Bispecific antibodies targeting BCMA and CD28

Abstract

The present invention relates to novel humanized BCMA antibodies, bispecific antibodies that specifically bind to BCMA and CD28, methods for producing the same, pharmaceutical compositions containing the same, and methods for using the same.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to novel humanized BCMA antibodies, bispecific antibodies that specifically bind to BCMA and CD28, methods for producing the same, pharmaceutical compositions containing these antibodies, and methods for using the same. [Background technology]

[0002] background Cancer immunotherapy is becoming an increasingly effective therapeutic option, potentially yielding dramatic and sustained responses in cancer types such as melanoma, non-small cell lung cancer, and renal cell carcinoma. This is primarily driven by the success of several immune checkpoint inhibitors, including anti-PD-1 (e.g., Merck's Keytruda; BMS's Opdivo), anti-CTLA-4 (e.g., BMS's Yervoy), and anti-PD-L1 (e.g., Roche's Tecentriq). While these drugs are likely to serve as standard care or as the backbone of combination therapy for many cancer types, only a small percentage of patients (<25%) benefit from such therapies. Furthermore, various cancers (prostate cancer, colorectal cancer, pancreatic cancer, sarcoma, non-triple-negative breast cancer, etc.) exhibit primary resistance to these immunomodulators. Several reports indicate that the absence of pre-existing anti-tumor T cells contributes to the lack of or insufficient response in some patients. In summary, despite the impressive anti-cancer effects of existing immunotherapies, there is a clear medical need to address the large cancer patient population and to develop therapies aimed at inducing and enhancing novel tumor-specific T-cell responses.

[0003] Multiple myeloma (MM) is one of the most common hematological malignancies, affecting approximately 75,000 new patients annually in the EU and the US, and still represents a high level of unmet medical need. Multiple myeloma (also known as plasma cell myeloma) is characterized by terminally differentiated plasma cells that secrete non-functional monoclonal immunoglobulins. When cancerous plasma cells accumulate in the bone marrow, they interfere with the production of normal blood cells, leading to a variety of symptoms and complications. Common symptoms of multiple myeloma include bone pain, particularly in the back or ribs, fatigue, weakness, frequent infections, weight loss, excessive thirst, and increased urination. Treatment options for multiple myeloma depend on a variety of factors, including the stage of the disease or the patient's overall health. In the short term, immunomodulatory agents such as lenalidomide and pomalidomide (ImiDs), as well as proteasome inhibitors such as carfilzomib or bortezomib, may remain the basis of first-line therapy for multiple myeloma (Moreau et al, The Lancet Oncology 2021, 22(3), e105-e118). However, these drugs do not specifically target pathogenic tumor cells, such as pathogenic plasma cells (PCs). Efforts have been made to selectively deplete plasma cells in multiple myeloma. The lack of surface proteins that specifically mark plasma cells has hindered the development of antibody or cell therapies for multiple myeloma. To date, there have been few successful cases of biologics, including daratumumab (anti-CD38) and elotuzumab (anti-CD319), and it should be noted that both antigens are also expressed in other normal tissues, including hematopoietic and immune effector cells, which may limit their long-term clinical use.

[0004] B-cell maturation antigen (BCMA), a transmembrane glycoprotein within the tumor necrosis factor receptor superfamily 17 (TNFRSF17), is expressed at significantly higher levels in all patient MM cells but not in other normal tissues except normal plasma cells. BCMA-chimeric antigen receptor (CAR) T cells have already demonstrated significant clinical activity in patients with relapsed / refractory MM (RRMM) who have received at least three prior therapies, including proteasome inhibitors and immunomodulatory agents (IMiDs). Further modalities, including anti-BCMA antibody-drug conjugates, have also achieved significant clinical responses in patients who have failed at least three prior therapies, including anti-CD38 antibodies, proteasome inhibitors, and immunomodulatory agents (Cho et al, Front Immunolog. 2018, 9, 1821). However, better treatment options for multiple myeloma are still needed.

[0005] CD28 is an established member of a subfamily of costimulatory molecules, characterized by a single transmembrane domain and a cytoplasmic domain containing an important signaling motif, bound to a pair of V-set immunoglobulin superfamily (IgSF) domains (Carreno and Collins, Annu Rev Immunol. 2002, 20, 29-53). Other members of the subfamily include ICOS, CTLA-4, PD1, PD1H, TIGIT, and BTLA (Chen and Flies, Nat Rev Immunol. 2013, 13(4), 227-42). CD28 expression is limited to T cells and is found in most of all naive T cell subsets and antigen-experienced T cell subsets, including subsets expressing PD-1 or CTLA-4. CD28 and CTLA-4 are highly homologous and compete for binding to the same B7 molecules CD80 and CD86, which are expressed on dendritic cells, B cells, macrophages, and tumor cells (Linsley et al., Proc Natl Acad Sci USA. 1990, 87(13), 5031-5). Because CTLA-4 has a higher affinity for B7 family ligands, it preferentially binds to ligands over CD28, enabling suppression of effector T cell responses (Engelhardt et al., J Immunol 2006, 177, 1052-1061). In contrast, PD-1 has been shown to inhibit CD28 signaling by partially dephosphorylating the cytoplasmic domain of CD28 (Hui et al., Science 2017, 355, 1428-1433). Ligation of CD28 by CD80 or CD86 on the surface of professional antigen-presenting cells is strictly required for functional de novo priming of naive T cells, subsequent clonal proliferation, cytokine production, target cell lysis, and long-term memory formation. CD28 ligand binding also promotes the expression of inducible costimulatory receptors such as OX-40, ICOS, and 4-1BB (Acuto and Michel, Nat Rev Immunol 2003, 3, 939-951).During CD28 ligation, disulfide-linked homodimers, the membrane-proximal YMNM motif, and the distal PYAP motif have been shown to form complexes with several kinases and adapter proteins (Boomer and Green, Cold Spring Harb Perspect Biol 2010, 2, a002436). These motifs are important for inducing IL2 transcription mediated by CD28-dependent activation of NFAT, AP-1, and NFκB family transcription factors (Fraser et al., Science 1991, 251, 313-316). However, further under-characterized sites for phosphorylation and ubiquitination are found within the cytoplasmic domain of CD28 (Esensten et al., Immunity 2016, 44, 973-988). As outlined, the CD28-initiated pathway plays a crucial role in promoting the proliferation and effector function of conventional T cells. CD28 ligation also enhances the anti-inflammatory function of regulatory T cells. CD28 co-stimulates T cells by partially enhancing signaling from T cell receptors, but has also been shown to mediate intrinsic signaling events (Acuto and Michel, 2003; Boomer and Green, 2010). Signaling specifically induced by CD28 controls many important aspects of T cell function, including phosphorylation and other post-translational modifications of downstream proteins (e.g., PI3K-mediated phosphorylation), transcriptional changes (e.g., Bcl-xL expression), epigenetic changes (e.g., IL-2 promoter), cytoskeletal remodeling (e.g., microtubule-forming center orientation), and alterations in glycolysis rates (e.g., glycolytic flow rate). CD28-deficient mice exhibit reduced responses to infectious pathogens, allograft antigens, graft-versus-host disease, contact hypersensitivity, and asthma (Acuto and Michel, 2003). The absence of CD28-mediated co-stimulation results in decreased T cell proliferation in vitro and in vivo, severe inhibition of germinal center formation and immunoglobulin isotype class switching, reduced T helper (Th) cell differentiation, and expression of Th2-type cytokines. The CD4-dependent cytotoxic CD8+ T cell response is also affected.Importantly, CD28-deficient naive T cells showed a reduced proliferation response, particularly at lower antigen concentrations. There is growing literature supporting the idea that activating CD28 on T cells has antitumor potential. Recent Devidence has demonstrated that the anticancer effects of PD-L1 / PD-1 and CTLA-4 checkpoint inhibitors are CD28-dependent (Kamphorst et al., Science 2017, 355, 1423-1427). Clinical studies investigating the therapeutic effects of CTLA-4 and PD-1 blockade have shown very promising results in patients with advanced melanoma and other cancers. Furthermore, infusion of genetically engineered T cells expressing an artificial chimeric T cell receptor containing an extracellular antigen recognition domain fused to the intracellular TCR signaling domain (CD3z) and intracellular costimulatory domain (CD28 and / or 4-1BB domain) has shown high response rates and sustained responses in B-cell cancer and other cancers.

[0006] CD28 agonist antibodies can be divided into two categories: (i) CD28 superagonist antibodies and (ii) conventional CD28 agonist antibodies. Normally, activation of naive T cells requires both the involvement of the T cell antigen receptor (TCR, signal 1) and CD28-mediated co-stimulatory signaling (signal 2). CD28 superagonists (CD28SA) are CD28-specific monoclonal antibodies that can autonomously activate T cells without the involvement of the T cell receptor (Hunig, Nat Rev Immunol 2012, 12, 317-318). In rodents, CD28SA activates conventional and regulatory T cells. CD28SA antibodies are therapeutically effective in multiple models of autoimmunity, inflammation, and transplantation. However, a Phase I study of the human CD28SA antibody TGN1412 resulted in a life-threatening cytokine storm in 2006. Follow-up studies suggest that toxicity was caused by drug errors due to differences in CD28 responsiveness between human T cells and T cells in preclinical animal models. Celalizumab (TGN1412 or TAB08) is currently being re-evaluated in open-label, multicenter dose-escalation studies in RA patients and patients with metastatic or unresectable advanced solid malignancies. Conventional agonist antibodies of CD28, e.g., clone 9.3, mimic the native ligand of CD28 and can enhance T cell activation only in the presence of T cell receptor signaling (signal 1). Published findings show that the binding epitope of an antibody significantly influences whether an agonist antibody is a superagonist or a conventional agonist (Beyersdorf et al., Ann. Rheum. Dis. 2005, 64, iv91-iv95). The superagonist TGN1412 binds to the lateral motif of CD28, while conventional agonist molecules bind near the ligand-binding epitope. As a result of the different binding epitopes, superagonist antibodies and conventional agonist antibodies differ in their ability to form linear complexes of CD28 molecules on the surface of T cells. More precisely, TGN1412 can efficiently form a linear array of CD28, which likely results in sufficiently aggregated signaling components to exceed the threshold for T cell activation.On the other hand, the conventional agonist 9.3 results in a complex with a non-linear structure. Attempts to convert conventional agonist conjugates based on the 9.3 clone have been previously published using recombinant bispecific single-chain antibodies against melanoma-associated proteoglycans and CD28 (Otz et al., Leukemia 2009, 23(1), 71-77). The reported bispecific single-chain antibodies have been reported to exhibit "superagonist" activity despite the use of the conventional CD28 agonist conjugate 9.3, based on the inherent tendency of bispecific single-chain antibodies to form multimeric constructs.

[0007] It has been found that combining a limit dose of anti-CD3 bispecific antibody, i.e., a T cell bispecific antibody (TCB), with an agonist anti-CD28 molecule results in better T cell activation. Given that CD28 is expressed at baseline on T cells in various tumor indications, and that activation of CD28 signaling enhances T cell receptor signaling, the combination of TCB molecules and tumor-targeted CD28 molecules may act synergistically to induce a potent and long-lasting antitumor response. International Publication No. 2020 / 127618A1 describes tumor-targeted agonist CD28 antigen-binding molecules, which list various tumor targets.

[0008] CD28 agonism in multiple myeloma may exert different biological functions on immune cells and MM plasma cells, respectively. CD28-mediated co-activation of T cells is expected to drive the antitumor response, while CD28 agonism on MM cells mediates pro-survival signaling through the regulation of PI3K / Akt, FoxO3a, and Bimm, which has been reported to induce chemotherapy resistance in multiple myeloma (Murray et al, Blood 2014, 123(24), 3770-3779). CD28 overexpression on newly diagnosed multiple myeloma plasma cells has been reported to correlate with worse clinical outcomes. However, CD28 activation inhibits myeloma cell proliferation (Bahlis et al., Blood 2007, 109(11), 5002-5010).

[0009] By agonizing CD28 in the presence of strong immune cell-mediated responses, such as T cell bispecific activation, efficient antitumor responses can be further enhanced. The inventors hereby provide a bispecific agonist CD28 antigen-binding molecule that specifically binds to BCMA. Enhancing the T cell response with a CD28 bispecific antibody targeting BCMA on myeloma cells may be an option for improving the treatment of multiple myeloma, and there is a need to provide a BCMA-targeted anti-CD28 antibody with advantageous properties. [Overview of the project]

[0010] summary This invention describes a novel BCMA-targeted bispecific agonist CD28 antigen-binding molecule that achieves tumor-dependent T cell activation and tumor cell death without requiring the formation of multimers. The bispecific CD28 antigen-binding molecule of this invention is characterized by comprising a monovalent binding to CD28 and a specific antigen-binding domain as defined herein that is capable of specific binding to BCMA. Furthermore, the bispecific CD28 antigen-binding molecule has an Fc domain composed of first and second subunits capable of stable association, each containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor. This inhibits Fc receptor-mediated crosslinking, and tumor-specific activation is achieved by crosslinking via the binding of the second antigen-binding domain capable of specific binding to BCMA.

[0011] Therefore, an antibody that specifically binds to B cell maturation antigen (BCMA), wherein the antibody includes a first antigen-binding domain, and the first antigen-binding domain is (i) Heavy chain variable region (V) including the heavy chain complementarity determination region of CDR-H1 (GYTFTNYWMH) of SEQ ID NO: 1, CDR-H2 (IIHPNSGSTNYNEKFQG) of SEQ ID NO: 2, and CDR-H3 (GIYDYPFAY) of SEQ ID NO: 3 H BCMA) and, (ii) Light chain variable region (V L BCMA) (a) A VL containing the light chain complementarity determination region of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASSLQS) of SEQ ID NO: 5, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6, or (b) A VL containing the light chain complementarity determination region of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASNLES) of SEQ ID NO: 7, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6, or (c) An antibody comprising a light chain variable region selected from the group consisting of VLs including the light chain complementarity determining regions of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASNLQS) of SEQ ID NO: 8, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6 is provided herein.

[0012] In one embodiment, an antibody that specifically binds to BCMA, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 (VH1a) and SEQ ID NO: 10 (VH1b), and / or V L An antibody is provided in which BCMA contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 (VL1f), 12 (VL1a), 13 (VL1b), 14 (VL1c), 15 (VL1d), and 16 (VL1e).

[0013] In one embodiment, an antibody that specifically binds to BCMA, wherein the antibody includes a first antigen-binding domain, and the first antigen-binding domain is (a) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 12 L Antibodies containing BCMA are provided.

[0014] In a particular embodiment, the first antigen-binding domain is a Fab molecule.

[0015] In one embodiment, the antibody that specifically binds to BCMA includes an Fc domain composed of a first and a second subunit.

[0016] In one embodiment, the antibody that specifically binds to BCMA includes a second antigen-binding domain that specifically binds to a second antigen, i.e., it is a bispecific antibody.

[0017] In one embodiment, the second antigen-binding domain that specifically binds to the second antigen is a Fab molecule, and the variable domains VL and VH or constant domains CL and CH1 of the Fab light chain and Fab heavy chain, particularly the variable domains VL and VH, are interchangeable.

[0018] In one embodiment, an antibody is provided that specifically binds to BCMA, comprising a first antigen-binding domain that specifically binds to BCMA, wherein the first antigen-binding domain is a Fab molecule, and in the constant domain CL, the amino acid at position 123 (numbered according to the Kabat EU index) is substituted with an amino acid selected from lysine (K), arginine (R), or histidine (H), and the amino acid at position 124 (numbered according to the Kabat EU index) is independently substituted with lysine (K), arginine (R), or histidine (H), and in the constant domain CH1, the amino acid at position 147 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D), and the amino acid at position 213 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index).

[0019] In all of these embodiments, the antibody that specifically binds to BCMA includes an Fc domain, and the Fc domain is IgG, particularly an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain.

[0020] In one aspect, the Fc domain comprises modifications that promote the association of the first and second subunits of the Fc domain. In one aspect, the Fc domain comprises a knob-into-hole modification. In one aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index).

[0021] In a further aspect, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector functions. In a particular aspect, the Fc domain is the Fc domain of the human IgG1 subclass and comprises the amino acid mutations L234A, L235A and P329G (numbering according to the Kabat EU index).

[0022] Further antibodies are provided herein that specifically bind to BCMA and comprise a second antigen-binding domain that specifically binds to CD28.

[0023] In one aspect, the second antigen-binding domain that specifically binds to CD28 comprises a heavy chain variable region (V H CD28) comprising the heavy chain complementarity determining regions of CDR-H1 of SEQ ID NO: 17, CDR-H2 of SEQ ID NO: 18, and CDR-H3 of SEQ ID NO: 19, and a light chain variable region (V L CD28) comprising the light chain complementarity determining regions of CDR-L1 of SEQ ID NO: 20, CDR-L2 of SEQ ID NO: 21, and CDR-L3 of SEQ ID NO: 22.

[0024] In one aspect, the second antigen-binding domain that specifically binds to CD28 comprises a heavy chain variable region (V H CD28) comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region (V L CD28) comprising the amino acid sequence of SEQ ID NO: 24 (v8).

