

[0159] The present invention includes methods comprising administering to an individual in need thereof a therapeutic composition comprising an anti-BCMA antibody or antigen-binding fragment thereof or a bispecific antigen-binding molecule that specifically binds CD3 and BCMA. The therapeutic composition may include any antibody or bispecific antigen-binding molecule as disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression "individual in need" means a human or non-human animal (e.g., an individual expressing a tumor or suffering from any cancer mentioned herein below) that exhibits one or more symptoms or signs of cancer or an individual that will additionally benefit from the inhibition or reduction of BCMA activity or the consumption of BCMA+ cells (e.g., multiple myeloma cells).

[0160] The antibodies and bispecific antigen binding molecules of the present invention (and therapeutic compositions comprising the same) are particularly suitable for treating any disease or condition in which stimulation, activation and / or targeting of an immune response would be beneficial. Specifically, 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 mitigate 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 methods of the present invention includes killing cells expressing BCMA in the presence of effector cells (e.g.) 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.

[0161] 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, methods are provided comprising 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. Analytical / diagnostic methods known in the art (such as tumor scanning, etc.) can be used to determine whether a patient carries multiple myeloma or another B cell lineage cancer.

[0162] The present invention also encompasses methods for treating residual cancer in an individual.As used herein, the term "residual cancer" means the presence or persistence of one or more cancer cells in an individual after treatment with an anti-cancer therapy.

[0163] According to certain aspects, the invention provides methods for treating a disease or condition associated with BCMA expression, such as multiple myeloma, comprising administering to an individual one or more anti-BCMA or bispecific antigen binding molecules described elsewhere herein after the individual has been determined to have multiple myeloma. For example, the invention comprises methods for treating multiple myeloma comprising administering to a 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 individual has received other immunotherapy or chemotherapy.

[0164] Combination therapies and formulations

[0165] 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 other therapeutic agents. Exemplary other therapeutic agents that can be combined with the antigen binding molecules of the present invention or administered in combination include, for example, anti-tumor agents (e.g., chemotherapeutic agents, including melphalan, vincristine (Ancopin), cyclophosphamide (Cancer Star), etoposide (VP-16), cranberries (Adlimycin), cranberries liposomes (Doxi), bendamustine (Terida) or any other known chemotherapeutic agents effective in treating plasma cell tumors of individuals). In some embodiments, the second therapeutic agent includes a steroid. In some embodiments, the second therapeutic agent includes targeted therapy, including thalidomide, lenalidomide and bortezomib, which is a therapy approved for the treatment of newly diagnosed patients. Lenalidomide, pomalidomide, bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab and daratumumab are examples of second therapeutic agents for the effective treatment of relapsed myeloma. In certain embodiments, the second therapeutic agent is a scheme including 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, including bortezomib (Vanke), carfilzomib (Keprolis), esazomib (Enleri). In certain embodiments, the second therapeutic agent may be a histone deacetylase inhibitor, such as panobinostat (Fairedak). In certain embodiments, the second therapeutic agent may be a monoclonal antibody, an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a checkpoint inhibitor, or a combination thereof. Other agents that may be advantageously administered in combination with the antigen binding molecules of the present invention include cytokine inhibitors, including small molecule cytokine inhibitors and antibodies binding 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 corresponding receptors. The pharmaceutical compositions of the invention (e.g., pharmaceutical compositions comprising an anti-BCMA x anti-CD3 bispecific antigen binding molecule as disclosed herein) may also be administered as part of a treatment regimen comprising one or more therapeutic combinations selected from monoclonal antibodies other than those described herein (which may interact with different antigens on the surface of plasma cells), bispecific antibodies (which have one arm that binds to an antigen on the surface of a tumor cell and another arm that binds to an antigen on a T cell), antibody drug conjugates, bispecific antibodies conjugated to an anti-tumor agent, checkpoint inhibitors (e.g., checkpoint inhibitors targeting PD-1 or CTLA-4), or combinations thereof. In certain embodiments, the checkpoint inhibitor may be selected from a PD-1 inhibitor, such as pembrolizumab (Crazyd), nivolumab (Opdivo), or simvastatin (REGN2810).In certain embodiments, the checkpoint inhibitor can be selected from PD-L1 inhibitors, such as atezolizumab (Tesenic), avelumab (Bavencia) or durvalumab (Infini). In certain embodiments, the checkpoint inhibitor can be selected from CTLA-4 inhibitors, such as ipilimumab (Ivoy). Other combinations that can be used in combination with the antibodies of the present invention are described above.

[0166] The present invention also comprises a therapeutic combination, which includes any antigen binding molecule mentioned herein and one or more inhibitors of the following: VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, urosoluble protein or any of the aforementioned cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribonuclease, 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 bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, mini antibodies and minimal recognition units). The antigen binding molecules of the present invention can also be administered and / or co-modulated in combination with antiviral agents, antibiotics, analgesics, corticosteroids and / or NSAIDs. 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.

[0167] Other therapeutically active ingredients may be administered immediately prior to, simultaneously with, or shortly after administration of the antigen-binding molecules of the present invention; (for the purposes of the present invention, such dosing regimens are considered administration of the antigen-binding molecules "in combination with other therapeutically active ingredients").

[0168] 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.

[0169] Dosage regimen

[0170] 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) may be administered to an individual 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 an individual. As used herein, "sequential administration" means that each dose of the antigen binding molecules is administered to an individual at different time points, such as on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes a method comprising sequentially administering a single initial dose of an antigen binding molecule to a patient, followed by one or more second doses of the antigen binding molecules, and optionally followed by one or more third doses of the antigen binding molecules.

[0171] The terms "initial dose", "secondary dose" and "tertiary dose" refer to the time sequence of administering the antigen binding molecules of the present invention. Therefore, the "initial dose" is the dose (also referred to as the "baseline dose") given at the beginning of the treatment regimen; the "secondary dose" is the dose given after the initial dose; and the "tertiary dose" is the dose given after the second dose. The initial, second and third doses may all contain the same amount of antigen binding molecules, but generally may differ from each other in terms of the frequency of administration. However, in certain embodiments, the amount of antigen binding molecules contained in the initial, second and / or third doses differs from each other during the treatment time course (e.g., up or down as needed). In certain embodiments, two or more (e.g., 2, 3, 4 or 5) doses are administered as a "loading dose" at the beginning of the treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0172] In an exemplary embodiment of the present invention, each second 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, 181 / 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 As used herein, the phrase "previous dose" means, in a sequence of multiple administrations, the dose of the antigen binding molecule administered to the patient prior to the next dose in the administration sequence, without an intervening dose.

[0173] The method according to this aspect of the invention may include administering any number of second 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.

[0174] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as the 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 to the patient at the same frequency as the 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 and / or third dose to the patient can vary within the treatment regimen time course. The frequency of administration can also be adjusted by the physician according to the needs of the individual patient after clinical examination during the treatment time course.

[0175] Diagnostic Uses of Antibodies

[0176] The anti-BCMA antibodies of the present invention can also be used to detect and / or measure BCMA or cells expressing BCMA in a sample (for example) for diagnostic purposes. For example, anti-BCMA antibodies or fragments thereof can be used to diagnose conditions or diseases characterized by abnormal expression of BCMA (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic analysis of BCMA may 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 used in combination with a secondary antibody that is itself detectably labeled for diagnostic applications. The detectable marker or reporter molecule may be a radioactive isotope, such as 3 H. 14 C. 32 P. 35 S or 125 I; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Another exemplary diagnostic use of the anti-BCMA antibodies of the present invention includes non-invasive identification and tracking of tumor cells in an individual (e.g., positron emission tomography (PET) imaging) for the purpose of 89 Zr labeling (such as 89 (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).

[0177] Samples that can be used in the BCMA diagnostic assay according to the present invention include any tissue or fluid sample that can be obtained from a patient that contains a detectable amount of BCMA protein or fragments thereof under normal or pathological conditions. In general, the BCMA level 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 initially establish a baseline or standard level of BCMA. This BCMA baseline level can then be compared to the BCMA level measured in a sample obtained from an individual suspected of having a BCMA-related disease (e.g., a tumor containing cells expressing BCMA) or condition.

[0178] Examples

[0179] The following examples are presented in order to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to the numbers 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 in degrees Celsius, and pressure is at or near atmospheric pressure.

[0180] Example 1: Generation of anti-BCMA antibodies

[0181] Anti-BCMA antibodies are obtained by immunizing genetically modified mice with human BCMA antigen (e.g., hBCMA, SEQ ID NO: 115) or by immunizing engineered mice comprising DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions with human BCMA antigen.

[0182] After immunization, spleen cells are collected from each mouse and either (1) fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for BCMA specificity or (2) sorted for B cells using human BCMA fragments as sorting agents that bind and identify reactive antibodies (antigen-positive B cells) (as described in US 2007 / 0280945A1).

[0183] Initially isolate chimeric antibodies to BCMA with human variable regions and mouse constant regions. Characterize and select antibodies for desired characteristics (including affinity, selectivity, etc.). If necessary, replace the mouse constant region with the desired human constant region (e.g., wild-type or modified IgG1 or IgG4 constant region) to produce fully human anti-BCMA antibodies. Although the selected constant region can be changed according to the specific use, high affinity antigen binding and target specificity characteristics are present in the variable region.

[0184] Anti-BCMA antibody heavy and light chain variable region amino acid and nucleic acid sequences: Table 1 lists 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.

[0185] Table 1: Amino Acid Sequence Identifiers

[0186]

[0187] Table 2: Nucleotide sequence identifiers

[0188]

[0189] Example 2: Generation of anti-CD3 antibodies

[0190] Anti-CD3 antibodies were generated as described in WO 2017 / 053856, which is incorporated herein by reference. Two of the anti-CD3 antibodies were selected from the products of the bispecific anti-BCMA x anti-CD3 antibodies according to the 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 invention can be found in, for example, WO 2014 / 047231.

[0191] Table 3: Amino Acid Sequence Identifiers

[0192]

[0193] Table 4: Nucleotide sequence identifiers

[0194]

[0195] Example 3: Generation of bispecific antibodies that bind BCMA and CD3

[0196] The present invention provides bispecific antigen binding molecules that bind CD3 and BCMA; the bispecific antigen binding molecules are also referred to herein as "anti-BCMA x anti-CD3 or anti-CD3 x BCMA or anti-BCMA x anti-CD3 bispecific molecules". The anti-BCMA portion of the anti-BCMA x anti-CD3 bispecific molecule is useful for targeting tumor cells expressing BCMA (also known as CD269), and the anti-CD3 portion of the bispecific molecule is useful for activating T cells. Simultaneous binding of BCMA on tumor cells and CD3 on T cells facilitates directed killing (cytolysis) of target tumor cells by activated T cells.

[0197] 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, 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 are used to construct the molecule (10082). In other cases, 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 mixed or paired with a variety of heavy chain arms can be used to construct a bispecific antibody.

[0198] Table 5: Overview of components of anti-BCMA x anti-CD3 bispecific antibodies

[0199]

[0200] Table 6 shows the amino acid sequence identifiers of the bispecific anti-BCMA x anti-CD3 antibodies exemplified herein.

[0201] Table 6: Amino acid sequences of anti-BCMA x anti-CD3 bispecific antibodies

[0202]

[0203] Example 4: Binding affinities and kinetic constants of anti-BCMA antibodies and anti-BCMA x anti-CD3 bispecific antibodies derived by surface plasmon resonance

[0204] The equilibrium dissociation constant (K) of 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 dilutions). For dimerization affinity, different concentrations of human BCMA expressed with a C-terminal mFc tag (human BCMA-MFC; SEQ ID NO: 108), extracellular domain of monkey BCMA expressed with a C-terminal mFc tag (monkey BCMA-MFC; SEQ ID NO: 112) or 30 nM BCMA expressed with a C-terminal mFc tag (mouse BCMA-MFC; SEQ ID NO: 114) were prepared in HBS-ET running buffer (ranging from 30 to 0.37 nM, 3-fold dilutions). Antigen samples were then injected at a flow rate of 30 μL / min over the captured anti-BCMA and anti-BCMA x anti-CD3 bispecific mAb surfaces. Antibody-reagent association 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. Kinetic association (k a ) and dissociation (k d ) rate constants were determined by fitting the real-time sensor profiles to a 1:1 binding model using Scrubber 2.0c curve fitting software. The binding dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) from the kinetic rate constants are calculated as follows:

[0205] and

[0206] As shown in Table 7, at 25°C, all anti-BCMA antibodies of the present invention bound to human BCMA-MMH with a 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, 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, 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, K D The values ​​ranged from 677 pM to 18.8 nM.

[0207] Table 7: Binding kinetic parameters of anti-BCMA monoclonal antibodies binding to human BCMA-MMH at 25°C

[0208]

[0209] Table 8: Binding kinetic parameters of anti-BCMA monoclonal antibodies binding to human BCMA-MFC at 25°C

[0210] Table 9: Binding kinetic parameters of anti-BCMA monoclonal antibodies binding to monkey BCMA-MMH at 25°C

[0211] Table 10: Binding kinetic parameters of anti-BCMA monoclonal antibodies binding to monkey BCMA-MFC at 25°C

[0212] Table 11: Binding kinetic parameters of anti-BCMA monoclonal antibodies binding to mouse BCMA-MFC at 25°C

[0213] Example 5: FACS binding of anti-BCMA x anti-CD3 bispecific antibodies to cells expressing human and cynomolgus macaque CD3

[0214] Flow cytometric analysis was used to determine the binding of BCMA x CD3 bispecific antibodies to human and cynomolgus CD3 (Jurkat cells, Jurkat cells engineered with mfCD3, primary human CD8+ and cynomolgus CD8+ T cells). Briefly, 1e05 cells / well were incubated with blocking solution (PBS + 1% filtered FBS + 5% mouse serum) and serial dilutions of BCMA x CD3 and control antibodies for 30 minutes on ice in the presence of FACS wash buffer. After incubation, cells were washed twice with cold FACS wash buffer (PBS + 1% filtered FBS) and bound antibodies were detected by incubation with Alexa647-conjugated anti-human secondary antibodies for another 30 minutes on ice. Wells containing no antibody or only secondary antibodies were used as controls. For detection of monkey and human T cells, a mixture of human and cynomolgus cross-reactive antibodies for CD4, CD8 and CD16 were added to the anti-human secondary antibodies. 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 selecting singlet events followed by side and forward scatter to select viable events. For monkey T cells, additional gating on CD8+ / CD16- cells was performed.

[0215] EC50 values ​​for FACS binding were calculated using 4-parameter nonlinear regression analysis in Prism software.

[0216] Jurkat cells are a T cell lymphoblast cell line that expresses human CD3. REGN5458 binds to human CD3 on Jurkat cells and primary human CD8+ T cells with a median EC50 of 1.50×10 -8 M and 3.20×10 -8 M. REGN5459 binds weakly to human CD3, with a median EC50 of 5.58 × 10 -7 M and the median EC50 for primary human CD8+ T cells was 4.71×10 -6 M. Using CRISPR / Cas9 technology, the Jurkat cell line was engineered to express cynomolgus macaque CD3ε and CD3δ chains instead of the human version. The median EC50 for REGN5458 binding to the mfCD3 engineered Jurkat cell line was 1.51×10 -8 M, and the median EC50 for binding to primary cynomolgus macaque CD8+ T cells was 4.66×10 -8 M. REGN5459 does not bind to cells expressing mfCD3.

[0217] No binding was observed for the negative isotype control antibody (designated mAb15260) on any of the cell lines.

[0218] Table 12: Binding to cells expressing CD3: Median EC50

[0219]

[0220] Example 6: FACS binding analysis to assess cell surface antigen binding

[0221] 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 in staining buffer (PBS without calcium and magnesium (Irving 9240) + 2% FBS (ATCC 30-2020)) at a density of 500,000 cells per well in 96-well V-bottom culture plates. 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 stained with LIVE / DEAD according to the manufacturer's instructions. TM Fixable green dead cell staining kit (Invitrogen, L34970) was used to distinguish live cells from dead cells. The cells were then washed and fixed for 25 minutes at 4°C using a 50% solution of BD Cytofix (BD, Cat#554655) diluted in PBS. The samples were flowed 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 and the EC 50Values ​​ranged from 27 to 83 nM. No detectable binding was observed on HEK293 cells.

[0222] Table 13: Binding to cells

[0223]

[0224] ND = Not determined due to non-S-shaped curve

[0225] Example 7: T cell activation via bispecific anti-BCMA x anti-CD3 antibodies in the presence of BCMA-expressing cells

[0226] The activity of the anti-BCMA x anti-CD3 bispecific antibodies 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 50 ul assay medium (RPMI medium with 10% FBS and 1% P / S / G) in a Thermo Nunclon delta 96 white microplate well (Thermo Scientific, Cat#136102). Three-fold serial dilutions of BCMA x CD3 bispecific antibody (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 to 6 hours. NFAT-luciferase activity was determined using Promega One-Glo (Cat#E6130) and Perkin Elmer Envision plate reader. RLU was plotted on a 12-point response curve using a four-parameter logistic equation in GraphPadPrism to calculate EC 50 The non-antibody treatment condition of each dose-response curve was also included in the analysis as a continuation of the three-fold serial dilution and represented as the lowest dose. The signal-to-noise ratio (S:N) was determined by taking the ratio of the highest RLU to the lowest RLU on the curve.