[0025] In a particular aspect, the antibody described herein comprises a V HV containing the amino acid sequences of BCMA and SEQ ID NO: 11 L The first antigen-binding domain contains BCMA, and V contains the amino acid sequence of SEQ ID NO: 23. H V containing the amino acid sequences of CD28 and SEQ ID NO: 24 L It includes a second antigen-binding domain containing CD28.

[0026] In one embodiment, the antibody comprises a first light chain containing the amino acid sequence of SEQ ID NO: 25, a first heavy chain containing the amino acid sequence of SEQ ID NO: 26, a second heavy chain containing the amino acid sequence of SEQ ID NO: 27, and a second light chain containing the amino acid sequence of SEQ ID NO: 28.

[0027] An antibody that specifically binds to B cell maturation antigen (BCMA) and CD28, (A) The first antigen-binding domain is (i) Heavy chain variable region (V H BCMA) (a) VH including the heavy chain complementarity determining regions of CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYADSVKG) of SEQ ID NO: 30, and CDR-H3 (DGYH) of SEQ ID NO: 31, and (b) A heavy chain variable region selected from the group consisting of VH including the heavy chain complementarity determination regions of CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYAAPVKG) of SEQ ID NO: 32, and CDR-H3 (DGYH) of SEQ ID NO: 31, (ii) Light chain variable region (V) including the light chain complementarity determination region of CDR-L1 (RASEDIRNGLA) of SEQ ID NO: 33, CDR-L2 (NANSLHT) of SEQ ID NO: 34, and CDR-L3 (EDTSKYPYT) of SEQ ID NO: 35 L A first antigen-binding domain including BCMA, (B)Antibodies comprising a second antigen-binding domain that specifically binds to CD28 are also provided herein.

[0028] In one embodiment, the antibody includes a first antigen-binding domain that specifically binds to (A)BCMA, and V HBCMA contains an amino acid sequence selected from the group consisting of SEQ ID NO: 36 (VH2a) and SEQ ID NO: 38 (VH1b), and / or V L BCMA contains an amino acid sequence selected from the group consisting of SEQ ID NO: 37 (VL2a) and SEQ ID NO: 39 (VL1a).

[0029] In one embodiment, an antibody that specifically binds to BCMA and CD28, wherein the antibody includes a first antigen-binding domain, and the first antigen-binding domain is (a) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 39 L Antibodies containing BCMA are provided.

[0030] In one embodiment, an antibody is provided that specifically binds to BCMA and CD28, wherein the first antigen-binding domain that binds to BCMA is a Fab molecule.

[0031] In one embodiment, the antibody that specifically binds to BCMA and CD28 includes an Fc domain composed of a first and a second subunit.

[0032] In one embodiment, the second antigen-binding domain that specifically binds to CD28 is a Fab molecule. In one embodiment, the second antigen-binding domain that specifically binds to CD28 is a Fab molecule, and the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain, particularly the variable domains VL and VH, are substituted for each other.

[0033] In one embodiment, an antibody is provided that specifically binds to BCMA and CD28, wherein the first antigen-binding domain that binds to BCMA is a Fab molecule, and in the constant domain CL, the amino acid at position 123 (numbered according to the Kabat EU index) is substituted with an amino acid selected from lysine (K), arginine (R), or histidine (H), and the amino acid at position 124 (numbered according to the Kabat EU index) is independently substituted with lysine (K), arginine (R), or histidine (H), and in the constant domain CH1, the amino acid at position 147 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D), and the amino acid at position 213 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index).

[0034] In all of these embodiments, the antibody that specifically binds to BCMA and CD28 includes an Fc domain, the Fc domain being IgG, particularly an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain.

[0035] In one embodiment, the Fc domain includes a modification that facilitates association between a first subunit and a second subunit of the Fc domain. In one embodiment, the Fc domain includes a knob-into-hole modification. In one embodiment, the first subunit of the Fc domain includes amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain includes amino acid substitutions Y349C, T366S and Y407V (Kabat EU index numbering).

[0036] In a further embodiment, the Fc domain includes one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor. In a particular embodiment, the Fc domain is the Fc domain of the human IgG1 subclass and includes amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index).

[0037] In all of these embodiments, the second antigen-binding domain that specifically binds to CD28 is a heavy chain variable region (V) that includes the heavy chain complementarity determining regions of CDR-H1 of SEQ ID NO: 17, CDR-H2 of SEQ ID NO: 18, and CDR-H3 of SEQ ID NO: 19. H CD28) and the light chain variable region (V) including the light chain complementarity determination region of CDR-L1 of SEQ ID NO: 20, CDR-L2 of SEQ ID NO: 21, and CDR-L3 of SEQ ID NO: 22 L CD28) and includes

[0038] In one particular embodiment, the second antigen-binding domain that specifically binds to CD28 is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 23. H CD28) and the light chain variable region containing the amino acid sequence of SEQ ID NO: 24 (V L Includes CD28).

[0039] In one embodiment, an antibody that specifically binds to BCMA and CD28 comprises a first antigen-binding domain, wherein the first antigen-binding domain contains the amino acid sequence of SEQ ID NO: 36. H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L The first antigen-binding domain contains BCMA, and V contains the amino acid sequence of SEQ ID NO: 23. H V containing the amino acid sequences of CD28 and SEQ ID NO: 24 L An antibody is provided that includes a second antigen-binding domain containing CD28.

[0040] In one embodiment, the antibody that specifically binds to BCMA and CD28 includes a first light chain containing the amino acid sequence of SEQ ID NO: 40, a first heavy chain containing the amino acid sequence of SEQ ID NO: 41, a second heavy chain containing the amino acid sequence of SEQ ID NO: 27, and a second light chain containing the amino acid sequence of SEQ ID NO: 28.

[0041] According to another aspect of the present invention, one or more isolated polynucleotides encoding the antibodies described herein are provided. The present invention further provides vectors, particularly expression vectors, comprising the isolated polynucleotides of the present invention, and host cells comprising the isolated nucleic acids or expression vectors of the present invention. In some aspects, the host cells are eukaryotic cells, particularly mammalian cells. In another aspect, a method is provided for producing antibodies that specifically bind to BCMA or bispecific BCMA antibodies described herein, comprising the steps of a) culturing the host cells described herein under conditions suitable for antibody expression, and optionally b) recovering antibodies that specifically bind to BCMA or bispecific BCMA antibodies. The present invention also encompasses antibodies or bispecific antibodies produced by the method of the present invention.

[0042] Further provided are pharmaceutical compositions comprising an antibody that specifically binds to BCMA as described herein or a bispecific BCMA antibody, and at least one pharmaceutically acceptable additive. In one embodiment, the pharmaceutical composition comprises an additional therapeutic agent.

[0043] Furthermore, the invention also includes antibodies that specifically bind to BCMA, the bispecific BCMA antibodies described herein, or pharmaceutical compositions containing bispecific BCMA antibodies, for use as pharmaceuticals.

[0044] In one embodiment, an antibody that specifically binds to BCMA, or a bispecific BCMA antibody as described herein, or a pharmaceutical composition, is provided for use in (a) activating T cells or (b) enhancing T cell effector function.

[0045] In one embodiment, an antibody that specifically binds to BCMA, or a bispecific BCMA antibody as described herein, or a pharmaceutical composition, is provided for use in the treatment of a disease. In one embodiment, the disease is cancer, in particular multiple myeloma (MM).

[0046] In one particular embodiment, an antibody that specifically binds to BCMA, or the bispecific BCMA antibody described herein, or a pharmaceutical composition, is provided for use in the treatment of cancer, particularly multiple myeloma. In another particular embodiment, an antibody that specifically binds to BCMA, or the bispecific BCMA antibody described herein, is provided for use in the treatment of cancer, the use of which is to be administered in combination with other agents for use in chemotherapeutic agents, radiotherapy and / or cancer immunotherapy. In one embodiment, the antibody that specifically binds to BCMA, or the bispecific BCMA antibody described herein, is for use in the treatment of cancer, the use of which is to be administered in combination with a T-cell activating anti-CD3 bispecific antibody. In one particular embodiment, the T-cell activating anti-CD3 bispecific antibody is an anti-GPRC5D / anti-CD3 antibody.

[0047] In a further embodiment, the present invention provides a method for inhibiting the proliferation of tumor cells in an individual, comprising administering to the individual an effective amount of an antibody that specifically binds to BCMA, or the bispecific BCMA antibody described herein, or a pharmaceutical composition of the present invention, to inhibit the proliferation of tumor cells. In another embodiment, the present invention provides a method for treating or delaying cancer in an individual, comprising administering to the individual an effective amount of an antibody that specifically binds to BCMA, or the bispecific BCMA antibody described herein, or a pharmaceutical composition of the present invention.

[0048] Also provided are the use of an antibody that specifically binds to BCMA or the bispecific BCMA antibody described herein for the manufacture of a pharmaceutical for the treatment of a disease in an individual requiring it, specifically for the manufacture of a pharmaceutical for the treatment of cancer, and a method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising the antibody that specifically binds to BCMA or the bispecific BCMA antibody of the present invention in a pharmaceutically acceptable form. In certain embodiments, the disease is cancer. In any of the above embodiments, the individual is a mammal, in particular a human. [Brief explanation of the drawing]

[0049] [Figure 1] Figures 1A and 1C show schematic diagrams of exemplary molecules described herein. Figure 1A shows a schematic diagram of a CD28 agonist antibody variant as a monovalent hu IgG1 PGLALA isotype ("Fc silent"). Figure 1B shows a 1+1 format bispecific BCMA-CD28 antigen-binding molecule, in which the VH and VL domains are exchanged (VH / VL crossfab) in the Fab molecule containing the CD28 antigen-binding domain, and specific amino acids in the CH1 and CL domains are exchanged (charged variant) in the Fab molecule containing the BCMA antigen-binding domain, enabling better pairing with the light chain. Figure 1C shows a 1+1 format bispecific BCMA-CD28 antigen-binding molecule. In Fab molecules containing the BCMA antigen-binding domain, the VH and VL domains are exchanged (VH / VL crossfab), while in Fab molecules containing the CD28 antigen-binding domain, specific amino acids in the CH1 and CL domains are exchanged (charged variant), enabling better pairing with the light chain. [Figure 2]Figures 2A and 2B show the binding of various BCMA-CD28 bispecific antigen-binding molecules to CD28-expressing CHO cells (CHO-k1-huCD28 cells). All BCMA-CD28 bispecific antigen-binding molecules were evaluated by flow cytometry and were able to bind to human CD28 on CHO-k1-huCD28 cells in a concentration-dependent manner. However, binding to human CD28 did not reach saturation due to the low affinity conjugates, and was comparable between the CD28v8 bispecific antibody (Figure 2A) and the CD28v15 bispecific antibody (Figure 2B), respectively. [Figure 3] Figures 3A and 3B show the binding of various BCMA-CD28 bispecific antigen-binding molecules to BCMA-expressing CHO cells (CHO-huBCMA cells). All BCMA-CD28 bispecific antigen-binding molecules were evaluated by flow cytometry and were able to bind to human BCMA in a concentration-dependent manner. Molecules with BCMA antibody PRs described in International Publication No. 2020 / 127618A1 had lower EC50 values ​​compared to the novel BCMA antibody molecules described herein, but the maximum binding (Emax) was comparable among all molecules tested. Figures 3C–3H show the binding of two BCMA-CD28 bispecific antigen-binding molecules, P1AG7191 and P1AG7207, as well as the BCMA-targeted CD3 T cell engager Alnuctamab, to CHO cells expressing human BCMA with the indicated point mutations. Figure 3C shows binding in the absence of BCMA, as well as human wt BCMA (Figure 3D), and the BCMA variants human BCMA P33S (Figure 3E), human BCMA P343del (Figure 3F), human BCMA R27P (Figure 3G), and human BCMA S30del (Figure 3H). Another BCMA-targeted CD3 T cell engager, Teclistamab, does not bind to two mutant BCMA variants, namely R27P (Figure 3G) and S30del (Figure 3H), while Elranatamab only weakly binds to BCMA with high concentrations of the R27P mutation. None of the bispecific molecules tested bind to BCMA-negative CHOk1 cells (Figure 3C). [Figure 4]Figures 4A–4F show dose-dependent activation of IL2 signaling in Jurkat IL2 reporter cells. Titration volumes (200.0–0.5 nM) of BCMA-CD28 v15 BsAb (Figures 4A–4C) and BCMA-CD28 v8 BsAb (Figures 4D–4F) were added to a mixture of target cells (NCI-H929) and effector cells (Jurkat IL2 reporters) along with three different concentrations (20, 200, and 2000 pM) of GPRC5D-TCB. [Figure 5] Figures 5A–5D provide an overview of the data obtained from the Jurkat NFkB reporter assay. Figure 5A shows the EC50 values, Figure 5B compares the efficacy of various BCMA-CD28 v15 BsAbs and untargeted CD28 v15 controls, Figure 5C shows the EC50 values, and Figure 5D compares the efficacy of various BCMA-CD28 v8 BsAbs and untargeted CD28 controls. Data were calculated from three independent experiments conducted in triplicate. The EC50 data in Figures 5A and 5C were aggregated from experiments using different concentrations of GPRC5D-TCB, and the same processing was applied to the efficacy data. All data are presented as mean ± standard deviation. [Figure 6] Figures 6A and 6B demonstrate that dose-dependent activation of CD8+ T cells via BCMA-CD28 v15 bispecific antibody (BsAb) occurs only in the presence of a primary signal (TCB) and BCMA expression. BCMA-expressing MM cell line NCI-H929 (Figure 6A) was co-cultured with healthy donor PBMCs (1:1 ratio). Alternatively, NCI-H929 BCMAko cells (knockout cells that do not express BCMA) were used as target cells (Figure 6B). The co-cultures were treated with GPRC5D-TCB (providing the primary signal) alone, or in combination with BCMA-CD28 BsAb or untargeted CD28 (negative reference). These were titrated from 500 nM to 0.03 nM (1:4 dilution step) and incubated for 4 days. Controls were left untreated or treated with BCMA-CD28 BsAb alone (three highest concentrations). The data is presented as the mean ± standard deviation of a triple series for one donor (representative of the three donors tested; BCMA ko N=1). [Figure 7] Figures 7A and 7B show dose-dependent activation of CD8+ T cells mediated by BCMA-CD28 v8 bispecific antibody (BsAb). Activation occurs only in the presence of a primary signal (TCB) and BCMA expression. BCMA-expressing MM cell line NCI-H929 (Figure 7A) was co-cultured with healthy donor PBMCs (1:1 ratio). Alternatively, NCI-H929 BCMAko cells (knockout cells that do not express BCMA) were used as target cells (Figure 7B). The co-cultures were treated with GPRC5D-TCB (providing the primary signal) alone, or in combination with BCMA-CD28 BsAb or untargeted CD28 (negative reference). These were titrated from 500 nM to 0.12 nM (1:4 dilution step) and incubated for 4 days. Controls were left untreated or treated with BCMA-CD28 BsAb alone. The data is presented as the mean ± standard deviation of a triple series for one donor (representative of the three donors tested; BCMA ko N=1). [Figure 8] Figures 8A and 8B demonstrate that dose-dependent activation of CD4+ T cells via BCMA-CD28 v8 bispecific antibody (BsAb) occurs only in the presence of a primary signal (TCB) and BCMA expression. BCMA-expressing MM cell line NCI-H929 (Figure 8A) was co-cultured with healthy donor PBMCs (1:1 ratio). Alternatively, NCI-H929 BCMAko cells (knockout cells that do not express BCMA) were used as target cells (Figure 8B). The co-cultures were treated with GPRC5D-TCB (providing the primary signal) alone, or in combination with BCMA-CD28 BsAb or untargeted CD28 (negative reference). These were titrated from 500 nM to 0.12 nM (1:4 dilution step) and incubated for 4 days. Controls were left untreated or treated with BCMA-CD28 BsAb alone (three highest concentrations). The data is presented as the mean ± standard deviation of a triple series for one donor (representative of the three donors tested; BCMA ko N=1). [Figure 9]Figures 9A and 9B show dose-dependent proliferation of CD4+ T cells mediated by BCMA-CD28 v8 bispecific antibody (BsAb). Proliferation occurs only in the presence of the first signal (TCB) and BCMA expression. BCMA-expressing MM cell line NCI-H929 (Figure 9A) was co-cultured with healthy donor PBMCs (1:1 ratio). Alternatively, NCI-H929 BCMAko cells (knockout cells that do not express BCMA) were used as target cells (Figure 9B). The co-cultures were treated with GPRC5D-TCB (providing the first signal) alone, or in combination with BCMA-CD28 BsAb or untargeted CD28 (negative reference). These were titrated from 500 nM to 0.12 nM (1:4 dilution step) and incubated for 4 days. Controls were left untreated or treated with BCMA-CD28 BsAb alone. The data is presented as the mean ± standard deviation of a triple series for one donor (representative of the three donors tested; BCMA ko N=1). [Figure 10] Figure 10 shows the results of an exemplary ex vivo test using bone marrow samples from primary MM patients described in Example 4.3. T cell activation was measured by flow cytometry, evaluating the upregulation of CD25 on CD8+ T cells when incubated with 1 nM GPRC5D-TCB for 96 hours, in or in the absence of a 200 nM indicated BCMA-CD28 bispecific antibody or a non-targeted negative reference molecule. All data shown refer to single-tube measurements for each condition. [Figure 11] Figure 11 shows the results of an exemplary ex vivo test using bone marrow samples from a primary MM patient. Tumor cell lysis (TCL) was measured by flow cytometry, where maleimide-positive cells with low FSCs were defined as dead cells, after incubation with 1 nM GPRC5D-TCB for 96 hours in the absence or presence of 400 nM indicated BCMA-CD28 bispecific antibody or a non-targeted negative reference molecule. All data shown refer to single-tube measurements for each condition. [Figure 12]Figures 12A–12D show the results of ex vivo experiments using bone marrow samples from patients with primary MM. T cell activation and degranulation were measured by flow cytometry to evaluate the upregulation of CD107A (see Figures 12C and 12D) on CD4+ (see Figures 12A and 12C) or CD8+ (see Figures 12B and 12D) T cells, respectively, after incubation with 10 nM GPRC5D-TCB for 96 hours in the absence or presence of 200 nM indicated BCMA-CD28 bispecific antibody or reference molecule. [Figure 13] Figures 13A and 13B show the results of ex vivo studies using BM MNCs from bone marrow samples of primary MM patients. Tumor cell lysis and T cell activation were measured by flow cytometry to assess the percentage of viable MM PCs (Figure 13A) or the upregulation of CD25 on CD8+ T cells (Figure 13B) after incubation with 0.01 nM GPRC5D-TCB for 96 hours in the absence or presence of 800 nM indicated BCMA-CD28 bispecific antibody or reference molecule. [Figure 14] Figures 14A to 14F show the results of in vivo experiments testing the efficacy of GPRC5D x CD3 monotherapy and its combination with BCMA-CD28 bispecific antibodies (10 mg / kg P1AG7215 and 10 mg / kg P1AG7282) in an NCI-H929 tumor model. Figures 14A to 14D show tumor growth inhibition in single animals, and Figure 14E shows the median (+ / -IQR) tumor volume per treatment group. Animals with a final tumor burden lower than the size at the start of treatment were defined as responders. The tumor volume at the end of the study (day 41) is shown for the GPRC5D x CD3 monotherapy group and the combination of both, and is shown as the median (+ / -IQR) in Figure 14F. [Figure 15]Figures 15A to 15L show the results of in vivo experiments testing the efficacy of GPRC5D x CD3 monotherapy and combinations with BCMA-CD28 var8 bispecific antibody P1AG7215 (20 mg / kg, 10 mg / kg, 5 mg / kg), BCMA-CD28 var15 bispecific antibody P1AG7282 (20 mg / kg, 10 mg / kg, 2 mg / kg), and BCMA(PR)-CD28 bispecific antibodies P1AE9053 (10 mg / kg) and P1AF7062 (10 mg / kg) in the NCI-H929 tumor model. Tumor growth inhibition is shown as a median (+ / -IQR) per treatment group in single animals (Figures 15A to 15J) (Figure 15K). Animals with a final tumor burden smaller than the size at the start of treatment were defined as responders. Statistical significance is indicated by an asterisk. In the NCI-H929 tumor model, the median (+ / -IQR) (C) tumor volume at the end of the study (day 40) is shown for the GPRC5D×CD3 monotherapy group and the combination therapy group. Figures 15A to 15J show tumor growth inhibition in single animals, and Figure 15K shows the median (+ / -IQR) tumor volume per treatment group. Animals with a final tumor burden lower than the size at the start of treatment were defined as responders. The median (+ / -IQR) tumor volume at the end of the study (day 41) is shown as the median (+ / -IQR) in Figure 15L for the GPRC5D×CD3 monotherapy group and the combination therapy group. [Figure 16]Figures 16A to 16G show the results of in vivo experiments testing the efficacy of GPRC5D×CD3 monotherapy (1 mg / kg) and combinations with BCMA-CD28 (20 and 30 mg / kg), P1AG7207 (20 and 30 mg / kg), and P1AG7191 (20 mg / kg), respectively, in the NCI-H929 tumor model. Figures 16A to 16E show tumor growth inhibition in single animals. Figure 16F shows the median (+ / -IQR) tumor volume per treatment group. Animals with a final tumor burden lower than the size at the start of treatment were defined as responders. Treatment with the combination of GPRC5D×CD3 and 20 mg / kg of P1AG7207 resulted in 4 responders, while treatment with the combination of GPRC5D×CD3 and 30 mg / kg of P1AG7207 resulted in 5 responders. Two responders were observed in the group treated with GPRC5D×CD3 and 20 mg / kg of P1AG7191. In Figure 16G, the tumor weight at the end of the study (day 57) is shown as the median + / - IQR for the GPRC5D×CD3 monotherapy group and the combination therapy group. [Figure 17] Figures 17A–17G show the results of in vivo experiments testing the efficacy of GPRC5D × CD3 monotherapy and combination therapy with BCMA-CD28 bispecific antibodies P1AG7191 (40, 10, and 1 mg / kg) and P1AE9053 (10 mg / kg) in an NCI-H929 tumor model. Tumor growth inhibition is shown as median (+ / -IQR) per treatment group (Figure 17A) and per animal (Figures 17B–17G). Animals with a final tumor burden below the size at the start of treatment were defined as responders. The two highest doses of P1AG7191 (40 and 10 mg / kg) delayed the time to tumor recurrence and inhibited tumor regrowth compared to monotherapy. [Figure 18] Figure 18 shows a comparison of the plasma concentration-time profiles of bispecific antibodies P1AG7282, P1AG7215, P1AG7191, and P1AG7207 measured in HuFcRN transgenic mice (PK test). [Figure 19]Figure 19 shows a comparative heatMAPPS representation of the results observed in the MAPPs assay (Example 6.3.1) for the bispecific antibodies P1AG7282, P1AG7215, P1AG7191, and P1AG7207. Clusters observed in each domain are highlighted. [Modes for carrying out the invention]