[0227] mAb25441D activated Jurkat / NFATLuc cells in the presence of BCMA expressing cells with EC50 ranging from 0.61 nM to 2.1 nM and S:N ranging from 8 to 123. mAb25442D activated Jurkat / NFATLuc cells in the presence of BCMA expressing cells with EC50 ranging from 2.6 nM to 11 nM and S:N ranging from 7 to 120. The BCMA x CD3 bispecific mAb25441D with a higher affinity CD3 binding arm was consistently more potent than mAb25442D with a lower affinity CD3 binding arm; however, the S:N of the two bispecific antibodies was similar. Neither antibody activated Jurkat / NFATLuc cells in the presence of HEK293 cells, and the control bispecific antibody did not significantly increase Jurkat reporter activity in any of the cell lines tested. The results are shown in Tables 14A and 14B below.

[0228] Table 14A: Activation of T cells

[0229]

[0230] Table 14B: Activation of T cells

[0231]

[0232] 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

[0233] 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).

[0234] Briefly, multiple myeloma (MM) cell lines (H929, MM1S, U266, MOLP8, and RPMI8226) and Quantum Simply Cellular beads were incubated with titrations of APC-conjugated anti-hBCMA-19F2 antibodies for 30 minutes at 4°C. Following 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 anti-BCMA 19F2 ABC for each cell line were interpolated from a standard curve generated from the channel intensity through a bead population at saturation.

[0235] Killing of BCMA-expressing target cells by resting human or cynomolgus macaque T cells was determined by flow cytometry. Briefly, human or cynomolgus macaque peripheral blood mononuclear cells (PBMCs) were plated at 1×10 6 Cells / mL were inoculated in medium supplemented with RPMI (human) or X-Vivo (cynomolgus macaque) and incubated overnight at 37°C to enrich lymphocytes by consuming adherent macrophages, dendritic cells, and some monocytes. The next day, target cells expressing BCMA were labeled with 1 μM of Violet CellTrace and co-cultured with PBMCs (effector / target cell 4:1 ratio) depleted of adherent cells and serial dilutions of BCMA x CD3 bispecific or control antibodies at 37°C. After 48 to 72 hours, cells were removed from the cell culture plate, stained with phenotyping antibodies and a mixture of live / dead cell viability dyes, and analyzed by FACS. In order to quantify the number of live target cells present in the wells, 20 μl of CountBright absolute counting beads were added to the wells just before collection. For the evaluation of killing specificity, cells were gated on the population labeled with the Violet CellTrace. The survival percentage of target cells was calculated as follows: target survival = (R1 / R2) x 100, where R1 = the absolute number of surviving target cells in the presence of effector cells and antibody, and R2 = the number of surviving target cells alone (cultured without effector cells or test antibody).

[0236] Human CD8+ T cells were gated as CD45+ / CD14- / CD4- / CD8+. Cynomolgus CD8+ T cells were gated as CD45+ / CD20- / CD14- / CD4- / CD8+ T cells, and activation was reported as the percentage of CD25+ or CD69+ T cells among total CD8+ T cells.

[0237] The EC50 values ​​of target cell survival and T cell activation were calculated using 4-parameter nonlinear regression analysis in Prism software.

[0238] The anti-BCMA x anti-CD3 bispecific antibody was tested for its ability to activate dormant human and cynomolgus macaque T cells to kill a panel of BCMA-expressing cells with varying surface BCMA levels. In the presence of dormant human T cells as effector cells, REGN5458-mediated killing of five different BCMA cell lines was at 7.07×10 -10 M to 3.45×10 -11 EC in the M range 50 REGN5459 showed a killing rate of 1.66×10 -9 M to 1.06×10 -10EC50 values ​​ranged from 4.4 to 1.3 M. EC50 values ​​for T cell activation, as measured by upregulation of CD25 on CD8+ T cells, were 50 Similar to killing EC 50 Appropriate T cell activation was observed in the presence of the 1-arm CD3 isotype control mAb 17664D, but only for the U266 cell line. No cytotoxicity was observed for the isotype controls tested.

[0239] BCMA x CD3 killing mediated by cynomolgus macaque T cells was tested only on the MM cell line H929. EC cytotoxicity mediated by REGN5458 and REGN5459 50 They are 2.34×10 -11 and 6.92×10 -11 No cytotoxicity or T cell activation was observed for the isotype control antibody mAb15260 in the context of human or cynomolgus monkey effector cells. The results are shown in Tables 15A, 15B and 16 below.

[0240] Table 15A: Median EC 50 , human effector cells

[0241]

[0242] Table 15B: Median EC 50 , human effector cells

[0243]

[0244] Table 16: Median EC 50 , cynomolgus macaque effector cells

[0245]

[0246] Example 9: FACS cytotoxicity analysis of autologous T cell-mediated killing of primary multiple myeloma blasts in the presence of anti-BCMA x anti-CD3 bispecific antibodies

[0247] To monitor specific killing of multiple myeloma cells by flow cytometry, bone marrow mononuclear cells (BMMCs) from multiple myeloma patients were seeded on human stromal cells (HS5) and incubated overnight at 37°C. Separately, matched patient peripheral blood mononuclear cells (PBMCs) were thawed and incubated at 37°C in supplemented RPMI medium at 1 × 10 6Cells / mL were cultured overnight to enrich lymphocytes by consuming adherent cells. The next day, BMMCs were co-cultured with untreated PBMCs depleted of adherent cells on stromal cells (HS5) and serial 10x dilutions of BCMA x CD3 bispecific or 1-arm CD3 isotype control (starting concentration 66.7 nM) at 37°C. Cells were removed from the cell culture plate on days 3, 4, or 7 and analyzed by FACS. In order to assess the specificity of killing, multiple myeloma cells were gated as single live CD90 negative (excluding stromal cells), CD2 negative, CD56 positive. CD45 was low on multiple myeloma cells in most samples except MM455. The percentage of live target cells was reported for the following calculation of adjusted survival rate: 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 antibody.

[0248] 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 among total CD4 or CD8 T cells.

[0249] The BCMA x CD3 bispecific antibodies were tested for their ability to direct killing of primary multiple myeloma blasts by autologous donor PBMCs. The maximum BCMA x CD3-mediated cytotoxicity of primary MM blasts ranged from 52% to 96%, with an EC50 of 9.89 × 10 -11 M to 3.67×10 -9 For REGN5459, the EC50 was 4.96×10 -10 M to 7.94×10 -8 M range. T cell activation was measured by assessing upregulation of CD25 on CD8+ T cells. The EC50 range for T cell activation was 3.23×10 -9 to 1.69×10 -10 Appropriate cytotoxicity and T cell activation were observed for the 1-arm CD3 (no target binding) isotype control. The results are shown in Tables 17A and 17B below.

[0250] Table 17A: MM % Lysis

[0251]

[0252] Table 17B: MM lysis EC50 and T cell activation

[0253]

[0254] Example 10: Anti-BCMA x anti-CD3 bispecific antibody prevents the growth of BCMA-expressing tumors (NCI-H929) in vivo in an allogeneic tumor model

[0255] To determine the in vivo efficacy of the BCMA x CD3 bispecific antibody (Ab), an allogeneic tumor study was performed in the immunodeficient NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ (NSG) mice were implanted subcutaneously with 10×10 6 0.5×10 NCI-H929 multiple myeloma cells expressing BCMA were added to 6 Mice (n=7 per group) were immediately dosed with PBS vehicle control, irrelevant anti-FelD1 bivalent isotype control Ab (REGN2759), CD3 binding control bispecific Ab (mAb17664D), BCMA x CD3 (G; REGN5458) bispecific Ab, or BCMA x CD3 (G20; REGN5459) bispecific Ab at a dose of 4 mg / kg. Mice were dosed with Ab twice weekly for a total of three weeks, and tumor growth was assessed over 40 days. Although BCMA was not significantly increased in mice treated with vehicle, isotype control, and CD3 binding control, the tumor growth was significantly decreased in mice treated with 40 days. + Tumors grew similarly, but all BCMA x CD3 Abs tested prevented tumor growth in vivo.

[0256] Implantation and measurement of syngeneic tumors: NSG mice were implanted with 10×10 6 0.5×10 NCI-H929 multiple myeloma cells expressing BCMA were added to 6 The mice (n=7 per group) were immediately dosed with PBS vehicle control, irrelevant anti-FelD1 bivalent isotype control Ab (REGN2759), CD3 binding control bispecific Ab (mAb17664D), BCMA x CD3 (G; REGN5458) bispecific Ab, or BCMA x CD3 (G20; REGN5459) bispecific Ab at a dose of 4 mg / kg. The mice were dosed with Ab twice a week for a total of three weeks. Tumor growth was measured with a caliper twice a week for the duration of the experiment. Mice were sacrificed on day 40 after tumor implantation.

[0257] Calculation of Syngeneic Tumor Growth and Inhibition: To determine tumor volume by external caliper, 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×width2 ) / 2.

[0258] BCMA x CD3 bispecific Ab prevents BCMA in allogeneic tumor models + In vivo growth of NCI-H929 tumors. The results are shown in Table 18 below.

[0259] Table 18: Average tumor size at each time point

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] Example 11: Anti-BCMA x anti-CD3 bispecific antibody prevents the growth of BCMA-expressing tumors (NCI-H929) in an allogeneic in vivo tumor model in a dose-dependent manner

[0269] To determine the in vivo efficacy of an anti-BCMA x anti-CD3 bispecific antibody (Ab), an allogeneic tumor study was performed in immunodeficient NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ (NSG) mice were implanted subcutaneously with 10×10 6 0.5×10 NCI-H929 human multiple myeloma tumor cells expressing BCMA were added to 6Mice (n=7 per group) were then immediately dosed with PBS vehicle control; CD3 binding control bispecific Ab (G; mAb17664D) at a dose of 4 mg / kg; CD3 binding control bispecific Ab (G20; REGN4460) at a dose of 4 mg / kg; BCMA x CD3 (G; REGN5458) bispecific Ab at a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg; or BCMA x CD3 (G20; REGN5459) bispecific Ab at a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. Mice were dosed with 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, but all anti-BCMA x anti-CD3 Abs tested prevented tumor growth in vivo in a dose-dependent manner.

[0270] Implantation and measurement of xenogeneic tumors: NSG mice were subcutaneously implanted with 10×10 6 0.5×10 NCI-H929 multiple myeloma cells expressing BCMA were added to 6 The mice (n=7 per group) were immediately dosed with PBS vehicle control, CD3 binding control bispecific Ab (G; mAb17664D), CD3 binding control bispecific Ab (G20; REGN4460), BCMA x CD3 (G; REGN5458) bispecific Ab, or BCMA x CD3 (G20; REGN5459) bispecific Ab. mAb17664D and REGN4460 were dosed at 4 mg / kg, while REGN5458 and REGN5459 were dosed at 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. The mice were dosed with Ab twice weekly for a total of seven doses. Tumor growth was measured with a caliper twice weekly for the duration of the experiment.

[0271] Calculation of allogeneic tumor growth and inhibition: To determine tumor volume by external caliper, 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.

[0272] The BCMA x CD3 bispecific Ab prevented BCMA in a dose-dependent manner in this allogeneic in vivo tumor model. +The results are shown in Table 19 below and are presented in Table 19. Figure 1 and Figure 2 In the description.

[0273] Table 19: Average tumor size at each time point

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288] Example 12: Anti-BCMA x anti-CD3 bispecific antibody reduces the size and prevents the growth of established BCMA-expressing tumors (NCI-H929) in an allogeneic in vivo tumor model in a dose-dependent manner

[0289] To determine the in vivo efficacy of an anti-BCMA x anti-CD3 bispecific antibody (Ab), an allogeneic tumor study was performed in immunodeficient NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ (NSG) mice were implanted subcutaneously with 10×10 6 0.5×10 NCI-H929 human multiple myeloma tumor cells expressing BCMA were added to 6 Tumors were grown and established for 5 days until they were approximately 70 mm in size.3 . Mice (n=7 to 8 per group) were then dosed with PBS vehicle control on day 5; CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 4 mg / kg; CD3-binding control bispecific Ab (G20; REGN4460) at a dose of 4 mg / kg; BCMA x CD3 (G; REGN5458) bispecific Ab at doses of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg; or BCMA x CD3 (G20; REGN5459) bispecific Ab at doses of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. Mice were dosed with 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, but all anti-BCMA x anti-CD3 Abs tested dose-dependently shrank established tumors in vivo and prevented tumor growth.

[0290] Implantation and measurement of xenogeneic tumors: NSG mice were subcutaneously implanted with 10×10 6 0.5×10 NCI-H929 multiple myeloma cells expressing BCMA were added to 6 Tumors were grown and established for 5 days until they were approximately 70 mm in size. 3 Mice (n=7 to 8 per group) were then dosed with PBS vehicle control, CD3 binding control bispecific Ab (G; mAb17664D), CD3 binding control bispecific Ab (G20; REGN4460), BCMA xCD3 (G; REGN5458) bispecific Ab, or BCMA x CD3 (G20; REGN5459) bispecific Ab on day 5. mAb17664D and REGN4460 were dosed at 4 mg / kg, while REGN5458 and REGN5459 were dosed at 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. Mice were dosed with Ab twice weekly for a total of seven doses. Tumor growth was measured with a caliper twice weekly for the duration of the experiment.

[0291] Calculation of allogeneic tumor growth and inhibition: To determine tumor volume by external caliper, 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.

[0292] The anti-BCMA x anti-CD3 bispecific antibody reduced established BCMA in a dose-dependent manner in this allogeneic in vivo tumor model. + The size of NCI-H929 tumors and prevented their growth. The results are shown in Table 20 below and are Figure 3 and 4 In the description.

[0293] Table 20: Average tumor size at each time point

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305] Example 13: Anti-BCMA x anti-CD3 bispecific antibody prevents the growth of BCMA-expressing tumors (MOLP-8) in an allogeneic in vivo tumor model in a dose-dependent manner

[0306] To determine the in vivo efficacy of an anti-BCMA x anti-CD3 bispecific antibody (Ab), an allogeneic tumor study was performed in immunodeficient NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ (NSG) mice were implanted subcutaneously with 5×10 6 1×10 MOLP-8 human multiple myeloma tumor cells expressing BCMA were added to 6Mice (n=7 per group) were then immediately dosed with PBS vehicle control; CD3 binding control bispecific Ab (G; mAb17664D) at a dose of 4 mg / kg; CD3 binding control bispecific Ab (G20; REGN4460) at a dose of 4 mg / kg; BCMA x CD3 (G; REGN5458) bispecific Ab at a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg; or BCMA x CD3 (G20; REGN5459) bispecific Ab at a dose of 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. Mice were dosed with 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, but all BCMA x CD3 Abs tested prevented tumor growth in vivo in a dose-dependent manner.

[0307] Implantation and measurement of xenogeneic tumors: NSG mice were subcutaneously implanted with 5×10 6 1×10 MOLP-8 multiple myeloma cells expressing BCMA were added to 6 The mice (n=7 per group) were immediately dosed with PBS vehicle control, CD3 binding control bispecific Ab (G; mAb17664D), CD3 binding control bispecific Ab (G20; REGN4460), BCMA x CD3 (G; REGN5458) bispecific Ab, or BCMA x CD3 (G20; REGN5459) bispecific Ab. mAb17664D and REGN4460 were dosed at 4 mg / kg, while REGN5458 and REGN5459 were dosed at 4 mg / kg, 0.4 mg / kg, or 0.04 mg / kg. The mice were dosed with Ab twice weekly for a total of seven doses. Tumor growth was measured by caliper twice weekly for the duration of the experiment.

[0308] Calculation of allogeneic tumor growth and inhibition: To determine tumor volume by external caliper, 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.

[0309] The anti-BCMA x anti-CD3 bispecific antibody prevented BCMA in a dose-dependent manner in this allogeneic in vivo tumor model +Growth of MOLP-8 tumors. The results are shown in Table 21 below and are Figure 5 and 6 In the description.