[0050] Detailed description of the invention definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly used in the art to which this invention pertains. In interpreting this specification, the following definitions apply, wherever singular terms are used, including their plural forms and vice versa.

[0051] As used herein, the term “antigen-binding molecule” refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules include antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and scaffold antigen-binding proteins.

[0052] As used herein, the terms “antigen-binding domain that binds to a tumor-associated antigen” or “a portion capable of specific binding to a tumor-associated antigen” refer to polypeptide molecules that specifically bind to the tumor-associated antigen BCMA. In one embodiment, the antigen-binding domain can activate BCMA-mediated signaling. In certain embodiments, the antigen-binding domain can direct the entity to which it binds (e.g., a CD28 antibody) to BCMA-expressing cells, such as certain types of tumor cells. Antigen-binding domains capable of specific binding to BCMA include antibodies and their fragments, as further defined herein. Furthermore, antigen-binding domains capable of specific binding to a tumor-associated antigen may include scaffold antigen-binding proteins, as further defined herein, such as binding domains based on designed repeat proteins or designed repeat domains (see, for example, International Publication No. 2002 / 020565).

[0053] With respect to antigen-binding molecules, i.e., antibodies or fragments thereof, the term “antigen-binding domain” refers to a portion of a molecule that contains a region that specifically binds to some or all of an antigen and is complementary to some or all of the antigen. An antigen-binding domain capable of specific antigen binding may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Specifically, an antigen-binding domain capable of specific antigen binding includes antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH). In another embodiment, an “antigen-binding domain capable of specific binding to tumor-associated antigens” may be a Fab fragment or a crossFab fragment. As used herein, the terms “first,” “second,” or “third” with respect to antigen-binding domains, etc., are used for convenience to distinguish when there are two or more parts of each type. The use of these terms is not intended to assign a particular order or orientation of parts unless expressly stated.

[0054] The term "antibody" as used herein is used in its broadest sense and encompasses a wide range of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0055] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous collection of antibodies, i.e., the individual antibodies constituting the collection are identical and / or bind to the same epitope, except for possible variant antibodies that contain, for example, naturally occurring mutations or arise during the production of the monoclonal antibody preparation, the presence of such variants generally in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on an antigen.

[0056] As used herein, the term “monospecific” antibody means an antibody having one or more binding sites, each binding to the same epitope of the same antigen. The term “bispecific” means that an antigen-binding molecule can specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule consists of two antigen-binding sites, each specific to a different antigenic determinant. However, a bispecific antigen-binding molecule may further include an antigen-binding site that binds to an additional antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells or on the same cell. Thus, the term “bispecific” according to the present invention may also include a trispecific molecule, such as a bispecific molecule comprising a CD28 antibody and two antigen-binding domains directed to two different target cell antigens.

[0057] As used herein, the term "~valent" indicates that an antigen-binding molecule that is specific to one antigenic determinant contains a specific number of binding sites that are specific to one antigenic determinant. Thus, the terms "divalent," "tetravalent," and "hexavalent" indicate that an antigen-binding molecule contains two, four, and six binding sites that are specific to a given antigenic determinant, respectively. In certain embodiments of the present invention, the bispecific antigen-binding molecule according to the present invention may be monovalent to a given antigenic determinant, i.e., have only one binding site to the antigenic determinant, or may be divalent or tetravalent to a given antigenic determinant, i.e., have two or four binding sites to the antigenic determinant, respectively.

[0058] In this specification, the terms “full-length antibody,” “complete antibody,” and “whole antibody” are used interchangeably to refer to antibodies having a structure substantially similar to that of a native antibody. “Native antibody” refers to naturally occurring immunoglobulin molecules with a variety of structures. For example, a native IgG class antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant domains. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a light chain constant domain (CL), also called light chain constant domains. The heavy chain of an antibody may be classified into one of five types called α(IgA), δ(IgD), ε(IgE), γ(IgG), or μ(IgM), some of which may be further divided into subtypes such as γ1(IgG1), γ2(IgG2), γ3(IgG3), γ4(IgG4), α1(IgA1), and α2(IgA2). The light chain of an antibody may be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of its constant domain.

[0059] An "antibody fragment" refers to a molecule other than a complete antibody, including a part of a complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, crossFab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and single-domain antibodies. For an overview of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For an overview of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994). See also International Publication No. 93 / 16185 and U.S. Patents No. 5,571,894 and 5,587,458. For Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues with increased in vivo half-lives, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments having two antigen-binding sites that may be bivalent or bispecific; see, for example, European Patent No. 404,097; International Publication No. 1993 / 01161; Hudson et al., Nat Med 9,129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90,6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA; e.g., U.S. Patent No. 6,248,516B1).Antibody fragments can be produced by a variety of techniques, including, but not limited to, the proteolytic digestion of complete antibodies as described herein, as well as production by recombinant host cells (e.g., E. coli or phages).

[0060] When a complete antibody is digested with papain, two identical antigen-binding fragments called "Fab" fragments are produced, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Therefore, as used herein, the terms "Fab fragment" or "Fab molecule" refer to the light chain fragment containing the variable light chain (VL) domain and the constant domain of the light chain (CL), and the antibody fragment containing the variable heavy chain (VH) domain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues, including one or more cysteines from the antibody hinge region, are added to the carboxyl terminus of the heavy chain CH1 domain. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domain possess free thiol groups. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. A "conventional Fab fragment" consists of a VL-CL light chain and a VH-CH1 heavy chain.

[0061] The terms "crossFab fragment," "xFab fragment," or "crossover Fab fragment" refer to Fab fragments in which either the variable or constant regions of the heavy and light chains are exchanged. Two different chain compositions are possible for crossover Fab molecules and are included in the bispecific antibodies of the present invention. In one case, the variable regions of the Fab heavy and light chains are replaced; that is, the crossover Fab molecule includes a peptide chain composed of a light chain variable (VL) domain and a heavy chain constant domain (CH1), and a peptide chain composed of a heavy chain variable (VH) domain and a light chain constant domain (CL). This crossover Fab molecule is called CrossFab. (VLVH)It is also called [another name]. On the other hand, when the constant regions of the Fab heavy chain and light chain are replaced, the crossover Fab molecule includes a peptide chain composed of a heavy chain variable domain (VH) and a light chain constant domain (CL), and a peptide chain composed of a light chain variable domain (VL) and a heavy chain constant domain (CH1). This crossover Fab molecule is called CrossFab. (CLCH1) It is also called [another name].

[0062] A "single-stranded Fab fragment" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following sequences from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL, and the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-stranded Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab molecules will be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (for example, at position 44 of the variable heavy chain and position 100 of the variable light chain, according to Kabat numbering).

[0063] A "crossover single-strand Fab fragment" or "x-scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following sequences from N-terminus to C-terminus: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL, where VH and VL together form an antigen-binding site that specifically binds to a certain antigen, and the linker is a polypeptide of at least 30 amino acids. Furthermore, these x-scFab molecules will be further stabilized by the generation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain according to Kabat numbering).

[0064] A "single-chain variable fragment (scFv)" is a linkage of the antibody's heavy chain (V) using a short linker peptide of 10 to approximately 25 amino acids. H ) and light chain (V L It is a fusion protein of the variable region of ). The linker is usually rich in glycine for flexibility, and also rich in serine or threonine for solubility, V H The N-terminus and V L It is possible to link the C-terminus of the protein to the VH domain, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. Furthermore, the antibody fragment is composed of a single-chain polypeptide characterized by the ability to associate with the VL domain, i.e., with the VH domain, or to associate with the VL domain, i.e., with the VH domain and bind to a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0065] "Scaffold antigen-binding proteins" are known in the art, and for example, fibronectin and engineered ankyrin repeat proteins (DARPin) have been used as alternative scaffolds for antigen-binding domains. See, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13:695-701 (2008). In one aspect of the present invention, the scaffold antigen-binding protein is CTLA-4 (epibody), lipocalin (anticalin), protein A-derived molecules, such as the Z-domain (afibody), A-domain (avimer / maxibody), serum transferrin (transbody); designed ankyrin repeat protein (DARPin), variable domain of antibody light or heavy chain (single-domain antibody, sdAb), variable domain of antibody heavy chain (nanobody, aVH), V NAR Fragment, fibronectin (adonectin), C-type lectin domain (tetranectin); variable domain (V) of a novel antigen receptor beta-lactamase. NAR Fragments), human gamma-crystallin or ubiquitin (affiliin molecules); selected from the group consisting of Knitz-type domains and microbodies of human protease inhibitors, such as Nottin family proteins, peptide aptamers and fibronectin (adonectin). CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is mainly CD4 +It is a CD28 family receptor expressed on T cells. Its extracellular domain has a variable domain-like Ig folding. The loop corresponding to the antibody's CDR can be substituted with heterologous sequences to give different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as organisms (e.g., U.S. Patent No. 7166697B1). Organisms are approximately the same size as the isolated variable region of an antibody (e.g., a domain antibody). For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipocalin is a family of extracellular proteins that carry small hydrophobic molecules such as steroids, pyrine, retinoids, and lipids. Lipocalin has a rigid beta-sheet secondary structure with many loops at the open end of a conical structure, and these loops can be engineered to bind to different target antigens. Antikarin is 160-180 amino acids in size and is derived from lipocalin. For further details, see Biochim Biophys Acta 1482:337-350 (2000), U.S. Patent No. 7,250297 B1, and U.S. Patent Publication No. 20070224633. Affibodies are scaffolds derived from protein A of Staphylococcus aureus and can be manipulated to bind to antigens. The domain consists of three helical bundles of approximately 58 amino acids. Libraries are generated by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 2004, 17, 455-462 and European Patent No. 1641818 A1. Avimers are multi-domain proteins derived from the A-domain scaffold family. The native domain of approximately 35 amino acids adopts a defined disulfide-bonded structure. Diversity is generated by shuffling the innate variations shown by the A-domain family. For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). Transferrin is a monomeric serum transport glycoprotein.Transferrin can be engineered to bind to different target antigens by inserting peptide sequences into permissible surface loops. An example of an engineered transferrin scaffold is a transbody. For further details, see J. Biol. Chem 274, 24066-24073 (1999). Engineered ankyrin repeat proteins (DARPin) are derived from ankyrin, a family of proteins that mediate the binding of intrinsic membrane proteins in the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha helices and a beta turn. A single ankyrin repeat can be engineered to bind to different target antigens by randomizing the residues in the first alpha helix and beta turn of each repeat. Its binding interface can be increased by increasing the number of modules (affinity maturation method). For further details, see J.Mol.Biol.332,489-503(2003), PNAS100(4),1700-1705(2003), and J.Mol.Biol.369,1015-1028(2007) and U.S. Patent Application Publication No. 20040132028A1. A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. The first single domain is derived from a variable domain of a camel-derived antibody heavy chain (nanobody or V). H (H fragment). Furthermore, the term single-domain antibody refers to an autonomous human heavy chain variable domain (aVH) or shark-derived V. NARThis includes fragments. Fibronectin is a scaffold that can be engineered to bind to antigens. Adnectin consists of a scaffold with the native amino acid sequence of the 10th domain of 15 repeat units of human fibronectin type III (FN3). Three loops at one end of the beta-sandwich can be engineered so that adnectin can specifically recognize the therapeutic target of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), U.S. Patent No. 20080139791, International Publication No. 2005056764, and U.S. Patent No. 6818418B1. Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop inserted into the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). The microbodies are derived from naturally occurring microproteins with a length of 25–50 amino acids and containing 3–4 cysteine ​​crosslinks. Examples of microproteins include KalataBI, conotoxin, and Nottin. The microproteins have loops that can be manipulated to contain up to 25 amino acids without affecting the overall folding of the microprotein. For further details on the manipulated Nottin domain, see International Publication No. 2008098796.

[0066] A reference molecule and an antibody that "binds to the same epitope" refer to an antigen-binding molecule that inhibits the binding of the reference molecule to its antigen by 50% or more in a competitive assay, and conversely, the reference molecule inhibits the binding of the antigen-binding molecule to its antigen by 50% or more in a competitive assay.

[0067] The term "antigen-binding domain" refers to a portion of an antigen-binding molecule that specifically binds to part or all of an antigen and contains a region complementary to part or all of the antigen. When the antigen is large, the antigen-binding molecule may bind to only a specific portion of the antigen, which is called an epitope. The antigen-binding domain may be provided, for example, by one or more variable domains (also called variable regions). Preferably, the antigen-binding domain includes an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0068] As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope” and refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds, forming an antigen-antigen complex (e.g., a three-dimensional structure consisting of a continuous amino acid region or discontinuous amino acids from different regions). Useful antigenic determinants may be found, for example, on the surface of tumor cells, virus-infected cells, other diseased cells, immune cells, free bodies in serum, and / or in the extracellular matrix (ECM). Proteins useful as antigens herein may be any native form of protein derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. In certain embodiments, the antigen is a human protein. When referring to a specific protein of the present invention, the term encompasses “full-length” untreated proteins and any form of protein obtained from cell treatment. The term also encompasses naturally occurring protein variants, such as splice variants or allele variants.

[0069] "Specific binding" means that the binding is selective for the antigen and can be distinguished from undesirable or nonspecific interactions. The ability of an antigen-binding molecule to bind to a particular antigen can be measured by enzyme-linked immunosorbent assay (ELISA), or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed with a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree of binding of an antigen-binding molecule to an unrelated protein is less than about 10% of the binding of the antigen-binding molecule to the antigen as measured, for example, by SPR. In certain embodiments, the molecule that binds to the antigen is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (Kd) of M.

[0070] "Affinity" or "binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding pair (e.g., an antigen). Unless otherwise stated herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of the binding pair (e.g., antibody and antigen). The affinity of molecule X for its binding pair Y can generally be expressed by its dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constants (koff and kon, respectively). Thus, equivalent affinities may include different rate constants, as long as the ratio of the rate constants is the same. Affinity can be measured by common methods known in the art, including those described herein. A specific method for measuring affinity is surface plasmon resonance (SPR).

[0071] As used herein, “activated T cell antigen” refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, that can induce T cell activation upon interaction with an antibody. Specifically, the interaction of an antibody with an activated T cell antigen can induce T cell activation by triggering a cascade of signaling in the T cell receptor complex. In certain embodiments, the activated T cell antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt number P07766 (version 189), NCBI RefSeq number NP_000724.1, SEQ ID NO. 167 for the human sequence, or UniProt number Q95LI5 (version 49), NCBI GenBank number BAB71849.1, SEQ ID NO. 168 for the cynomolgus monkey [Macaca fascicularis] sequence).

[0072] As used herein, “T cell activation” refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and are described herein.

[0073] The term "T cell effector function" refers to the activity of T cells, which play a crucial role in the adaptive immune system. T cells play a role in initiating and regulating the body's immune response to foreign invaders such as viruses or bacteria and tumor cells. Effector function refers to the various activities performed by T cells to eliminate these invaders, including the release of cytokines, stimulation of other cells, and the direct attack and elimination of infected cells.

[0074] As used herein, “tumor-associated antigen” or TAA refers to an antigenic determinant presented on the surface of target cells, such as cancer cells, tumor stromal cells, malignant B lymphocytes, or melanoma cells within a tumor. In certain embodiments, the target cell antigen is an antigen on the surface of a tumor cell. In a particular embodiment, the TAA is a BCMA.

[0075] The term "BCMA" refers to the B cell maturation antigen, also known as tumor necrosis factor receptor superfamily member 17 (TNFRS17) or CD269. It is a type III transmembrane protein lacking a signal peptide and containing a cysteine-rich extracellular domain. BCMA ligands include B cell activator (BAFF) and proliferation-inducing ligand (APRIL), with APRIL exhibiting higher affinity for BCMA. BCMA is preferentially expressed by mature B lymphocytes, with minimal expression in hematopoietic stem cells or non-hematopoietic tissues, and is essential for the survival of long-lived myeloid plasma cells. Membrane-bound BCMA undergoes gamma-secretase-mediated shedding from the cell surface, potentially leading to reduced circulation of soluble BCMA (sBCMA) and decreased activation of surface BCMA by APRIL and BAFF. BCMA is significantly overexpressed in all patient MM cells but not in other normal tissues except normal plasma cells. BCMA, along with two related TNFR superfamily B cell activator receptors (BAFF-R) and transmembrane activators, calcium regulators, and cyclophyllin ligand interactors (TACIs), critically regulates B cell proliferation and survival, as well as maturation and differentiation into plasma cells. These three functionally related receptors support the long-term survival of B cells at different stages of development by binding to their congener ligands, BAFF and / or APRIL. As used herein, BCMA refers to any BCMA protein derived from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human BCMA is shown in UniProt (www.uniprot.org) accession number Q02223 (SEQ ID NO: 99).

[0076] "CD28" (differentiation cluster 28, Tp44) refers to any CD28 protein derived from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. CD28 is expressed in T cells and provides co-stimulatory signals necessary for T cell activation and survival. In addition to the T cell receptor (TCR), CD28-mediated T cell stimulation can provide potent signals for the production of various interleukins. CD28 is a receptor for the CD80 (B7.1) and CD86 (B7.2) proteins and is the only B7 receptor constitutively expressed in naive T cells. The amino acid sequence of human CD28 is shown in UniProt (www.uniprot.org) accession number P10747 (SEQ ID NO: 100).

[0077] An "agonist antibody" refers to an antibody that possesses agonist function against a given receptor. Generally, when an agonistic ligand (factor) binds to a receptor, the tertiary structure of the receptor protein changes, and the receptor is activated (if the receptor is a membrane protein, this usually transmits cell proliferation signals, etc.). If the receptor is of the type that forms dimers, an agonist antibody can dimerize the receptor at the appropriate distance and angle, and therefore can exert the same effect as the ligand. A suitable anti-receptor antibody can mimic the receptor dimerization performed by the ligand and therefore can be an agonist antibody.

[0078] A "CD28 agonist antibody" or "conventional CD28 agonist antibody" is an antibody that mimics the CD28 natural ligand (CD80 or CD86), which plays a role in enhancing T cell activation in the presence of T cell receptor signaling ("signal 2"). Two signals are required for complete T cell activation. Under physiological conditions, "signal 1" arises from the interaction between the T cell receptor (TCR) molecule and the peptide / major histocompatibility complex (MHC) on antigen-presenting cells (APCs), while "signal 2" is brought about by engagement with a costimulatory receptor, such as CD28. CD28 agonist antibodies can co-stimulate T cells (signal 2). Furthermore, when combined with molecules that have specificity for the TCR complex, they can induce T cell proliferation and cytokine secretion, but CD28 agonist antibodies cannot completely activate T cells without additional stimulation of the TCR. However, there is a subclass of CD28-specific antigen-binding molecules known as so-called CD28 superagonist antibodies. A "CD28 superagonist antibody" is a CD28 antibody that can completely activate T cells without additional stimulation of the TCR. CD28 superagonist antibodies can induce T cell proliferation and cytokine secretion without prior activation of T cells (Signal 1).

[0079] The term "variable domain" or "variable region" refers to a domain in the antibody heavy or light chain involved in the binding of antigen-binding molecules to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, and each domain contains four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th, WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0080] As used herein, the terms “hypervariable region” or “HVR” refer to each of the regions of antigen-binding variable domains within a sequence that are hypervariable and determine antigen-binding specificity, such as “complementarity-determining regions” (CDRs). Generally, an antigen-binding domain contains six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Illustrative CDRs as used herein include: (a) Hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs present in amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J.Mol.Biol.262:732-745 (1996)).

[0081] Unless otherwise specified, the CDR is determined according to Kabat et al., as described above. Those skilled in the art will understand that the notation of the CDR can be determined according to Chothia, McCallum, or any other scientifically recognized nomenclature. Kabat et al. also defined a numbering system for variable region sequences applicable to any antibody. Those skilled in the art can clearly assign this system of “Kabat numbering” to any variable region sequence without relying on experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system described in Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). Unless otherwise specified, references to the numbering of the positions of specific amino acid residues in the antibody variable region follow the Kabat numbering system.

[0082] As used herein, the term “affinity maturation” in the context of antigen-binding molecules (e.g., antibodies) refers to an antigen-binding molecule derived from a reference antigen-binding molecule, which binds to the same antigen as the reference antibody, preferably to the same epitope, and has a higher affinity for the antigen than the reference antigen-binding molecule, for example, through mutation. Affinity maturation generally involves modification of one or more amino acid residues in one or more CDRs of the antigen-binding molecule. Typically, an affinity-matured antigen-binding molecule binds to the same epitope as the original reference antigen-binding molecule.

[0083] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences generally appear in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0084] For the purposes of this specification, “acceptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived” from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0085] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0086] The "class" of an antibody refers to the type of constant domain or constant region contained in its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0087] A “humanized” antibody refers to a chimeric antibody containing amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the HVR (e.g., CDR) corresponds to those of a non-human antibody, and all or substantially all of the FR corresponds to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. “Humanized” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of “humanized antibodies” as encompassed in the present invention are those in which the constant region has been further modified or altered from the constant region of the original antibody to produce the properties according to the present invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0088] A “human” antibody is one that is produced by a human or human cell, or has an amino acid sequence corresponding to the amino acid sequence of an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues. In particular, a “human” antibody or “humanized” antibody includes a human-derived constant region containing human CH1, CH2, CH3 and / or CL domains, especially the constant region of an IgG isotype, more specifically the constant region of an IgG1 isotype.

[0089] The term "CL domain" refers to the constant portion of an antibody light chain polypeptide. Exemplary sequences of human constant domains are shown in SEQ ID NOs: 101 and 102 (human kappa and lambda CL domains, respectively).

[0090] The term "CH1 domain" refers to the portion of the antibody heavy chain polypeptide that extends from approximately EU position 118 to EU position 215 (Kabat's EU numbering system). In one embodiment, the CH1 domain has the amino acid sequence ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV (SEQ ID NO: 103). Typically, the segment having the amino acid sequence EPKSC (SEQ ID NO: 104) then ligates the CH1 domain to the hinge region.

[0091] The term "hinge region" refers to the portion of the antibody heavy chain polypeptide that binds the CH1 and CH2 domains in the wild-type antibody heavy chain, for example, from approximately position 216 to approximately position 230, or from approximately position 226 to approximately position 230, based on the Kabat EU numbering system. Hinge regions of other IgG subclasses can be determined by aligning them with hinge region cysteine ​​residues of the IgG1 subclass sequence. Hinge regions are typically dimeric molecules composed of two polypeptides with identical amino acid sequences. Hinge regions generally contain up to 25 amino acid residues and are flexible, allowing the associated target binding site to move independently. Hinge regions can be subdivided into three domains: upper, middle, and lower (see, e.g., Roux, et al., J.Immunol. 161 (1998) 4083).

[0092] In one embodiment, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 105), where X is either S or P. In another embodiment, the hinge region has the amino acid sequence HTCPXCP (SEQ ID NO: 106), where X is either S or P. In yet another embodiment, the hinge region has the amino acid sequence CPXCP (SEQ ID NO: 107), where X is either S or P.

[0093] As used herein, the terms “Fc domain” or “Fc region” are used to define the C-terminal region of an antibody heavy chain that includes at least a portion of the constant region. This term includes the native sequence Fc region and variant Fc regions. In relation to molecules already defined by a Fab fragment (including the CH1 domain), the term “Fc domain” may refer only to the IgG CH2 domain and the IgG CH3 domain.

[0094] The "CH2 domain" in the human IgG Fc region typically extends from amino acid residue approximately EU position 231 to amino acid residue approximately EU position 340 (Kabat's EU numbering system). In one embodiment, the CH2 domain has the amino acid sequence APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQESTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAK (SEQ ID NO: 108). The CH2 domain is unique in that it is not closely paired with other domains. Rather, two N-linked branched glycans interpose between the two CH2 domains in the complete native Fc region. It is hypothesized that the glycans may act as a substitute for domain-to-domain pairing and contribute to the stabilization of the CH2 domain. Burton, Mol.Immunol.22(1985)161-206. In one embodiment, the sugar chain is bound to the CH2 domain. The CH2 domain in this specification may be a native sequence CH2 domain or a variant CH2 domain.

[0095] The "CH3 domain" represents a portion of the antibody heavy chain polypeptide that includes the C-terminal residue region of the CH2 domain in the Fc region and extends approximately from EU position 341 to EU position 446 (Kabat EU numbering system). In one embodiment, the CH3 domain has the amino acid sequence GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 109). The CH3 region as used herein may be a native CH3 domain or a variant CH3 domain (for example, a CH3 domain having a "nob" introduced on one chain and a "hole" introduced on the other chain; see U.S. Patent No. 5,821,333 expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0096] The “knob-into-hole” technique is described, for example, in U.S. Patents 5,731,168 and 7,695,936, Ridgway et al., Prot Eng 9,617-621 (1996), and Carter, J Immunol Meth 248,7-15 (2001). Generally, this method involves introducing a projection ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the projection may be positioned within the cavity to promote heterodimerization and inhibit homodimerization. The projection is constructed by replacing a smaller amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the projection is created at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In specific embodiments, the knob modification includes the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification includes the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In more specific embodiments, the subunit of the Fc domain containing the knob modification further includes the amino acid substitution S354C, and the subunit of the Fc domain containing the hole modification further includes the amino acid substitution Y349C. The introduction of these two cysteine ​​residues forms a disulfide bridge between the two subunits of the Fc region, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0097] The “region corresponding to the Fc region of immunoglobulins” is intended to include allelic variants of the naturally occurring Fc region of immunoglobulins, as well as variants that result in substitution, addition, or deletion, but with modifications that do not substantially reduce the effector function of the immunoglobulin (e.g., the ability to mediate antibody-dependent cell-mediated cytotoxicity). For example, one or more amino acids may be deleted from the N-terminus or C-terminus of the Fc region of immunoglobulins without substantially losing biological function. Such variants may be selected according to general rules known in the art to minimize the impact on activity (see, for example, Bowie, JU et al., Science 247:1306-10 (1990)).

[0098] The term "wild-type Fc domain" refers to an amino acid sequence identical to that of an Fc domain found in nature. Wild-type human Fc domains include the natural human IgG1 Fc region (non-A and A allotypes), the natural human IgG2 Fc region, the natural human IgG3 Fc region, and the natural human IgG4 Fc region, as well as their naturally occurring variants. Wild-type Fc regions are shown in SEQ ID NO: 110 (IgG1, Caucasian allotype), SEQ ID NO: 111 (IgG1, African American allotype), SEQ ID NO: 112 (IgG2), SEQ ID NO: 113 (IgG3), and SEQ ID NO: 114 (IgG4).

[0099] The term “variant (human) Fc domain” refers to an amino acid sequence that differs from the “wild-type” (human) Fc domain amino acid sequence by at least one “amino acid mutation.” In one embodiment, the variant Fc region has at least one amino acid mutation compared to the native Fc region, for example, about 1 to about 10 amino acid mutations, and in one embodiment, it has about 1 to about 5 amino acid mutations in the native Fc region. In one embodiment, the (variant) Fc region has at least about 95% homology with the wild-type Fc region. A particular variant Fc domain disclosed herein is a human IgG1 heavy chain constant region having mutations L234A, L235A and P329G, comprising the amino acid sequence of SEQ ID NO: 115.

[0100] The term "effector function" refers to the function of biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0101] Effector functions dependent on Fc receptor binding can be mediated by the interaction between the Fc region of an antibody and the Fc receptor (FcR), a specialized cell surface receptor on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the elimination of antibody-coated pathogens through phagocytosis by immune complexes, and the lysis of corresponding antibody-coated red blood cells and various other cellular targets (e.g., tumor cells) via antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Van de Winkel, J. Gand Anderson, CL, J. Leukoc. Biol. 49(1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes, and the Fc receptor for an IgG antibody is called FcγR. Fc receptor binding is described, for example, in Ravetch, J.V. and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P.J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J.E., et al., Ann. Hematol. 76 (1998) 231-248.