[0310] Table 21: Average tumor size at each time point

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324] Example 14: Anti-BCMA x anti-CD3 bispecific antibody delays growth of BCMA-expressing tumors (MOLP-8) in an allogeneic in vivo tumor model

[0325] To determine the in vivo efficacy of an anti-BCMA x anti-CD3 bispecific antibody (Ab), an allogeneic tumor study was performed. scid Iq tm1Wjl / SzJ(NSG) mice were injected intraperitoneally with 4×10 6 On day 0, mice were intravenously administered 2×10 6 BCMA engineered to also express firefly luciferase +MOLP-8 human multiple myeloma tumor cells (MOLP-8 luciferase cells). Mice (n=5 per group) were then immediately administered a CD3 binding control bispecific Ab (G; mAb17664D) at a dose of 4 mg / kg or a BCMA x CD3 (G; REGN5458) bispecific Ab at a dose of 4 mg / kg. 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 provided with 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 grew progressively, but BCMA x CD3 Ab treatment with REGN5458 delayed tumor growth in vivo.

[0326] Implantation and measurement of allogeneic tumors: On day 11, the immunodeficient NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ(NSG) mice were injected intraperitoneally with 5×10 6 On day 0, 2×10 6 BCMA + MOLP-8 luciferase cells. Mice (n = 5 per group) were then immediately given a CD3 binding control bispecific Ab (G; mAb17664D) at a dose of 4 mg / kg or a BCMA x CD3 (G; REGN5458) bispecific Ab at a dose of 4 mg / kg. Mice were given these Abs twice more on days 3 and 7, for a total of three doses. Tumor growth was assessed over 48 days by measuring tumor BLI in anesthetized animals. As a positive control, a group of mice (n = 5) were provided with only MOLP-8 luciferase cells without PBMCs or antibodies. To measure background BLI levels, a group of mice (n = 5) were untreated and did not receive tumors, PBMCs or antibodies.

[0327] Measurement of allogeneic 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 by Living Image software at a field of view at D, an object height of 1.5 cm, and a medium merge level with automatic exposure time. BLI signals were extracted using Living Image software: a region of interest was drawn around each tumor mass and photon intensity was recorded as p / s / cm2 / sr.

[0328] The anti-BCMA x anti-CD3 bispecific antibody REGN5458 delays BCMA in this allogeneic in vivo tumor model + Growth of MOLP-8 luciferase tumors. The results are shown in Table 22 below.

[0329] Table 22: Average tumor size at each time point (in terms of radiation rate)

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340] Example 15: Anti-BCMA x anti-CD3 bispecific antibody reduces tumor (OPM-2) burden to background levels in vivo

[0341] To determine the in vivo efficacy of an anti-BCMA x anti-CD3 bispecific antibody (Ab), an allogeneic tumor study was performed. scid Iq tm1Wjl / SzJ (NSG) mice were intravenously administered 2×10 6BCMA engineered to also express firefly luciferase + OPM-2 human multiple myeloma tumor cells (OPM-2 luciferase cells). On day 10, mice were injected intraperitoneally with 4×10 6 Human peripheral blood mononuclear cells (PBMCs) from normal healthy donors. On day 21, mice (n=5 per group) were given a dose of 0.4 mg / kg of a CD3 binding control bispecific Ab (G; mAb17664D), 0.4 mg / kg of a BCMA x CD3 (G; REGN5458) bispecific Ab, or 0.4 mg / kg of a BCMA x CD3 (G20; REGN5459) bispecific Ab. Mice were given these Abs twice more on days 25 and 28 for a total of three doses. Tumor growth was assessed on day 61 by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, one group of mice (n=5) was provided with 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. Although BCMA + OPM-2 luciferase tumors grew progressively, but BCMAx CD3 Ab treatment with REGN5458 and REGN5459 reduced tumor burden to background levels in most animals.

[0342] Implantation and measurement of allogeneic tumors: on day 0, cells were implanted into immunodeficient NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ (NSG) mice were intravenously administered 2×10 6 BCMA engineered to also express firefly luciferase + OPM-2 human multiple myeloma tumor cells (OPM-2 luciferase cells). On day 10, mice were injected intraperitoneally with 4×10 6Human peripheral blood mononuclear cells (PBMCs) from normal healthy donors. On day 21, mice (n=5 per group) were given a dose of 0.4 mg / kg of a CD3 binding control bispecific Ab (G; mAb17664D), 0.4 mg / kg of a BCMA x CD3 (G; REGN5458) bispecific Ab, or 0.4 mg / kg of a BCMA x CD3 (G20; REGN5459) bispecific Ab. Mice were given these Abs twice more on days 25 and 28 for a total of three doses. Tumor growth was assessed on day 61 by measuring tumor bioluminescence (BLI) in anesthetized animals. As a positive control, one group of mice (n=5) was provided with 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.

[0343] Measurement of allogeneic 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 by Living Image software at a field of view at D, an object height of 1.5 cm, and a medium merge level with automatic exposure time. BLI signals were extracted using Living Image software: a region of interest was drawn around each tumor mass and photon intensity was recorded as p / s / cm2 / sr.

[0344] Although BCMA + OPM-2 luciferase tumors gradually grew, but BCMA x CD3 Ab treatment with REGN5458 and REGN5459 reduced tumor burden to background levels in most animals. The results are shown in Table 23 below and are Figure 7 In the description.

[0345] Table 23: Average tumor size at each time point (in terms of radiation rate)

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357] Example 16: BCMA x CD3 bispecific antibody suppresses the growth of allogeneic homogeneous tumors in vivo in a dose-dependent manner

[0358] To determine the in vivo efficacy of the anti-BCMA x anti-CD3 bispecific antibody (Ab), a xenogeneic tumor study was performed in mice expressing human CD3. C57BL / 6 mice expressing human CD3deg but not murine CD3deg (CD3 humanized mice) were implanted subcutaneously with 0.5×10 6 B16 melanoma cells engineered to express full-length human BCMA (B16 / BCMA cells) or 1×10 6 MC38 colon carcinoma cells that have been engineered to express full-length human BCMA (MC38 / BCMA). Mice (n=7 per group) were then immediately administered a CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 0.4 mg / kg or a BCMA x CD3 (G; REGN5458) bispecific Ab at a dose of 0.4 mg / kg or 0.04 mg / kg. Mice were administered these Abs twice more on days 4 and 7 for a total of three doses, and tumor growth was assessed during the experiment. While B16 / BCMA tumors and MC38 / BCMA tumors grew in mice treated with the CD3-binding control, BCMA x CD3 REGN5458 was able to suppress the growth of both tumor lines in vivo in a dose-dependent manner.

[0359] Implantation and measurement of allogeneic tumors: C57BL / 6 mice expressing human CD3deg but not murine CD3deg (CD3 humanized mice) were subcutaneously implanted with 0.5×10 6 B16F10 melanoma cells engineered to express full-length human BCMA (B16 / BCMA cells) or 1×10 6MC38 colon carcinoma cells that have been engineered to express full-length human BCMA (MC38 / BCMA). Mice (n=7 per group) were then immediately administered a CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 0.4 mg / kg or a BCMA x CD3 (G; REGN5458) bispecific Ab at a dose of 0.4 mg / kg or 0.04 mg / kg. These Abs were administered to mice twice more on days 4 and 7 for a total of three doses, and tumor growth was assessed during the experiment.

[0360] Calculation of allogeneic tumor growth and inhibition: To determine tumor volume by external caliper, 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.

[0361] While B16 / BCMA tumors and MC38 / BCMA tumors grew in mice treated with CD3 binding control, BCMAxCD3 REGN5458 was able to suppress the growth of both tumor lines in vivo in a dose-dependent manner. The results are shown in Table 24 below.

[0362] Table 24: Average tumor size at each time point

[0363]

[0364]

[0365]

[0366]

[0367] Example 17: Epitope Mapping of REGN5458 Binding to BCMA by Hydrogen-Deuterium Exchange

[0368] H / D exchange epitope mapping mass spectrometry (HDX-MS) was performed to determine the amino acid residues of BCMA (recombinant human BCMA, amino acid sequence of SEQ ID NO: 115) that interacted with REGN5458 (BCMA x CD3 bispecific antibody). A general description of the H / D exchange method is described in, for example, Ehring (1999) Analytical Biochemistry 267(2): 252-259; and Engen and Smith (2001) Analytical Chemistry 73: 256A-265A.

[0369] HDX-MS experiments were performed on an integrated HDX / MS platform consisting of a Leaptec HDX PAL system for deuterium labeling and quenching, a Waters Acquity M-Class (Auxiliary solvent manager) for sample digestion and loading, a Waters Acquity M-Class (μBinary solvent manager) for analytical gradients, and a Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

[0370] 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 of 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 of D2O labeling solution at 20°C for each time point in duplicate (e.g., non-deuterated control = 0 seconds; deuterium labeling lasted for 5 minutes and 10 minutes). The deuteration reaction was quenched by adding 100 μL of pre-cooled quenching buffer (0.5 M TCEP-HCl, 8 M urea and 1% formic acid) to each sample and incubated at 20°C for 5 minutes. The quenched sample was then injected into a Waters HDXManager for online pepsin / protease XIII digestion. The digested peptides were separated by a C8 column (1.0 mm×50 mm, NovaBioassays) with a 13-minute gradient of 10% to 32% B (mobile phase A: 0.5% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The eluted peptides were analyzed by a Q Exactive HF mass spectrometer in LC-MS / MS or LC-MS mode.