[0102] Cross-linking of the Fc region of IgG antibodies with receptors (FcγRs) not only regulates immune complex clearance and antibody production, but also induces a wide variety of effector functions, including phagocytosis, antibody-dependent cell-mediated cytotoxicity, and the release of inflammatory mediators. In humans, three classes of FcγRs are characterized by the following: -FcγRI(CD64) binds to monomeric IgG with high affinity and is expressed in macrophages, monocytes, neutrophils, and eosinophils. Modification of at least one amino acid residue E233-G236, P238, D265, N297, A327, and P329 (numbered according to Kabat's EU index) of the Fc region IgG reduces binding to FcγRI. Substitution of IgG2 residues at positions 233-236 with IgG1 and IgG4 reduced binding to FcγRI to 1 / 1000th, and the human monocyte response to antibody-sensitized erythrocytes disappeared (Armour, KL, et al., Eur. J. Immunol. 29 (1999) 2613-2624). -FcγRII(CD32) binds to complex IgG with moderate to low affinity and is widely expressed. This receptor can be divided into two subtypes: FcγRIIA and FcγRIIB. FcγRIIA is present in many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and is thought to be able to activate the killing process. FcγRIIB appears to be involved in the inhibitory process and is present in B cells, macrophages, mast cells, and eosinophils. In B cells, it appears to function to suppress further immunoglobulin production and isotype switching, such as to the IgE class. In macrophages, FcγRIIB inhibits phagocytosis via FcγRIIA. In eosinophils and mast cells, the B form may help suppress the activation of these cells by preventing IgE from binding to another receptor. Decreased binding to FcγRIIA is observed, for example, in antibodies consisting of an IgG Fc region with mutations in at least one amino acid residue E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414 (numbered according to Kabat's EU index). -FcγRIII(CD16) binds to IgG with moderate to low affinity and exists in two types. FcγRIIIA is present in NK cells, macrophages, eosinophils, and some monocytes and T cells and mediates ADCC. FcγRIIIB is highly expressed in neutrophils. Decreased binding to FcγRIIIA is observed, for example, in antibodies containing an IgG Fc region with mutations in at least one amino acid residue E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376 (numbered according to Kabat's EU index).

[0103] The mapping of the binding site of human IgG1 to the Fc receptor, the above-mentioned mutation sites, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al. J. Biol. Chem. 276 (2001) 6591-6604.

[0104] The term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which antibodies or derivatives containing an Fc region specifically bind, typically via a protein moiety located at the N-terminus of the Fc region. As used herein, the term "ADCC reduction" is defined as either a decrease in the number of target cells lysed at a given time at a given antibody concentration in the culture medium surrounding the target cells, due to the ADCC mechanism as defined above, and / or an increase in the antibody concentration in the culture medium surrounding the target cells, necessary to achieve the lysis of a given number of target cells at a given time, due to the ADCC mechanism. ADCC reduction is defined as ADCC mediated by the same antibody, produced by the same type of host cells using the same standard production, purification, formulation, and storage methods (known to those skilled in the art), but without manipulation. For example, ADCC reduction mediated by an antibody containing an amino acid substitution in its Fc domain that reduces ADCC is the same as ADCC mediated by the same antibody without this amino acid substitution in its Fc domain. Appropriate assays for measuring ADCC are known in the art (see, for example, PCT Publication No. 2006 / 082515 or PCT Publication No. 2012 / 130831). For example, the ability of an antibody to induce the initial steps mediated by ADCC is investigated by measuring its binding to Fcγ receptor-expressing cells, such as cells recombinantly expressing FcγRI and / or FcγRIIA, or NK cells (which essentially express FcγRIIIA). In particular, binding to FcγR in NK cells is measured.

[0105] An "activated Fc receptor" is an Fc receptor that, following involvement by the Fc region of an antibody, triggers a signaling event that stimulates receptor-hosting cells to perform effector functions. Examples of activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activated Fc receptor is human FcγRIIIa (SEQ ID NO: 116, UniProt accession number P08637, version 141).

[0106] An "ectodomain" is a domain of a membrane protein that extends into the extracellular space (i.e., the space outside the target cell). Ectodomains are typically the regions that initiate contact with the protein surface and lead to signal transduction.

[0107] The term "peptide linker" refers to a peptide containing one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n (SG4) n Or G4 (SG4) n It is a peptide linker, where "n" is generally a number from 1 to 5, typically 2 to 4, especially 2, and the peptide is selected from the group consisting of GGGGS (SEQ ID NO: 117), GGGGSGGGG (SEQ ID NO: 198), GGGGSGGGGS (SEQ ID NO: 118), SGGGGSGGGG (SEQ ID NO: 119), and GGGGSGGGGSGGGG (SEQ ID NO: 120), but also includes sequences GSPGSSSSGS (SEQ ID NO: 121), (G4S)3 (SEQ ID NO: 122), (G4S)4 (SEQ ID NO: 123), GSGSGSGS (SEQ ID NO: 124), GSGSGNGS (SEQ ID NO: 125), GGSGSGSG (SEQ ID NO: 126), GGSGSG (SEQ ID NO: 127), GGSG (SEQ ID NO: 128), GGSGNGSG (SEQ ID NO: 129), GGNGSGSG (SEQ ID NO: 130), and GGNGSG (SEQ ID NO: 131). Particularly interesting peptide linkers are (G4S)(SEQ ID NO: 117), GGGGSGGGG(SEQ ID NO: 198), (G4S)2 or GGGGSGGGGS(SEQ ID NO: 118), (G4S)3(SEQ ID NO: 122), and (G4S)4(SEQ ID NO: 123).

[0108] As used herein, the term "amino acid" refers to a group of naturally occurring carboxy-α-amino acids, including alanine (three-letter abbreviation: ala, one-letter abbreviation: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0109] "Fusion" or "linking" means that the constituent components (e.g., one polypeptide and another polypeptide) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0110] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences, introducing gaps as necessary to achieve maximum percentage sequence identity, and disregarding conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art of a person skilled in the art, using, for example, commonly available computer software such as BLAST, BLAST-2, ALIGN.SAWI, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. However, as used herein, the amino acid sequence identity percentage value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.In situations where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity % of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (or, a given amino acid sequence A may be described as having or containing a particular amino acid sequence identity % to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 × X / Y, where X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the amino acid sequence identity % of A to B will be different from the amino acid sequence identity % of B to A. Unless otherwise specified, all amino acid sequence identity % values ​​used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.

[0111] In certain embodiments, amino acid sequence variants of BCMA antibodies or bispecific BCMA antibodies provided herein are envisioned. For example, it may be desirable to improve the binding affinity and / or other biological properties of the BCMA antibody or bispecific BCMA antibody. Amino acid sequence variants of BCMA antibodies or bispecific BCMA antibodies may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the molecule, or by peptide synthesis. Such modifications include, for example, deletions from residues in the amino acid sequence of the antibody, and / or insertions into residues in the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be performed to reach the final construct, insofar as the final construct has the desired characteristics (e.g., antigen binding). Sites targeted for substitutional mutagenesis include CDRs and frameworks (FRs). Conservative substitutions are given in Table B under the heading "Preferred Substitutions" and are further described below with reference to amino acid side chain classes (1) to (6). By introducing amino acid substitutions into the target molecule, the product can be screened for desired activities, such as retention / improvement of antigen binding, reduction of immunogenicity, and improvement of ADCC and CDC. [Table 1]

[0112] Amino acids can be classified according to their general side-chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0113] Non-conservative substitution involves swapping one member of one of these classes with one of another.

[0114] The term “amino acid sequence variant” includes substantial variants in which amino acid substitutions exist in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized antibody or a human antibody). Generally, variants selected for further study and obtained will have alterations (e.g., improvements) to specific biological properties (e.g., increased affinity, decreased immunogenicity) and / or substantially retain specific biological properties of the parent antigen-binding molecule compared to the parent antigen-binding molecule. Exemplary substitution variants are affinity-matured antibodies and can be readily generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, one or more CDR residues are mutated, the variant antigen-binding molecule is presented on a phage, and screened for specific biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that these changes do not substantially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made within a CDR. A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or target residue groups (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at the positions of amino acids that exhibit functional sensitivity to the initial substitution. Alternatively, or in addition to this, the crystal structure of the antigen-antigen binding molecule complex is used to identify contact sites between the antibody and the antigen. Such contact residues and adjacent residues may be targeted as candidates for substitution or removed. Variants may be screened to determine whether they contain the desired properties.

[0115] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of insertions include BCMA antibodies or bispecific BCMA antibodies with N-terminal or C-terminal fusion to polypeptides that increase the serum half-life of CD28 antigen-binding molecules.

[0116] In certain embodiments, the BCMA antibodies or bispecific BCMA antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Glycosylated variants of a molecule can be conveniently obtained by modifying the amino acid sequence so that one or more glycosylation sites are created or removed. If the agonist ICOS-binding molecule contains an Fc domain, the carbohydrate bound to the Fc domain can be modified. Native antibodies produced by mammalian cells typically contain branched-type bibranched oligosaccharides, which are generally bound to Asn297 of the CH2 domain of the Fc region by an N-bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the bibranched oligosaccharide structure. In some embodiments, the oligosaccharide in the BCMA antibody or bispecific BCMA antibody may be modified to produce variants having specific improved properties. In one embodiment, a variant of the BCMA antibody or bispecific BCMA antibody is provided having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. Such a fucosylated variant may have improved ADCC function. See, for example, U.S. Patent Publication 2003 / 0157108 (Presta, L.) or U.S. Patent 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Further variants of the BCMA antibody or bispecific BCMA antibody include those having a bifid oligosaccharide, for example, a bifid oligosaccharide bound to the Fc region bifid by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. See, for example, International Publication No. 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided.Such antibody variants may possess improved CDC functionality and are described, for example, in International Publication No. 1997 / 30087 (Patel et al.), International Publication No. 1998 / 58964 (Raju, S.), and International Publication No. 1999 / 22764 (Raju, S.).

[0117] In certain embodiments, it may be desirable to produce cysteine-modified variants of the CD28 antigen-binding molecule of the present invention, for example, "thioMAb" in which one or more residues of the molecule are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the molecule. By substituting these residues with cysteine, a reactive thiol group may be positioned at an accessible site of the antibody, which can then be used to conjugate the antibody to other parts, such as a drug portion or a linker-drug portion, to produce an immunoconjugate. In certain embodiments, one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. The cysteine-modified antigen-binding molecule may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0118] In certain embodiments, the BCMA antibodies or bispecific BCMA antibodies provided herein may be modified to include further non-proteinoid moieties that are known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers bound to the antibody may vary, and if more than one polymer is bound, the polymers may be the same molecule or different molecules. Generally, the number and / or types of polymers used in derivatization are not limited but may be determined with consideration to specific properties or functions of the antibody to be improved, whether the bispecific antibody derivative will be used therapeutically under specific conditions, etc. In another embodiment, a conjugate of an antibody and a non-protein portion that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein portion is a carbon nanotube (Kam, NWet al., Proc. Natl. Acad. Sci. USA 102(2005) 11600-11605). The radiation may have any wavelength, not limited but including wavelengths that are not harmful to normal cells but heat the non-protein portion to a temperature that kills cells proximal to the antibody non-protein portion.In another embodiment, an immunoconjugate of a BCMA antibody or a bispecific BCMA antibody provided herein can be obtained. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, such as a small molecule drug.

[0119] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or other types of bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments present in a polynucleotide, such as a DNA or RNA fragment. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by their base sequence, which represents the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented 5' to 3'. In this specification, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules may be linear or cyclic. Furthermore, the term nucleic acid molecule includes sense strands and antisense strands, as well as both single-stranded and double-stranded forms. In addition, nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may or may not be modified.For example, mRNA can be chemically modified to improve the stability of the RNA vector and / or the expression of the encoded molecule, allowing the mRNA to be injected into a target to generate antibodies in vivo (see, for example, Stadler et al. (2017) Nature Medicine 23:815-817, or European Patent No. 2 101 823B1).

[0120] The term "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA that has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of this invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that originally contained polynucleotide molecules, but the polynucleotide molecules are extrachromosomal or located at chromosomal locations different from their native chromosomal locations. Isolated RNA molecules include the in vivo or in vitro RNA transcripts of this invention, in positive and negative strand forms, and double-stranded forms. Furthermore, isolated polynucleotides or nucleic acids of this invention include such molecules produced by synthesis. Furthermore, polynucleotides or nucleic acids may be regulatory elements such as promoters, ribosome-binding sites, or transcription terminators, or may contain regulatory elements.

[0121] A nucleic acid or polynucleotide having a nucleotide sequence that is, for example, at least 95% "identical" to the reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference nucleotide sequence, except that the nucleotide sequence of the polynucleotide may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These modifications to the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or somewhere between these terminal positions, and may be individually scattered among the residues in the reference sequence, or scattered among one or more consecutive groups in the reference sequence. In practice, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the present invention can be conventionally determined using a known computer program, such as the one described above for polypeptides (e.g., ALIGN-2).

[0122] The term "expression cassette" refers to a polynucleotide, generated by recombination or synthesis, comprising a set of specific nucleic acid elements that enable the transcription of a particular nucleic acid in target cells. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, in particular, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the present invention comprises a polynucleotide sequence or fragment thereof encoding the bispecific antigen-binding molecule of the present invention.

[0123] The terms “vector” or “expression vector” are synonymous with “expression construct” and refer to a DNA molecule used to introduce and induce the expression of a specific gene to which it operably binds within a target cell. This term includes not only vectors as self-replicating nucleic acid structures, but also vectors integrated into the genome of the host cell to which the vector has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector enables the transcription of large amounts of stable mRNA. Once the expression vector enters the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation mechanism. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence or fragment thereof encoding the bispecific antigen-binding molecule of the present invention.

[0124] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and also include the offspring of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the number of passages. The offspring may not have nucleic acid content that is exactly the same as that of the parent cell and may contain mutations. In this specification, mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included. Host cells are any type of cell line that can be used to generate the bispecific antigen-binding molecules of the present invention. Examples of host cells include cultured cells, e.g., mammalian cultured cells, e.g., to name just a few, CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, but also include cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues.

[0125] The "effective dose" of a drug refers to the amount of that drug required to cause a certain physiological change in the cells or tissues to which it is administered.

[0126] The "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to the amount effective at the dosage and for the period required to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the side effects of a disease.

[0127] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., non-human primates such as humans and monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0128] The term "pharmaceutical composition" refers to a preparation in a form in which the biological activity of the active ingredient contained therein is effective, and which does not contain additional constituents that are unacceptably toxic to the subject to whom the preparation is administered.

[0129] "Pharmaceutically acceptable additives" refer to components other than the active ingredient in a pharmaceutical composition that are non-toxic to the subject. Pharmaceutically acceptable additives include, but are not limited to, buffering agents, stabilizing agents, preservatives, and the like.

[0130] The term "package insert" is used to refer to the instructions customarily included in the commercial packaging of a therapeutic product and contains information about indications, usage, dosage, administration, combination therapy, contraindications and / or precautions regarding the use of the therapeutic product.

[0131] As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of an individual being treated and can be performed for prophylaxis or during the course of a clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating a medical condition, and remission or improvement of prognosis. In some embodiments, the molecules of the invention are used to delay the onset of a disease or to slow the progression of a disease.

[0132] The term "therapy" refers to any protocol, method, and / or agent that can be used for the prevention, management, treatment, and / or amelioration of a disease (or symptoms associated therewith) or cancer. In certain aspects, the terms "therapies" and "therapy" refer to biological therapies, supportive therapies, and / or other therapies known to those of skill in the art such as physicians that are useful for the prevention, management, treatment, and / or amelioration of a disease or cancer.

[0133] The terms "combination therapy" or "co - administration" as described herein encompass co - administration (where two or more therapeutic agents are included in the same or separate formulations) and separate administrations, in which case administration of the antibodies reported herein can occur before, simultaneously with, and / or after administration of one or more additional therapeutic agents, preferably one or more antibodies.

[0134] The term "cancer" refers to or describes a physiological condition in mammals typically characterized by uncontrolled cell growth / proliferation. Therefore, as used herein, the term cancer refers to proliferative disorders such as carcinomas, lymphomas (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma), blastomas, sarcomas, and leukemias. In particular, the term cancer refers to lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar carcinoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer. This includes cancer, colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular carcinoma, biliary tract cancer, neoplasms of the central nervous system (CNS), spinal axial tumor, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma (including any refractory form of any of the above-mentioned cancers), or one or more combinations of the above-mentioned cancers. In one embodiment, cancer is a solid tumor. In another embodiment, cancer is a blood cancer, particularly leukemia, most specifically acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). In a preferred embodiment, the term cancer refers to any cancer in which BCMA is expressed. More preferably, cancer is multiple myeloma (MM).

[0135] Exemplary novel BCMA antibody Novel antibodies and / or antibody fragments that specifically bind to B cell maturation antigen (BCMA) are provided herein. Novel antibodies and / or antibody fragments that specifically bind to the extracellular domain of human BCMA containing the amino acid sequence of SEQ ID NO: 132 are provided. Therefore, these antibodies specifically bind to human BCMA.

[0136] These antibodies can bind to human BCMA and cynomolgus monkey BCMA.