[0371] The LC-MS / MS data of non-deuterated BCMA samples were searched using the Byonic search engine (Protein Metrics) for a database containing BCMA and its random sequences. The search parameters (in ELN) were set to default values ​​using nonspecific enzymatic digestion and human glycosylation as shared variable modifications. The list of identified peptides was then imported into HDX Workbench software (version 3.3) to calculate the deuterium absorption of 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 deuterium absorption (D) and deuterium absorption percentage (% D):

[0372]

[0373] A total of 8 peptides from hBCMA.hFc were identified from hBCMA.hFc alone and hBCMA.hFc complexed 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 exhibiting a differential D-absorption percentage value higher than 4.2% (3 times the average SD) was defined as significantly protected. For hBCMA.hFc, the peptide corresponding to amino acids 1 to 43 of SEQ ID NO: 106 (MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNA; SEQ ID NO: 121) was significantly protected by REGN5458. hBCMA.mmH (REGN2744, amino acid sequence of SEQ ID NO: 106) was used to confirm that these residues were protected by REGN5458.

[0374] Table 25: Selected BCMA.hFc peptides have significant protective effects after binding to REGN5458

[0375]

[0376] Example 18: FACS Binding Analysis of BCMA x CD3 Bispecific Antibodies and Other BCMA Antibodies on Multiple Myeloma Cell Lines After Overnight Incubation with Anti-BCMA Antibodies

[0377] Flow cytometry analysis was used to determine the effect of overnight incubation of multiple myeloma cell lines with anti-BCMA antibodies on surface BCMA levels. MM cell lines (H929, Molp8, U266, and MM1.S) were washed twice and cultured at 37°C in R10 medium (RPMI + 10% FBS + pen / strep / glut) containing 66.7 or 667 nM anti-BCMA antibodies, DAPT (γ-secretase inhibitor), or culture medium alone. After 18 hours, wells were washed with cold FACS wash (PBS + 1% filtered FBS) and resuspended in 667 nM of the same anti-BCMA antibody in cold staining buffer (Miltenyi 130-091-221) on ice for 30 minutes. After incubation, cells were washed twice with cold FACS wash solution (PBS + 1% filtered FBS) and bound antibodies were detected by incubation with appropriate anti-human secondary antibodies (anti-hIgG or anti-HIS) for another 30 to 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 ab or DAPT by the MFI of stained cells incubated overnight in culture medium alone.

[0378] BCMA is rapidly cleaved from the cell surface by the enzyme γ-secretase. Cultivation with γ-secretase inhibitors (such as DAPT) overnight prevents BCMA cleavage that causes increased BCMA levels on the cell surface. Tables 26 to 29 report the increase in median fluorescence intensity (MFI) of BCMA on cells cultivated overnight in anti-BCMA antibodies or DAPT compared to cells cultivated only in culture medium. It was observed that cultivation with DAPT overnight increased the BCMA levels detected on H929, Molp8, U266, and MM.1S by 2.3 to 4 times, 2.4 to 8.6 times, 5.3 to 9.0 times, and 11.9 times, respectively.

[0379] It is also important to note that incubation of MM cell lines with 66.7 or 667 nM REGN5458 or the parental bivalent anti-BCMA antibody mAb21581 overnight similarly resulted in an increase in surface BCMA levels detected by FACS, indicating that binding of the anti-BCMA antibodies prevents cleavage of BCMA by γ-secretase. The antibody-induced increase in surface BCMA varied by cell line, with greater fold increases on Molp8 and MM1S cells compared to H929 or U266. The phenomenon is not limited to REGN5458, as it was also observed with other in-house BCMA antibodies.

[0380] Table 26: Fold change in MFI compared to cells grown in medium alone (NCI-H929)

[0381]

[0382] Table 27: Fold change in MFI compared to cells grown in medium alone (Molp8)

[0383]

[0384] Table 28: Fold change in MFI compared to cells grown in medium alone (U266)

[0385]

[0386] Table 29: Fold change in MFI compared to cells grown in medium alone (MM1S)

[0387]

[0388] Example 19: Autologous T cell-mediated killing of human and cynomolgus macaque plasma cells in the presence of BCMA x CD3 bispecific antibody

[0389] Evaluation of CD138 enrichment by non-stimulatory autologous T cell-specific killing by flow cytometry + Human or cynomolgus macaque plasma cells. Human or cynomolgus macaque bone marrow aspirates and blood were provided within 24 hours of collection. EasySep Human CD138 was used according to the manufacturer's instructions. + Positive Selection Kit Enrich 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 tracking dye. After labeling, 1×10 4 CD138 enriched +Plasma cells were seeded in round-bottom 96-well plates at an E:T ratio of 10:1 with Vybrant CFDA-SE-labeled PBMCs and serial dilutions of REGN5458, CD3-binding control bsAb, or BCMA-binding control mAb in complete medium for 72 hours. 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).

[0390] 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 autologous plasma cell cytotoxicity. EC values ​​for cytotoxicity and percentage of T cell activation for each donor were 50 The values ​​are summarized in Table 30.

[0391] 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. 50 The values ​​were 42.8 pM and 191 pM, respectively, and produced 91% and 89% maximum cytotoxicity percentages, respectively. Meanwhile, REGN5458 mediated T cell activation in a concentration-dependent manner in the presence of human plasma cells from donors 1 and 2, CD8 + T cell activated EC 50 The values ​​were 214pM and 860pM, respectively, and the maximum CD8 + The percentage of T cell activation was 2% and 36%, respectively. Cytotoxicity of plasma cells in both donors and CD8 in donor 2 alone was observed at nanomolar concentrations of CD3 binding control. + Increased T cell activation. No effects on cytotoxicity or T cell activation were observed in either donor at any concentration tested in the case of BCMA binding control.

[0392] REGN5458 mediated cytotoxicity in primary cynomolgus macaque plasma cells from both donors in a concentration-dependent manner; the 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. 50In both donors, REGN5458 treatment resulted in increased plasma cell cytotoxicity (maximum cytotoxicity percentages were 94% and 91% for donors 1 and 2, respectively). At the same time, REGN5458 mediated T cell activation in a concentration-dependent manner in the presence of cynomolgus macaque plasma cells from donors 1 and 2, with CD4 + T cell activated EC 50 The values ​​were 28.1 nM and 18.1 nM, respectively, and CD8 + T cell activated EC 50 The values ​​were 22.4 nM and 76.7 nM, respectively. For donors 1 and 2, the obtained CD4 + The maximum T cell activation percentages of T cells were 9% and 16%, respectively, and CD8 + The maximum T cell activation percentages of T cells were 12% and 17%, respectively.

[0393] No target cell killing was observed in any of the cell lines evaluated with the BCMA binding control at any concentration tested. Some target cell killing and T cell activation in the presence of plasma cells from donor 2 was observed with the CD3 binding control at nanomolar concentrations.

[0394] Table 30: EC values ​​for cytotoxicity and T cell activation percentage for each donor 50 value

[0395]

[0396] a Each donor was tested for autologous plasma cells.

[0397] Example 20: Anti-BCMA x anti-CD3 bispecific antibody synergizes with anti-PD-1 antibody to enhance anti-tumor efficacy in vivo

[0398] To determine whether the BCMA x CD3 bispecific antibody (Ab) synergizes with PD-1 blockade to provide superior in vivo anti-tumor efficacy, a xenogeneic tumor study was performed in mice expressing human CD3. The results demonstrated that the combination of REGN5458 plus PD-1 blockade provided superior anti-tumor efficacy than either REGN5458 or PD-1 blockade alone.

[0399] Implantation and measurement of allogeneic tumors: C57BL / 6 mice expressing human CD3deg but not murine CD3deg (CD3 humanized mice) were subcutaneously implanted with 1×10 6MC38 colon carcinoma cells that have been engineered to express full-length human BCMA (MC38 / BCMA). Tumors were allowed to establish for 3 days, at which time mice (n=6 or 7 per group) were given 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 BCMA x CD3 (G; REGN5458) bispecific Ab and 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). The specific treatment groups are shown in Table 31 below.