[0137] These antibodies, when measured by surface plasmon resonance (SPR), have a K content of less than 5 nM. D The value binds to the human BCMA extracellular domain (ECD of the amino acid sequence of SEQ ID NO: 132) (see Example 1.3).

[0138] As demonstrated in Example 3, the novel antibody can also bind to mutant human BCMA variants hu BCMA_R27P (SEQ ID NO: 210), Hu BCMA_S30del (SEQ ID NO: 211), hu BCMA_P33S (SEQ ID NO: 212), and hu BCMA_P34del (SEQ ID NO: 213). Therefore, the novel antibody is unaffected by point mutations in the BCMA ECD and retains its therapeutic activity as observed with other BCMA-targeting molecules such as Elranatamab and Teclistamab (Lee et al., Nature Medicine 2023, 29, 2295-2306).

[0139] The novel antibody can be produced in large quantities and with high titers, (at an aggregation temperature T agg They are further characterized by high thermal stability (as measured by [method]) or a high degree of human-likeness, and therefore potentially low immunogenicity in the human body. The proportion of human-likeness of VH and VL sequences compared to human germline sequences can be determined by the method described in Abhinandan, KR and Martin, Andrew CR2007, J.Mol.Biol.2007, 369, 852-862.

[0140] In one embodiment, the antibody specifically binds to B cell maturation antigen (BCMA), and the antibody is: (i) Heavy chain variable region (V) including the heavy chain complementarity determination region of CDR-H1 (GYTFTNYWMH) of SEQ ID NO: 1, CDR-H2 (IIHPNSGSTNYNEKFQG) of SEQ ID NO: 2, and CDR-H3 (GIYDYPFAY) of SEQ ID NO: 3 H BCMA) and, (ii) Light chain variable region (V L BCMA) (a) A VL containing the light chain complementarity determination region of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASSLQS) of SEQ ID NO: 5, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6, or (b) A VL containing the light chain complementarity determination region of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASNLES) of SEQ ID NO: 7, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6, or (c) An antibody comprising a light chain variable region selected from the group consisting of VLs including the light chain complementarity determining regions of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASNLQS) of SEQ ID NO: 8, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6 is provided herein.

[0141] In one embodiment, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, and the antibody contains an amino acid sequence selected from the group consisting of SEQ ID NO: 9 (VH1a) and SEQ ID NO: 10 (VH1b). H V containing an amino acid sequence selected from the group consisting of BCMA and / or SEQ ID NOs: 11 (VL1f), 12 (VL1a), 13 (VL1b), 14 (VL1c), 15 (VL1d), and 16 (VL1e) L Includes BCMA.

[0142] In one embodiment, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, and the antibody (or antigen-binding domain) is, (a) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 12 L BCMA, or (c) V containing the amino acid sequence of SEQ ID NO: 9H V containing the amino acid sequences of BCMA and SEQ ID NO: 13 L BCMA, or (d) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 14 L BCMA, or (e) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 15 L BCMA, or (f) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 16 L BCMA, or (g) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 135 L BCMA, or (h) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 136 L BCMA, or (i) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L BCMA, or (j) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 12 L BCMA, or (k) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 13 L BCMA, or (l) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 14 L BCMA, or (m) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 15 L BCMA, or (n) V containing the amino acid sequence of SEQ ID NO: 10 HV comprising BCMA and the amino acid sequence of SEQ ID NO: 16 L BCMA, or (o) V comprising the amino acid sequence of SEQ ID NO: 10 H V comprising BCMA and the amino acid sequence of SEQ ID NO: 135 L BCMA, or (p) V comprising the amino acid sequence of SEQ ID NO: 10 H V comprising BCMA and the amino acid sequence of SEQ ID NO: 136 L Comprises BCMA.

[0143] In one aspect, the BCMA antibody (or antigen-binding domain that specifically binds to BCMA) is (a) V comprising the amino acid sequence of SEQ ID NO: 9 H V comprising BCMA and the amino acid sequence of SEQ ID NO: 11 L BCMA, or (b) V comprising the amino acid sequence of SEQ ID NO: 9 H V comprising BCMA and the amino acid sequence of SEQ ID NO: 12 L BCMA, or (c) V comprising the amino acid sequence of SEQ ID NO: 9 H V comprising BCMA and the amino acid sequence of SEQ ID NO: 13 L BCMA, or (d) V comprising the amino acid sequence of SEQ ID NO: 9 H V comprising BCMA and the amino acid sequence of SEQ ID NO: 14 L BCMA, or (e) V comprising the amino acid sequence of SEQ ID NO: 9 H V comprising BCMA and the amino acid sequence of SEQ ID NO: 15 L BCMA, or (f) V comprising the amino acid sequence of SEQ ID NO: 9 H V comprising BCMA and the amino acid sequence of SEQ ID NO: 16 L BCMA, or (g) V comprising the amino acid sequence of SEQ ID NO: 9 H V comprising BCMA and the amino acid sequence of SEQ ID NO: 135 L BCMA, or (h) V comprising the amino acid sequence of SEQ ID NO: 9 H V comprising BCMA and the amino acid sequence of SEQ ID NO: 136 LIt includes BCMA.

[0144] In one aspect, the BCMA antibody (or antigen-binding domain that specifically binds to BCMA) is (i) a V containing the amino acid sequence of SEQ ID NO: 10 H BCMA and a V containing the amino acid sequence of SEQ ID NO: 11 L BCMA, or (j) a V containing the amino acid sequence of SEQ ID NO: 10 H BCMA and a V containing the amino acid sequence of SEQ ID NO: 12 L BCMA, or (k) a V containing the amino acid sequence of SEQ ID NO: 10 H BCMA and a V containing the amino acid sequence of SEQ ID NO: 13 L BCMA, or (l) a V containing the amino acid sequence of SEQ ID NO: 10 H BCMA and a V containing the amino acid sequence of SEQ ID NO: 14 L BCMA, or (m) a V containing the amino acid sequence of SEQ ID NO: 10 H BCMA and a V containing the amino acid sequence of SEQ ID NO: 15 L BCMA, or (n) a V containing the amino acid sequence of SEQ ID NO: 10 H BCMA and a V containing the amino acid sequence of SEQ ID NO: 16 L BCMA, or (o) a V containing the amino acid sequence of SEQ ID NO: 10 H BCMA and a V containing the amino acid sequence of SEQ ID NO: 135 L BCMA, or (p) a V containing the amino acid sequence of SEQ ID NO: 10 H BCMA and a V containing the amino acid sequence of SEQ ID NO: 136 L It includes BCMA.

[0145] In a specific aspect, a BCMA antibody (or antigen-binding domain that specifically binds to BCMA) is provided, and the antibody (or antigen-binding domain) is (a) a V containing the amino acid sequence of SEQ ID NO: 9 H BCMA and a V containing the amino acid sequence of SEQ ID NO: 11 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 12 L Includes BCMA.

[0146] More specifically, the BCMA antibody (or the antigen-binding domain that specifically binds to BCMA) contains the amino acid sequence of SEQ ID NO: 9. H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L Includes BCMA.

[0147] In another embodiment, an antibody that specifically binds to B cell maturation antigen (BCMA), wherein the antibody is (i) Heavy chain variable region (V H BCMA) (a) VH including the heavy chain complementarity determining regions of CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYADSVKG) of SEQ ID NO: 30, and CDR-H3 (DGYH) of SEQ ID NO: 31, and (b) A heavy chain variable region selected from the group consisting of VH including the heavy chain complementarity determination regions of CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYAAPVKG) of SEQ ID NO: 32, and CDR-H3 (DGYH) of SEQ ID NO: 31, (ii) Light chain variable region (V) including the light chain complementarity determination region of CDR-L1 (RASEDIRNGLA) of SEQ ID NO: 33, CDR-L2 (NANSLHT) of SEQ ID NO: 34, and CDR-L3 (EDTSKYPYT) of SEQ ID NO: 35 L Antibodies comprising BCMA are provided herein.

[0148] In one embodiment, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, and the antibody (or antigen-binding domain) is the heavy chain variable region (V) of CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYADSVKG) of SEQ ID NO: 30, and CDR-H3 (DGYH) of SEQ ID NO: 31. HBCMA) and the light chain variable region (V) of CDR-L1 (RASEDIRNGLA) of SEQ ID NO: 33, CDR-L2 (NANSLHT) of SEQ ID NO: 34, and CDR-L3 (EDTSKYPYT) of SEQ ID NO: 35 L Includes BCMA.

[0149] In one embodiment, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, and the antibody (or antigen-binding domain) is the heavy chain variable region (V) of CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYAAPVKG) of SEQ ID NO: 32, and CDR-H3 (DGYH) of SEQ ID NO: 31. H BCMA) and the light chain variable region (V) of CDR-L1 (RASEDIRNGLA) of SEQ ID NO: 33, CDR-L2 (NANSLHT) of SEQ ID NO: 34, and CDR-L3 (EDTSKYPYT) of SEQ ID NO: 35 L Includes BCMA.

[0150] In one embodiment, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, and the antibody (or antigen-binding domain) contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 (VH2a), 38 (VH1b), 48 (VH1a), 49 (VH1a_Y292D), 50 (VH1c_hu CDR2), and 51 (VH1a_W197Y). H V containing an amino acid sequence selected from the group consisting of BCMA and / or SEQ ID NOs: 37 (VL2a), 39 (VL1a), 52 (VL1a_L2_GL), 53 (VL2a_L2_GL), 54 (VL1a_L1_pGL), 55 (VL1a_N651A), 56 (VL1a_N651A_N695S), 57 (VL1a_H698Q), 58 (VL1a_T699S), and 59 (VL1a_H698Q_T699S) L Includes BCMA.

[0151] In one embodiment, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, and the antibody (or antigen-binding domain) contains an amino acid sequence selected from the group consisting of SEQ ID NO: 36 (VH2a) and SEQ ID NO: 38 (VH1b). H V containing an amino acid sequence selected from the group consisting of BCMA and / or SEQ ID NO: 37 (VL2a) and SEQ ID NO: 39 (VL1a) L Includes BCMA.

[0152] In one embodiment, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, and the antibody (or antigen-binding domain) is, (a) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 39 L BCMA, or (c) V containing the amino acid sequence of SEQ ID NO: 48 H V containing the amino acid sequences of BCMA and SEQ ID NO: 52 L BCMA, or (d) V containing the amino acid sequence of SEQ ID NO: 49 H V containing the amino acid sequences of BCMA and SEQ ID NO: 39 L BCMA, or (e) V containing the amino acid sequence of SEQ ID NO: 49 H V containing the amino acid sequences of BCMA and SEQ ID NO: 52 L BCMA, or (f) V containing the amino acid sequence of SEQ ID NO: 49 H V containing the amino acid sequences of BCMA and SEQ ID NO: 58 L BCMA, or (g) V containing the amino acid sequence of SEQ ID NO: 49 H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L BCMA, or (h) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 54L BCMA, or (i) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 55 L BCMA, or (j) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 56 L BCMA, or (k) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 58 L BCMA, or (l) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L BCMA, or (m) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 39 L BCMA, or (n) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 54 L BCMA, or (o) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 58 L Includes BCMA.

[0153] In one particular embodiment, a BCMA antibody (or an antigen-binding domain that specifically binds to BCMA) is provided, and the antibody (or antigen-binding domain) is, (a) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 39 L Includes BCMA.

[0154] More specifically, the BCMA antibody contains the amino acid sequence of SEQ ID NO: 36.H BCMA and V containing the amino acid sequence of SEQ ID NO: 37 L Includes BCMA.

[0155] In one embodiment, the antibody that specifically binds to BCMA is a full-length antibody, particularly an antibody of the human IgG1 subclass. In a particular embodiment, the antibody that specifically binds to BCMA includes amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index).

[0156] Novel bispecific CD28 agonist antibodies containing BCMA antibodies This patent application also provides novel BCMA antibodies comprising a second antigen-binding domain that specifically binds to a second antigen, particularly CD28. These bispecific CD28 agonist antibodies possess advantageous properties such as superior manufacturability, stability, binding affinity, biological activity, targeting efficiency, reduced toxicity, a wider dose range that can be administered to patients, and the potential for improved efficacy that may be observed therein. The novel bispecific CD28 agonist antibodies comprise an Fc domain composed of first and second subunits capable of stable association, each containing one or more amino acid substitutions, which reduces the binding affinity and / or effector function (Fc silent) of the antigen-binding molecule to the Fc receptor, thus avoiding nonspecific crosslinking via the Fc receptor. Instead, they comprise a specific antigen-binding domain capable of specific binding to BCMA, which would cause crosslinking at the tumor site. Surprisingly, the inventors have found that, based on their binding properties, the BCMA antigen-binding domains described herein possess advantageous properties that make them more readily usable in a bispecific format. Furthermore, these bispecific agonist CD28 antigen-binding molecules containing BCMA antigen-binding domains were found to possess improved functionality and ability to increase T cell activation, particularly in the presence of T cell-activating anti-CD3 bispecific antibodies. Thus, enhancement of tumor-specific T cell activation is achieved.

[0157] In one embodiment, a bispecific antibody that specifically binds to B cell maturation antigen (BCMA) and CD28, (A) The first antigen-binding domain is (i) Heavy chain variable region (V) including the heavy chain complementarity determination region of CDR-H1 (GYTFTNYWMH) of SEQ ID NO: 1, CDR-H2 (IIHPNSGSTNYNEKFQG) of SEQ ID NO: 2, and CDR-H3 (GIYDYPFAY) of SEQ ID NO: 3 H BCMA) and, (ii) Light chain variable region (V L BCMA) (a) A VL containing the light chain complementarity determination region of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASSLQS) of SEQ ID NO: 5, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6, or (b) A VL containing the light chain complementarity determination region of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASNLES) of SEQ ID NO: 7, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6, or (c) A first antigen-binding domain comprising a light chain variable region selected from the group consisting of VLs including the light chain complementarity determining regions of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASNLQS) of SEQ ID NO: 8, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6, (B) An antibody comprising a second antigen-binding domain that specifically binds to CD28 is provided herein.

[0158] In one embodiment, an antibody that specifically binds to BCMA and CD28, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9 (VH1a) and SEQ ID NO: 10 (VH1b) H V containing an amino acid sequence selected from the group consisting of BCMA and / or SEQ ID NOs: 11 (VL1f), 12 (VL1a), 13 (VL1b), 14 (VL1c), 15 (VL1d), and 16 (VL1e) L Antibodies containing BCMA are provided.

[0159] In one embodiment, the antibody specifically binds to BCMA and CD28, (a) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 12 L BCMA, or (c) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 13 L BCMA, or (d) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 14 L BCMA, or (e) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 15 L BCMA, or (f) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 16 L BCMA, or (g) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 135 L BCMA, or (h) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 136 L BCMA, or (i) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L BCMA, or (j) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 12 L BCMA, or (k) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 13 L BCMA, or (l) V containing the amino acid sequence of SEQ ID NO: 10H V containing the amino acid sequences of BCMA and SEQ ID NO: 14 L BCMA, or (m) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 15 L BCMA, or (n) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 16 L BCMA, or (o) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 135 L BCMA, or (p) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 136 L Antibodies containing BCMA are provided.

[0160] In one embodiment, the antibody that specifically binds to BCMA and CD28 is: (a) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 12 L BCMA, or (c) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 13 L BCMA, or (d) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 14 L BCMA, or (e) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 15 L BCMA, or (f) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 16L BCMA, or (g) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 135 L BCMA, or (h) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 136 L Includes BCMA.

[0161] In one embodiment, the antibody that specifically binds to BCMA and CD28 is: (i) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L BCMA, or (j) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 12 L BCMA, or (k) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 13 L BCMA, or (l) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 14 L BCMA, or (m) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 15 L BCMA, or (n) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 16 L BCMA, or (o) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 135 L BCMA, or (p) V containing the amino acid sequence of SEQ ID NO: 10 H V containing the amino acid sequences of BCMA and SEQ ID NO: 136 L Includes BCMA.

[0162] In one particular embodiment, an antibody that specifically binds to BCMA and CD28, (a) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 12 L Antibodies containing BCMA are provided.

[0163] More specifically, antibodies that specifically bind to BCMA and CD28 include the amino acid sequence of SEQ ID NO: 9. H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L Includes BCMA.

[0164] In all of the embodiments referred to above herein, the first antigen-binding domain that specifically binds to BCMA may be a Fab molecule. In some embodiments, the first antigen-binding domain that specifically binds to BCMA may be a cross-Fab molecule, i.e., a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1, particularly the variable domains VL and VH, of the Fab light chain and Fab heavy chain are substituted for each other.

[0165] In a particular embodiment, the first antigen-binding domain that specifically binds to BCMA is a Fab molecule, in which, in the constant domain CL, the amino acid at position 123 (numbered according to the Kabat EU index) is substituted with an amino acid selected from lysine (K), arginine (R), or histidine (H), and the amino acid at position 124 (numbered according to the Kabat EU index) is independently substituted with lysine (K), arginine (R), or histidine (H); in the constant domain CH1, the amino acid at position 147 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D), and the amino acid at position 213 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index).