[0400] Table 31: Treatment Groups

[0401] Group Bispecific treatment Antibody n 1 H4SH17664D (0.24 mg / kg) Same type (4mg / kg) 7 2 H4SH17664D (0.24 mg / kg) RPM1-14 (4 mg / kg) 7 3 REGN5458 (0.04 mg / kg) Same type (4mg / kg) 7 4 REGN5458 (0.04 mg / kg) RPM1-14 (4 mg / kg) 7 5 REGN5458 (0.24 mg / kg) Same type (4mg / kg) 6 6 REGN5458 (0.24 mg / kg) RPM1-14 (4 mg / kg) 6

[0402] Mice were given these Abs two more times on days 7 and 11 for a total of three doses, and tumor growth was assessed during the experiment.

[0403] Calculation of allogeneic tumor growth and inhibition: To determine tumor volume by external caliper, 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.

[0404] The results demonstrated that the combination of REGN5458 plus PD-1 blockade provided superior anti-tumor efficacy than REGN5458 or PD-1 blockade alone. Specifically, the results demonstrated that the combination of BCMA x CD3 bispecific antibody and anti-PD-1 antibody produced a statistically significant synergistic therapeutic effect in inhibiting tumor growth on day 24 (the last day of collecting data for all treatment groups) (Table 32, 0.04 mg / kg of BCMA x CD3 and 4 mg / kg of anti-PD-1). Using two-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 two-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). Increasing the dose of BCMA x CD3 bispecific antibody (0.24 mg / kg) in combination with PD-1 blockade in this experiment produced tumor inhibition similar to the lower bispecific antibody dose plus PD-1 blockade. Synergy with lower doses of bispecific antibody was demonstrated to be advantageous because the use of lower doses reduces the risk of any adverse side effects. Similarly, the combination of BCMA x CD3 bispecific antibody with anti-PD-1 antibody demonstrated a synergistic therapeutic effect on the number of tumor-free mice at the end of the experiment (day 28) at both doses of bispecific antibody (0.04 mg / kg and 0.24 mg / kg), as shown in Table 33.

[0405] Table 32: Average tumor size at each time point

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414] Table 33: Tumor-free mice at the end of the experiment

[0415]

[0416] Example 21: Anti-BCMA x anti-CD3 bispecific antibody synergizes with anti-PD-1 antibody to enhance anti-tumor efficacy in vivo

[0417] Similar results were obtained in the second experiment, consistent with those discussed above in Example 20, except that the number of mice per group = 10, and the higher dose of BCMA x CD3 REGN5458 was 0.4 mg / kg. The specific treatment groups for the second experiment are shown in Table 34 below.

[0418] Table 34: Treatment Groups

[0419] Group Bispecific treatment Antibody n 1 H4SH17664D (0.4 mg / kg) Same type (4mg / kg) 10 2 H4SH17664D (0.4 mg / kg) RPM1-14 (4 mg / kg) 10 3 REGN5458 (0.04 mg / kg) Same type (4mg / kg) 10 4 REGN5458 (0.04 mg / kg) RPM1-14 (4 mg / kg) 10 5 REGN5458 (0.4 mg / kg) Same type (4mg / kg) 10 6 REGN5458 (0.4 mg / kg) RPM1-14 (4 mg / kg) 10

[0420] The results demonstrated that the combination of REGN5458 plus PD-1 blockade provided superior anti-tumor efficacy than REGN5458 or PD-1 blockade alone. Specifically, the results demonstrated that the combination of BCMA x CD3 bispecific antibody and anti-PD-1 antibody produced a synergistic therapeutic effect in inhibiting tumor growth on day 21 (the last day of collecting data for all treatment groups) (Table 35, 0.04 mg / kg of BCMA x CD3 and 4 mg / kg of anti-PD-1). Using a two-way ANOVA test on day 21, p<0.0001 between (i) the combination of REGN5458 (0.04 mg / kg) + isotype and REGN5458 (0.04 mg / kg) + anti-PD-1 antibody (Group 3 vs. Group 4); (ii) the combination of anti-PD-1 and REGN5458 (0.04 mg / kg) + anti-PD-1 antibody (Group 2 vs. Group 4); (iii) the combination of anti-PD-1 and REGN5458 (0.4 mg / kg) + anti-PD-1 antibody (Group 2 vs. Group 6). As discussed above in Example 20, increasing the dose of BCMA x CD3 bispecific antibody (0.4 mg / kg) in combination with PD-1 blockade in this experiment produced tumor inhibition similar to that of the lower bispecific antibody dose in combination with PD-1 blockade. Synergy with lower doses of bispecific antibodies was demonstrated to be advantageous because the use of lower doses reduces the risk of any adverse side effects. Similarly, the combination of BCMA x CD3 bispecific antibody and anti-PD-1 antibody demonstrated a synergistic therapeutic effect on the number of tumor-free mice at both doses of bispecific antibody (0.04 mg / kg and 0.4 mg / kg) at the end of the experiment (day 25), as shown in Table 36.

[0421] Table 35: Average tumor size at each time point

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429] Table 36: Tumor-free mice at the end of the experiment

[0430]

[0431] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to the modifications described herein, will become apparent to those skilled in the art from the foregoing description. The modifications are intended to fall within the scope of the appended claims.

Claims

1. An isolated bispecific antigen-binding molecule that binds to human B-cell maturation antigen (BCMA) and human CD3, wherein the bispecific antigen-binding molecule comprises: (a) a first antigen binding domain that specifically binds to human BCMA, wherein the first antigen binding domain comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 68, 70, and 72, respectively, and the LCVR comprises LCDR1, LCDR2, and LCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively; and (b) a second antigen binding domain that specifically binds to human CD3, wherein the second antigen binding domain comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 92, 94, and 96, respectively, and the LCVR comprises LCDR1, LCDR2, and LCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively.

2. An isolated bispecific antigen-binding molecule that binds to human B-cell maturation antigen (BCMA) and human CD3, wherein the bispecific antigen-binding molecule comprises: (a) a first antigen binding domain that specifically binds to human BCMA, wherein the first antigen binding domain comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 68, 70, and 72, respectively, and the LCVR comprises LCDR1, LCDR2, and LCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively; and (b) a second antigen binding domain that specifically binds to human CD3, wherein the second antigen binding domain comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 100, 102, and 104, respectively, and the LCVR comprises LCDR1, LCDR2, and LCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively.

3. The isolated bispecific antigen binding molecule of claim 1, wherein: (a) the first antigen binding domain comprises a HCVR having an amino acid sequence of SEQ ID NO: 66 and a LCVR having an amino acid sequence of SEQ ID NO: 82; and (b) the second antigen binding domain comprises a HCVR having an amino acid sequence of SEQ ID NO: 90 and a LCVR having an amino acid sequence of SEQ ID NO:

82.

4. The isolated bispecific antigen binding molecule of claim 2, comprising: (a) the first antigen binding domain comprises a HCVR having an amino acid sequence of SEQ ID NO: 66 and a LCVR having an amino acid sequence of SEQ ID NO: 82; and (b) the second antigen binding domain comprises a HCVR having an amino acid sequence of SEQ ID NO: 98 and a LCVR having an amino acid sequence of SEQ ID NO:

82.

5. The isolated bispecific antigen binding molecule according to any one of claims 1 to 4, which is a bispecific antibody.

6. The isolated bispecific antigen binding molecule of claim 5, wherein the bispecific antibody comprises a human IgG heavy chain constant region.

7. The isolated bispecific antigen binding molecule of claim 5, wherein the bispecific antibody comprises a chimeric hinge that reduces Fcɣ receptor binding relative to a wild-type hinge of the same isotype.

8. The isolated bispecific antigen binding molecule of claim 6, wherein the human IgG heavy chain constant region is of the isotype IgG1.

9. The isolated bispecific antigen binding molecule of claim 6, wherein the human IgG heavy chain constant region is of the isotype IgG4. 10 . A pharmaceutical composition comprising the bispecific antigen binding molecule according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier or diluent. 11 . A nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antigen binding molecule according to any one of claims 1 to 9.

12. An expression vector comprising the nucleic acid molecule according to claim 11. 13 . A host cell comprising the bispecific antigen binding molecule according to any one of claims 1 to 9 , the nucleic acid molecule according to claim 11 , or the expression vector according to claim 12 .