[0166] In any of the embodiments referred to above in this specification, the second antigen-binding domain that specifically binds to CD28 may be a Fab molecule. In certain embodiments, the second antigen-binding domain that specifically binds to CD28 is a cross-Fab molecule, i.e., a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain, or the constant domains CL and CH1, particularly the variable domains VL and VH, are substituted for each other.

[0167] In one embodiment, the second antigen-binding domain that specifically binds to CD28 is a heavy chain variable region (V) that includes the heavy chain complementarity determining regions of CDR-H1 of SEQ ID NO: 17, CDR-H2 of SEQ ID NO: 18, and CDR-H3 of SEQ ID NO: 19. H CD28) and the light chain variable region (V) including the light chain complementarity determination region of CDR-L1 of SEQ ID NO: 20, CDR-L2 of SEQ ID NO: 21, and CDR-L3 of SEQ ID NO: 22 L CD28) and includes. In one embodiment, the second antigen-binding domain that specifically binds to CD28 is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 23. H CD28) and the light chain variable region (V) containing the amino acid sequence of sequence number 24(v8). L CD28) and includes

[0168] In a particular embodiment, an antibody that specifically binds to BCMA and CD28, comprising the amino acid sequence of SEQ ID NO: 9, V H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L The first antigen-binding domain contains BCMA, and V contains the amino acid sequence of SEQ ID NO: 23. H V containing the amino acid sequences of CD28 and SEQ ID NO: 24 L An antibody is provided that includes a second antigen-binding domain containing CD28.

[0169] In one embodiment, an antibody that specifically binds to BCMA and CD28 comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 25, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 26, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 27, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 28. In a particular embodiment, an antibody that specifically binds to BCMA and CD28 is provided, comprising a first light chain containing the amino acid sequence of SEQ ID NO: 25, a first heavy chain containing the amino acid sequence of SEQ ID NO: 26, a second heavy chain containing the amino acid sequence of SEQ ID NO: 27, and a second light chain containing the amino acid sequence of SEQ ID NO: 28.

[0170] In another embodiment, an antibody that specifically binds to BCMA and CD28, comprising the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 12 L The first antigen-binding domain contains BCMA, and V contains the amino acid sequence of SEQ ID NO: 23. H V containing the amino acid sequences of CD28 and SEQ ID NO: 24 L An antibody is provided that includes a second antigen-binding domain containing CD28.

[0171] In one embodiment, an antibody that specifically binds to BCMA and CD28 comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 92, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 26, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 27, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 28. In one embodiment, an antibody that specifically binds to BCMA and CD28 is provided, comprising a first light chain containing the amino acid sequence of SEQ ID NO: 92, a first heavy chain containing the amino acid sequence of SEQ ID NO: 26, a second heavy chain containing the amino acid sequence of SEQ ID NO: 27, and a second light chain containing the amino acid sequence of SEQ ID NO: 28.

[0172] In a further embodiment, an antibody that specifically binds to B cell maturation antigen (BCMA) and CD28, (A) The first antigen-binding domain is (i) Heavy chain variable region (V H BCMA) (a) VH including the heavy chain complementarity determining regions of CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYADSVKG) of SEQ ID NO: 30, and CDR-H3 (DGYH) of SEQ ID NO: 31, and (b) A heavy chain variable region selected from the group consisting of VH including the heavy chain complementarity determination regions of CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYAAPVKG) of SEQ ID NO: 32, and CDR-H3 (DGYH) of SEQ ID NO: 31, (ii) Light chain variable region (V) including the light chain complementarity determination region of CDR-L1 (RASEDIRNGLA) of SEQ ID NO: 33, CDR-L2 (NANSLHT) of SEQ ID NO: 34, and CDR-L3 (EDTSKYPYT) of SEQ ID NO: 35 L A first antigen-binding domain including BCMA, (B) An antibody is provided comprising a second antigen-binding domain that specifically binds to CD28.

[0173] In one embodiment, an antibody that specifically binds to BCMA and CD28, comprising the heavy chain variable region (V) of CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYADSVKG) of SEQ ID NO: 30, and CDR-H3 (DGYH) of SEQ ID NO: 31. H BCMA) and the light chain variable region (V) of CDR-L1 (RASEDIRNGLA) of SEQ ID NO: 33, CDR-L2 (NANSLHT) of SEQ ID NO: 34, and CDR-L3 (EDTSKYPYT) of SEQ ID NO: 35 L Antibodies containing BCMA are provided.

[0174] In one embodiment, an antibody that specifically binds to BCMA and CD28, comprising a heavy chain variable region (V) including CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYAAPVKG) of SEQ ID NO: 32, and CDR-H3 (DGYH) of SEQ ID NO: 31. H BCMA) and the light chain variable region (V) including CDR-L1 (RASEDIRNGLA) of SEQ ID NO: 33, CDR-L2 (NANSLHT) of SEQ ID NO: 34, and CDR-L3 (EDTSKYPYT) of SEQ ID NO: 35 L Antibodies containing BCMA are provided.

[0175] In one embodiment, an antibody that specifically binds to BCMA and CD28, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 (VH2a), 38 (VH1b), 48 (VH1a), 49 (VH1a_Y292D), 50 (VH1c_hu CDR2), and 51 (VH1a_W197Y). H V containing an amino acid sequence selected from the group consisting of BCMA and / or SEQ ID NOs: 37 (VL2a), 39 (VL1a), 52 (VL1a_L2_GL), 53 (VL2a_L2_GL), 54 (VL1a_L1_pGL), 55 (VL1a_N651A), 56 (VL1a_N651A_N695S), 57 (VL1a_H698Q), 58 (VL1a_T699S), and 59 (VL1a_H698Q_T699S) LAntibodies containing BCMA are provided.

[0176] In one embodiment, an antibody that specifically binds to BCMA and CD28, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36 (VH2a) and SEQ ID NO: 38 (VH1b) H V containing an amino acid sequence selected from the group consisting of BCMA and / or SEQ ID NO: 37 (VL2a) and SEQ ID NO: 39 (VL1a) L Antibodies containing BCMA are provided.

[0177] In one embodiment, the antibody specifically binds to BCMA and CD28, (a) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 39 L BCMA, or (c) V containing the amino acid sequence of SEQ ID NO: 48 H V containing the amino acid sequences of BCMA and SEQ ID NO: 52 L BCMA, or (d) V containing the amino acid sequence of SEQ ID NO: 49 H V containing the amino acid sequences of BCMA and SEQ ID NO: 39 L BCMA, or (e) V containing the amino acid sequence of SEQ ID NO: 49 H V containing the amino acid sequences of BCMA and SEQ ID NO: 52 L BCMA, or (f) V containing the amino acid sequence of SEQ ID NO: 49 H V containing the amino acid sequences of BCMA and SEQ ID NO: 58 L BCMA, or (g) V containing the amino acid sequence of SEQ ID NO: 49 H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L BCMA, or (h) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 54L BCMA, or (i) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 55 L BCMA, or (j) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 56 L BCMA, or (k) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 58 L BCMA, or (l) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L BCMA, or (m) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 39 L BCMA, or (n) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 54 L BCMA, or (o) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 58 L Antibodies containing BCMA are provided.

[0178] In one particular embodiment, an antibody that specifically binds to BCMA and CD28, (a) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 39 L Antibodies containing BCMA are provided.

[0179] More specifically, antibodies that specifically bind to BCMA and CD28 include the amino acid sequence of SEQ ID NO: 36.H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L Includes BCMA.

[0180] In all of the embodiments referred to above herein, the first antigen-binding domain that specifically binds to BCMA may be a Fab molecule. In some embodiments, the first antigen-binding domain that specifically binds to BCMA may be a cross-Fab molecule, i.e., a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1, particularly the variable domains VL and VH, of the Fab light chain and Fab heavy chain are substituted for each other.

[0181] In a particular embodiment, the first antigen-binding domain that specifically binds to BCMA is a Fab molecule, in which, in the constant domain CL, the amino acid at position 123 (numbered according to the Kabat EU index) is substituted with an amino acid selected from lysine (K), arginine (R), or histidine (H), and the amino acid at position 124 (numbered according to the Kabat EU index) is independently substituted with lysine (K), arginine (R), or histidine (H); in the constant domain CH1, the amino acid at position 147 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D), and the amino acid at position 213 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index).

[0182] In any of the embodiments described above, the second antigen-binding domain that specifically binds to CD28 may be a Fab molecule. In certain embodiments, the second antigen-binding domain that specifically binds to CD28 may be a cross-Fab molecule, i.e., a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain, or the constant domains CL and CH1, particularly the variable domains VL and VH, are substituted for each other.

[0183] In one embodiment, the second antigen-binding domain that specifically binds to CD28 is a heavy chain variable region (V) that includes the heavy chain complementarity determining regions of CDR-H1 of SEQ ID NO: 17, CDR-H2 of SEQ ID NO: 18, and CDR-H3 of SEQ ID NO: 19. H CD28) and the light chain variable region (V) including the light chain complementarity determination region of CDR-L1 of SEQ ID NO: 20, CDR-L2 of SEQ ID NO: 21, and CDR-L3 of SEQ ID NO: 22 L CD28) and includes. In one embodiment, the second antigen-binding domain that specifically binds to CD28 is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 23. H CD28) and the light chain variable region (V) containing the amino acid sequence of sequence number 24(v8). L CD28) and includes

[0184] In one particular embodiment, an antibody that specifically binds to BCMA and CD28, comprising the amino acid sequence of SEQ ID NO: 36. H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L The first antigen-binding domain contains BCMA, and V contains the amino acid sequence of SEQ ID NO: 23. H V containing the amino acid sequences of CD28 and SEQ ID NO: 24 L An antibody is provided that includes a second antigen-binding domain containing CD28.

[0185] In one embodiment, an antibody that specifically binds to BCMA and CD28 comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 40, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 41, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 27, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 28. In a particular embodiment, an antibody that specifically binds to BCMA and CD28 is provided, comprising a first light chain containing the amino acid sequence of SEQ ID NO: 40, a first heavy chain containing the amino acid sequence of SEQ ID NO: 41, a second heavy chain containing the amino acid sequence of SEQ ID NO: 27, and a second light chain containing the amino acid sequence of SEQ ID NO: 28.

[0186] In another embodiment, an antibody that specifically binds to BCMA and CD28, comprising the amino acid sequence of SEQ ID NO: 38, V H V containing the amino acid sequences of BCMA and SEQ ID NO: 39 L The first antigen-binding domain contains BCMA, and V contains the amino acid sequence of SEQ ID NO: 23. H V containing the amino acid sequences of CD28 and SEQ ID NO: 24 L An antibody is provided that includes a second antigen-binding domain containing CD28.

[0187] In one embodiment, an antibody that specifically binds to BCMA and CD28 comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 94, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 93, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 27, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 28. In a particular embodiment, an antibody that specifically binds to BCMA and CD28 is provided, comprising a first light chain containing the amino acid sequence of SEQ ID NO: 94, a first heavy chain containing the amino acid sequence of SEQ ID NO: 93, a second heavy chain containing the amino acid sequence of SEQ ID NO: 27, and a second light chain containing the amino acid sequence of SEQ ID NO: 28.

[0188] In any of the above embodiments, the antibody that specifically binds to BCMA and CD28 includes an Fc domain. In one embodiment, the Fc domain is IgG, in particular an IgG1Fc domain. In a particular embodiment, the Fc domain is a human Fc domain, in particular a human IgG1 Fc domain. In any of the above embodiments, the Fc domain includes one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor. In a particular embodiment, it includes amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index).

[0189] Fc domain modification that reduces Fc receptor binding and / or effector function. The Fc domain of antibodies that specifically bind to BCMA and CD28 consists of a pair of polypeptide chains containing the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is dimer, with each subunit containing the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. The Fc domain confers favorable pharmacokinetic properties to the antigen-binding molecule of the present invention, including a long serum half-life and a good tissue-to-blood distribution ratio, which contribute to good accumulation in target tissues. On the other hand, this may lead to undesirable targeting of the bispecific antibodies of the present invention against cells expressing the Fc receptor, rather than the preferred antigen-containing cells.

[0190] Therefore, the Fc domain of antibodies that specifically bind to BCMA and CD28 exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to the native IgG1 Fc domain. In one embodiment, the Fc domain does not substantially bind to the Fc receptor and / or does not induce effector function. In a particular embodiment, the Fc receptor is the Fcγ receptor. In one embodiment, the Fc receptor is the human Fc receptor. In a specific embodiment, the Fc receptor is the activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the Fc domain does not induce effector function. Reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cell-mediated cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0191] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0192] In one particular embodiment, an antibody is provided that specifically binds to BCMA and CD28, wherein the Fc region comprises one or more amino acid substitutions that reduce binding to the Fc receptor, particularly to the Fcγ receptor. In another embodiment, an antibody is provided that specifically binds to BCMA and CD28, wherein the Fc region comprises one or more amino acid substitutions, and the ADCC induced by the antibody is reduced to 0-20% of the ADCC induced by an antibody containing the wild-type human IgG1 Fc region.

[0193] In one embodiment, the Fc domain of the antibody described herein contains one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to the Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In particular, the Fc domain contains amino acid substitutions at positions E233, L234, L235, N297, P331 and P329 (EU numbering). Specifically, the Fc domain contains amino acid substitutions at positions 234 and 235 (EU numbering) and / or 329 (EU numbering) of the IgG heavy chain. More specifically, antibodies are provided that specifically bind to BCMA and CD28 and contain an Fc domain having amino acid substitutions L234A, L235A and P329G ("P329G LALA", EU numbering) of the IgG heavy chain. The amino acid substitutions L234A and L235A refer to so-called LALA mutations. The amino acid substitution combination "P329G LALA" almost completely eliminates Fcγ receptor binding of the human IgG1 Fc domain and is described in International Patent Application Publication 2012 / 130831A1, which also describes a method for preparing such a mutant Fc domain and a method for determining its properties, such as Fc receptor binding or effector function.

[0194] Fc domains with reduced Fc receptor binding and / or effector function include those in which one or more Fc domain residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Patent No. 6,737,056). Such Fc variants include so-called "DANA" Fc variants with substitutions at alanine residues 265 and 297, as well as Fc variants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581).

[0195] In another embodiment, the Fc domain is the IgG4 Fc domain. Compared to the IgG1 antibody, the IgG4 antibody has reduced binding affinity to the Fc receptor and reduced effector function. In a more specific embodiment, the Fc domain is the IgG4 Fc domain containing an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. In a more specific embodiment, the Fc domain is the IgG4 Fc domain containing amino acid substitutions L235E, S228P, and P329G (EU numbering). The binding properties of such IgG4 Fc domain variants and their Fcγ receptors are also described in International Publication No. 2012 / 130831.

[0196] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of coding DNA sequences, PCR, gene synthesis, etc. Correct nucleotide changes can be confirmed, for example, by sequencing.

[0197] Binding to Fc receptors can be readily identified, for example, by ELISA or surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare), and Fc receptors can be obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain or a cell-activating antibody containing an Fc domain to an Fc receptor can be evaluated using cell lines known to express a specific Fc receptor, such as human NK cells expressing the FcγIIIa receptor.

[0198] The effector function of the Fc domain, or the antigen-binding molecule of the present invention containing the Fc domain, can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for evaluating the ADCC activity of the molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al., Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166,1351-1361 (1987). Alternatively, non-radioactive assay methods can be employed (see, for example, ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition to the above, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, for example, as disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0199] In some embodiments, the binding of the Fc domain to complement components, particularly C1q, is reduced. Therefore, in some embodiments where the Fc domain is designed to have reduced effector function, this reduction in effector function includes a reduction in CDC activity. C1q binding assays may be performed to determine whether the bispecific antibodies of the present invention can bind to C1q and thus possess CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and International Publication Nos. 2005 / 100402. CDC assays may also be performed to assess complement activation (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0200] In a particular embodiment, the Fc domain exhibiting reduced binding affinity to the Fc receptor and / or reduced effector function compared to the native IgG1 Fc domain is a human IgG1 Fc domain containing the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain containing the amino acid substitutions S228P, L235E and optionally P329G (numbered according to the Kabat EU index). More specifically, it is a human IgG1 Fc domain containing the amino acid substitutions L234A, L235A and P329G (numbered according to the Kabat EU index).

[0201] Fc domain modification that promotes heterodimerization Antibodies that specifically bind to BCMA and CD28 contain different antigen-binding sites fused to one or the other of two subunits of the Fc domain, and therefore the two subunits of the Fc domain may be contained in two non-identical polypeptide chains. Several combinations of the two polypeptides are possible through recombinant co-expression of these polypeptides and subsequent dimerization. To increase the yield and purity of the bispecific antigen-binding molecules of the present invention in recombinant production, it is advantageous to introduce modifications to the Fc domain of the antibodies described herein that promote the association of the desired polypeptides.

[0202] Accordingly, in certain embodiments, antibodies are provided that specifically bind to BCMA and CD28, comprising an Fc domain composed of first and second subunits capable of stable association, wherein the modification includes a modification in which the Fc domain promotes the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is located within the CH3 domain of the Fc domain. Accordingly, in one embodiment, the modification is located within the CH3 domain of the Fc domain.