14. A set of nucleic acid molecules encoding a bispecific antigen binding molecule that binds to human B cell maturation antigen (BCMA) and human CD3, wherein the set of nucleic acid molecules comprises: (a) a first nucleic acid molecule encoding a heavy chain variable (HCVR), wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 68, 70, and 72, respectively; (b) a second nucleic acid molecule encoding a HCVR, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 92, 94, and 96, respectively; and (c) a third nucleic acid molecule encoding a light chain variable region (LCVR), wherein the LCVR comprises LCDR1, LCDR2, and LCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively, The HCVR encoded by the first nucleic acid molecule pairs with the LCVR encoded by the third nucleic acid molecule to form an antigen binding domain that specifically binds to human BCMA; and the HCVR encoded by the second nucleic acid molecule pairs with the LCVR encoded by the third nucleic acid molecule to form an antigen binding domain that specifically binds to human CD3.

15. A set of nucleic acid molecules according to claim 14, wherein: (a) the first nucleic acid molecule encodes a HCVR consisting of the amino acid sequence of SEQ ID NO: 66; (b) the second nucleic acid molecule encodes a HCVR consisting of the amino acid sequence of SEQ ID NO: 90; and (c) the third nucleic acid molecule encodes a LCVR consisting of the amino acid sequence of SEQ ID NO:

82.

16. The set of nucleic acid molecules of claim 14, wherein the bispecific antigen binding molecule is a bispecific antibody, the first nucleic acid molecule encodes a first heavy chain, the second nucleic acid molecule encodes a second heavy chain, and the third nucleic acid molecule encodes a light chain.

17. The set of nucleic acid molecules of claim 16, wherein the first heavy chain, the second heavy chain, or both the first heavy chain and the second heavy chain comprise a human IgG heavy chain constant region.

18. The set of nucleic acid molecules of claim 17, wherein the first heavy chain, the second heavy chain, or both the first heavy chain and the second heavy chain comprise a human IgG1 heavy chain constant region.

19. The set of nucleic acid molecules of claim 17, wherein the first heavy chain, the second heavy chain, or both the first heavy chain and the second heavy chain comprise a human IgG4 heavy chain constant region.

20. The set of nucleic acid molecules according to claim 15, wherein the first nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

65.

21. A set of nucleic acid molecules according to claim 15, wherein the second nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

89.

22. A set of nucleic acid molecules according to claim 15, wherein the third nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

81.

23. A set of nucleic acid molecules encoding a bispecific antigen binding molecule that binds to human B cell maturation antigen (BCMA) and human CD3, wherein the set of nucleic acid molecules comprises: (a) a first nucleic acid molecule encoding HCVR, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 68, 70, and 72, respectively; (b) a second nucleic acid molecule encoding a HCVR, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 100, 102, and 104, respectively; and (c) a third nucleic acid molecule encoding a LCVR, wherein the LCVR comprises LCDR1, LCDR2, and LCDR3 domains consisting of the amino acid sequences of SEQ ID NOs: 84, 86, and 88, respectively, The HCVR encoded by the first nucleic acid molecule pairs with the LCVR encoded by the third nucleic acid molecule to form an antigen binding domain that specifically binds to human BCMA; and the HCVR encoded by the second nucleic acid molecule pairs with the LCVR encoded by the third nucleic acid molecule to form an antigen binding domain that specifically binds to human CD3.

24. A set of nucleic acid molecules according to claim 23, wherein: (a) the first nucleic acid molecule encodes a HCVR consisting of the amino acid sequence of SEQ ID NO: 66; (b) the second nucleic acid molecule encodes a HCVR consisting of the amino acid sequence of SEQ ID NO: 98; and (c) the third nucleic acid molecule encodes a LCVR consisting of the amino acid sequence of SEQ ID NO:

82.

25. The set of nucleic acid molecules of claim 23, wherein the bispecific antigen binding molecule is a bispecific antibody, the first nucleic acid molecule encodes a first heavy chain, the second nucleic acid molecule encodes a second heavy chain, and the third nucleic acid molecule encodes a light chain.

26. The set of nucleic acid molecules of claim 25, wherein the first heavy chain, the second heavy chain, or both the first heavy chain and the second heavy chain comprise a human IgG heavy chain constant region.

27. The set of nucleic acid molecules of claim 26, wherein the first heavy chain, the second heavy chain, or both the first heavy chain and the second heavy chain comprise a human IgGl heavy chain constant region.

28. The set of nucleic acid molecules of claim 26, wherein the first heavy chain, the second heavy chain, or both the first heavy chain and the second heavy chain comprise a human IgG4 heavy chain constant region.

29. A set of nucleic acid molecules according to claim 24, wherein the first nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

65.

30. A set of nucleic acid molecules according to claim 24, wherein the second nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

97.

31. A set of nucleic acid molecules according to claim 24, wherein the third nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

81.

32. A set of nucleic acid molecules encoding a bispecific antibody that binds to human B cell maturation antigen (BCMA) and human CD3, wherein the set of nucleic acid molecules comprises: (a) a first nucleic acid molecule encoding a first heavy chain, wherein the first heavy chain comprises a HCVR consisting of the amino acid sequence of SEQ ID NO: 66; (b) a second nucleic acid molecule encoding a second heavy chain, wherein the second heavy chain comprises a HCVR consisting of the amino acid sequence of SEQ ID NO: 90; (c) a third nucleic acid molecule encoding a light chain, wherein the light chain comprises a LCVR consisting of the amino acid sequence of SEQ ID NO: 82, The HCVR of the first heavy chain encoded by the first nucleic acid molecule is paired with the LCVR of the light chain encoded by the third nucleic acid molecule to form an antigen binding domain that specifically binds to human BCMA; and the HCVR of the second heavy chain encoded by the second nucleic acid molecule is paired with the LCVR of the light chain encoded by the third nucleic acid molecule to form an antigen binding domain that specifically binds to human CD3.

33. A set of nucleic acid molecules encoding a bispecific antibody that binds to human B cell maturation antigen (BCMA) and human CD3, wherein the set of nucleic acid molecules comprises: (a) a first nucleic acid molecule encoding a first heavy chain, wherein the first heavy chain comprises a HCVR consisting of the amino acid sequence of SEQ ID NO: 66; (b) a second nucleic acid molecule encoding a second heavy chain, wherein the second heavy chain comprises a HCVR consisting of the amino acid sequence of SEQ ID NO: 98; (c) a third nucleic acid molecule encoding a light chain, wherein the light chain comprises a LCVR consisting of the amino acid sequence of SEQ ID NO: 82, The HCVR of the first heavy chain encoded by the first nucleic acid molecule is paired with the LCVR of the light chain encoded by the third nucleic acid molecule to form an antigen binding domain that specifically binds to human BCMA; and the HCVR of the second heavy chain encoded by the second nucleic acid molecule is paired with the LCVR of the light chain encoded by the third nucleic acid molecule to form an antigen binding domain that specifically binds to human CD3.

34. An expression vector or a set of expression vectors comprising a set of nucleic acid molecules according to any one of claims 14-22.

35. A host cell comprising the expression vector or a set of expression vectors according to claim 34.

36. A host cell comprising a set of nucleic acid molecules according to claim 32.

37. An expression vector or a set of expression vectors comprising a set of nucleic acid molecules according to any one of claims 23-31.

38. A host cell comprising the expression vector or a set of expression vectors according to claim 37.

39. A host cell comprising a set of nucleic acid molecules according to claim 33.

40. Use of the isolated bispecific antigen binding molecule of any one of claims 1 to 9 or the pharmaceutical composition of claim 10 in the preparation of a medicament for treating multiple myeloma expressing B-cell maturation antigen (BCMA).

41. The use according to claim 40, wherein the medicament is administered in combination with a second therapeutic agent or treatment regimen.

42. The use according to claim 41, wherein the second therapeutic agent or treatment regimen comprises a chemotherapeutic drug, an immunomodulator, a proteasome inhibitor, a histone deacetylase inhibitor, radiotherapy, stem cell transplantation, different bispecific antibodies that interact with different tumor cell surface antigens and T cell or immune cell antigens, antibody drug conjugates, PD-1, PD-L1 or CTLA-4 checkpoint inhibitors or a combination thereof.

43. The use according to claim 41, wherein the second therapeutic agent or treatment regimen comprises a DNA alkylating agent.

44. Use of the isolated bispecific antigen binding molecule of any one of claims 1 to 9 or the pharmaceutical composition of claim 10 in the preparation of a medicament for treating multiple myeloma expressing BCMA in combination with an anti-PD-1 antibody or antigen-binding fragment thereof.

45. The use according to claim 44, wherein the anti-PD-1 antibody or antigen-binding fragment is an anti-PD-1 antibody.

46. ​​The use according to claim 45, wherein the anti-PD-1 antibody is simvastatin.

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