[0203] In a specific embodiment, the modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. Thus, an antibody is provided that specifically binds to BCMA and CD28 and comprises an Fc domain composed of first and second subunits capable of stable association, each containing one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor, wherein the first subunit of the Fc domain contains a knob and the second subunit of the Fc domain contains a hole by the knob-into-hole method. In a particular embodiment, the first subunit of the Fc domain contains amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain contains amino acid substitutions Y349C, T366S and Y407V (Kabat EU index numbering).

[0204] The knob-into-hole technique is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996); and Carter, J Immunol Meth 248,7-15 (2001). Generally, this method involves introducing a projection ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide. As a result, the projection may be positioned within the cavity to promote heterodimerization and inhibit homodimerization. The projection is constructed by replacing a smaller amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the projection is created at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0205] Accordingly, in one embodiment, in the CH3 domain of the first subunit of the Fc domain of an antibody that specifically binds to BCMA and CD28, an amino acid residue is replaced with an amino acid residue having a larger side-chain volume, thereby generating a repositionable protrusion within the CH3 domain of the first subunit within a cavity in the CH3 domain of the second subunit; and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side-chain volume, thereby generating a cavity within the CH3 domain of the second subunit, within which the protrusion in the CH3 domain of the first subunit is repositionable. The protrusion and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In certain embodiments, the threonine residue at position 366 in the CH3 domain of the first subunit of the Fc domain is replaced with a tryptophan residue (T366W), and the tyrosine residue at position 407 in the CH3 domain of the second subunit of the Fc domain is replaced with a valine residue (Y407V). In one embodiment, the threonine residue at position 366 in the second subunit of the Fc domain is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0206] In a further embodiment, the serine residue at position 354 in the first subunit of the Fc domain is further replaced with a cysteine ​​residue (S354C), and the tyrosine residue at position 349 in the second subunit of the Fc domain is further replaced with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues leads to the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter (2001), J Immunol Methods 248, 7-15 (2001)). In a specific embodiment, the first subunit of the Fc domain contains amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain contains amino acid substitutions Y349C, T366S and Y407V (Kabat EU index numbering).

[0207] In alternative embodiments, modifications that facilitate the association of the first and second subunits of the Fc domain include modifications that intervene in the electrostatic maneuvering effect, as described, for example, in PCT International Publication No. 2009 / 089004. Generally, this method involves the substitution of one or more amino acid residues by charged amino acid residues at the interface of the two Fc domain subunits such that homodimerization is electrostatically undesirable, but heterodimerization is electrostatically desirable.

[0208] The C-terminus of the heavy chain of the antibodies reported herein may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two of the C-terminal amino acid residues are removed. In one preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus ending with P. In one preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one of the embodiments reported herein, the CD28 antigen-binding molecule containing a heavy chain with the C-terminal CH3 domain specified herein contains a C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one of the embodiments reported herein, the CD28 antigen-binding molecule containing a heavy chain with the C-terminal CH3 domain specified herein contains a C-terminal glycine residue (G446, numbered according to the Kabat EU index).

[0209] Modifications in the Fab domain In one embodiment, an antibody is provided that specifically binds to BCMA and CD28, characterized by monovalent binding to BCMA and CD28, comprising (a) a first antigen-binding domain capable of specific binding to BCMA, (b) a second antigen-binding domain capable of specific binding to CD28, and (c) an Fc domain composed of first and second subunits capable of stable association including one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor, wherein the second antigen-binding domain capable of specific binding to CD28 is a Fab fragment, and in the Fab fragment, either the variable domains VH and VL or the constant domains CH1 and CL are exchanged according to Crossmab technology.

[0210] In another embodiment, an antibody is provided that specifically binds to BCMA and CD28, characterized by monovalent binding to BCMA and CD28, comprising (a) a first antigen-binding domain capable of specific binding to BCMA, (b) a second antigen-binding domain capable of specific binding to CD28, and (c) an Fc domain composed of first and second subunits capable of stable association including one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor, wherein the first antigen-binding domain capable of specific binding to BCMA is a Fab fragment, and in the Fab fragment, either the variable domains VH and VL or the constant domains CH1 and CL are exchanged according to Crossmab technology.

[0211] Multispecific antibodies with domain substitution / exchange in one binding arm (CrossMabVH-VL or CrossMabCH-CL) are described in detail in International Publication No. 2009 / 080252 and Schaefer, W. et al., PNAS, 108(2011)11187-1191. These multispecific antibodies significantly reduce byproducts resulting from mismatches between the light chain for the primary antigen and the incorrect heavy chain for the secondary antigen (compared to approaches without such domain exchange).

[0212] In one embodiment, the present invention relates to an antibody that specifically binds to BCMA and CD28, comprising (a) a first antigen-binding domain capable of specific binding to BCMA, (b) a second antigen-binding domain capable of specific binding to CD28, and (c) an Fc domain composed of first and second subunits capable of stable association including one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor, wherein in the Fab fragment capable of specific binding to CD28, the variable domains VL and VH are substituted for each other such that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In another embodiment, the present invention relates to an antibody that specifically binds to BCMA and CD28, comprising (a) a first antigen-binding domain capable of specific binding to BCMA, (b) a second antigen-binding domain capable of specific binding to CD28, and (c) an Fc domain composed of first and second subunits capable of stable association including one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor, wherein in the Fab fragment capable of specific binding to BCMA, the variable domains VL and VH are substituted for each other such that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

[0213] In another embodiment, to further improve correct pairing, an antibody that specifically binds to BCMA and CD28 (characterized by monovalent binding to CD28) comprises (a) a first antigen-binding domain capable of specific binding to BCMA, (b) a second antigen-binding domain capable of specific binding to CD28, and (c) an Fc domain composed of first and second subunits capable of stable association, including one or more amino acid substitutions that reduce the binding affinity and / or effector function of the antigen-binding molecule to the Fc receptor, and may contain different charged amino acid substitutions (so-called "charged residues"). These modifications are introduced into the cross- or non-cross-linked CH1 and CL domains. In a particular embodiment, the present invention relates to an antibody that specifically binds to BCMA and CD28, wherein in one of the CL domains, the amino acid at position 123 (EU numbering) is replaced with arginine (R), the amino acid at position 124 (EU numbering) is replaced with lysine (K), and in one of the CH1 domains, the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are replaced with glutamic acid (E). In a particular embodiment, in the CL domain of a Fab fragment capable of specific binding to CD28, the amino acid at position 123 (EU numbering) is replaced with arginine (R), the amino acid at position 124 (EU numbering) is replaced with lysine (K), and in the CH1 domain of a Fab fragment capable of specific binding to CD28, the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are replaced with glutamic acid (E).

[0214] Polynucleotides The present invention further provides isolated polynucleotides encoding BCMA antibodies or antibodies that specifically bind to BCMA and CD28 as described herein. One or more isolated polynucleotides encoding BCMA antibodies or antibodies that specifically bind to BCMA and CD28 may be expressed as a single polynucleotide encoding a complete antigen-binding molecule, or as a plurality (e.g., two or more) of polynucleotides that are co-expressed. Polypeptides encoded by co-expressed polynucleotides may associate, for example, via disulfide bonds or other means to form a functional antigen-binding molecule. For example, the light chain portion of an immunoglobulin may be encoded by a separate polynucleotide derived from the heavy chain portion of an immunoglobulin. When co-expressed, the heavy chain polypeptide associates with the light chain polypeptide to form an immunoglobulin. In some embodiments, the isolated polynucleotide encodes an entire antibody that specifically binds to BCMA and CD28 as described herein. In other embodiments, the isolated polynucleotide encodes a polypeptide contained in an antibody that specifically binds to BCMA and CD28 as described herein. In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is, for example, RNA in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.

[0215] Recombination method The BCMA antibodies described herein, or antibodies that specifically bind to BCMA and CD28, may be obtained, for example, by solid peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the antibody that specifically binds to BCMA and CD28, or its polypeptide fragment, e.g., those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such polynucleotides can be readily isolated and sequenced using conventional procedures. In one embodiment, a vector, preferably an expression vector, containing one or more of the polynucleotides described herein is provided. Using methods well known to those skilled in the art, an expression vector containing the coding sequence of the antibody (fragment), along with appropriate transcription / translation control signals, can be constructed. Such methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1989). An expression vector may be a plasmid, part of a virus, or a nucleic acid fragment. An expression vector comprises an expression cassette in which a polynucleotide encoding an antibody or its polypeptide fragment (i.e., the coding region) is operably bound to a promoter and / or other transcriptional or translational regulatory elements and cloned. As used herein, “coding region” is a part of a nucleic acid consisting of codons that are translated into amino acids. “Stop codons” (TAG, TGA, or TAA) are not translated into amino acids but may be considered part of the coding region (if present). However, any adjacent sequences, such as promoters, ribosome-binding sites, transcription terminators, introns, and 5' and 3' untranslated regions, are not part of the coding region.Two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector) or in separate polynucleotide constructs (e.g., on separate (different) vectors). Furthermore, any vector may contain a single coding region or two or more coding regions. For example, the vector of the present invention may encode one or more polypeptides that are separated into a final protein post-translation or concurrently with translation by proteolytic cleavage. Furthermore, the vector, polynucleotide, or nucleic acid of the present invention may encode heterologous coding regions that are either fused to or unfused to the antibody of the present invention, or a polynucleotide encoding a polypeptide fragment thereof, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, special elements or motifs, such as secretory signal peptides or heterologous functional domains. An operable association is when the coding region of a gene product, such as a polypeptide, associates with one or more regulatory sequences in such a way that the expression of the gene product is under the influence or control of one or more regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter bound to it) are "operably bound" if the induction of promoter function results in the transcription of mRNA encoding a desired gene product, and the nature of the linkage between the two DNA fragments does not interfere with the ability of an expression regulatory sequence to direct gene product expression or the ability of the DNA template to be transcribed. Therefore, a promoter region can be said to be operably bound to a polypeptide-encoding nucleic acid if the promoter can result in the transcription of that nucleic acid. A promoter may be a cell-specific promoter that directs substantial transcription of DNA only in a given cell. Besides promoters, other transcriptional regulatory elements, such as enhancers, operators, repressors, and transcription termination signals, can operably bind to polynucleotides that direct cell-specific transcription.

[0216] Suitable promoters and other transcriptional regulatory regions are disclosed herein. Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, e.g., promoters and enhancer segments derived from cytomegalovirus (e.g., the initial promoter and intron A), Simian virus 40 (e.g., the initial promoter), and retroviruses (e.g., Roussarcoma virus). Other transcriptional regulatory regions include those derived from vertebrate genes, e.g., actin, heat shock proteins, bovine growth hormone and rabbit α-globin, and other sequences capable of regulating gene expression in eukaryotic cells. Further suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., promoter-induced tetracycline). Similarly, various translational regulatory elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and viral elements (in particular, intrasequence ribosome entry sites, i.e., IRES, also known as CITE sequences). Furthermore, the expression cassette may also include other features such as chromosomal integration elements, including, for example, the origin of replication and / or the long terminal repeat sequence (LTR) of a retrovirus or the inverted terminal sequence (ITR) of an adeno-associated virus (AAV).

[0217] The polynucleotides and nucleic acid coding regions described herein may be coupled with additional coding regions encoding secretory peptides or signal peptides that direct the secretion of polypeptides encoded by the polynucleotides of the present invention. For example, if the secretion of an antibody or a polypeptide fragment thereof is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the antibody or polypeptide fragment thereof as described herein. According to the signaling hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein when the transport of the growing protein chain across the coarse endoplasmic reticulum begins. Those skilled in the art recognize that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce the secretory or “mature” form of the polypeptide. In certain embodiments, native signal peptides, such as immunoglobulin heavy or light chain signal peptides, are used, or functional derivatives of their sequences that retain the ability to induce the secretion of polypeptides operably bound thereto are used. Alternatively, heterologous mammalian signal peptides or functional derivatives thereof may be used. For example, the wild-type leader sequence may be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase. DNA encoding a short protein sequence, which can be used to facilitate subsequent purification (e.g., histidine tagging) or to assist in the labeling of the antibodies described herein, may be included in or at the end of the polynucleotide encoding the antibody or polypeptide fragment described herein.

[0218] In further embodiments, host cells comprising one or more polynucleotides described herein are provided. In specific embodiments, host cells comprising one or more vectors described herein are provided. The polynucleotides and vectors may each incorporate, individually or in combination, any of the features described herein in relation to the polynucleotides and vectors, respectively. In one embodiment, the host cell comprises a vector comprising a polynucleotide encoding (or a portion thereof) of an antibody disclosed herein (e.g., transformed or transfected therewith). As used herein, the term “host cell” refers to any type of cell line that can be manipulated to produce the fusion protein or fragment thereof of the present invention. Host cells suitable for replicating antigen-binding molecules and for assisting the expression of antigen-binding molecules are well known in the art. Such cells can be appropriately transfected or transduced with a specific expression vector, and large quantities of vector-containing cells can be grown and seeded in a large fermenter to obtain sufficient quantities of antibody for clinical application. Suitable host cells include prokaryotic microorganisms (e.g., Escherichia coli) or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, polypeptides may be produced within bacteria, especially when glycosylation is not required. After expression, polypeptides may be isolated from the bacterial cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains with "humanized" glycosylation pathways that produce polypeptides with partially or completely human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006).

[0219] Suitable host cells for (glycosylated) polypeptide expression can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for generating antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human fetal kidney cell line (e.g., 293 cells or 293T cells described in Graham et al., J Gen Virol 36, 59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol Reprod 23, 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); and TRI cells (e.g., Mather et al., Annals NYAcad Sci). These include MRC 5 cells and FS4 cells (described in 383,44-68 (1982)). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77,4216 (1980)), and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0.For an overview of specific mammalian host cells suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, to name just a few, but also cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphocytes (e.g., Y0, NS0, Sp20 cells). Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing polypeptides containing either the heavy or light chain of an immunoglobulin can be engineered to express the other part of the immunoglobulin chain as well, so that the expressed product is an immunoglobulin having both the heavy and light chains.

[0220] In one embodiment, a method is provided for producing an antibody or polypeptide fragment thereof that specifically binds to BCMA and CD28, comprising culturing a host cell containing a polynucleotide encoding the antibody or polypeptide fragment provided herein under conditions suitable for expressing the antibody or polypeptide fragment thereof, and recovering the antibody or polypeptide fragment described herein from the host cell (or host cell culture medium).

[0221] In certain embodiments, an antigen-binding domain capable of specific binding to a BCMA (e.g., a Fab fragment) forming part of an antibody includes at least an immunoglobulin variable region capable of binding to an antigen. The variable region may form part of an antibody or fragment thereof that is naturally occurring or not naturally occurring, and may be derived from them. Methods for producing polyclonal and monoclonal antibodies are well known in the art (see, for example, Harlow and Lane, "Antibodies, a laboratory manual," Cold Spring Harbor Laboratory, 1988). Antibodies not naturally occurring may be constructed using peptide solid-phase synthesis, produced recombinantly (e.g., as described in U.S. Patent No. 4,186,567), or obtained by screening a combinatorial library containing variable heavy and variable light chains (see, for example, U.S. Patent No. 5,969,108 to McCaffery).

[0222] Immunoglobulins from any animal species can be used for the methods described herein. Useful, non-limiting immunoglobulins may be of mouse, primate, or human origin. If the antibody is intended for use in humans, chimeric immunoglobulins in which the constant region of the immunoglobulin is of human origin may be used. Humanized or fully human forms of immunoglobulins can also be prepared according to methods well known in the art (see, for example, U.S. Patent No. 5,565,332 for Winter). Humanization can be achieved by a variety of methods. Such methods include, but are not limited to, (a) transplanting a non-human (e.g., donor antibody) CDR into the framework and constant regions of a human (e.g., recipient antibody) that either retains or does not retain important framework residues (e.g., those important for maintaining good antigen-binding affinity or antibody function); (b) transplanting only non-human specificity-determining regions (SDR or a-CDR; residues important for antibody-antigen interaction) into the human framework and constant regions; or (c) transplanting the entire non-human variable domain, but "cloaking" them with human-like sections by replacing surface residues.Humanized antibodies and their production methods are outlined, for example, in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and also in Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44,65-92 (1988); Verhoeyen et al., Science 239,1534-1536 (1988); Padlan, Molec Immunol 31(3),169-217 (1994); Kashmiri et al., Methods 36,25-34 (2005) (describes SDR(a-CDR) grafting); Padlan, Mol Immunol 28,489-498 (1991) (describes "resurfacing"); Dall'Acqua et al., Methods 36,43-60 (2005) (describes "FR shuffling"); and Osbourn et al., Methods 36,61-68 (2005) and Klimka et al., Br J Cancer Further details are provided in 83,252-260 (2000) (which describes an "inducible selection" approach to FR shuffling). The specific immunoglobulins according to the present invention are human immunoglobulins. Human antibodies and human variable regions can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol. 5,368-74 (2001) and Lonberg, Curr Opin Immunol 20,450-459 (2008).Human variable regions can form part of human monoclonal antibodies produced by hybridoma technology, and may be derived from such antibodies (see, for example, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions can also be prepared by administering immunogens to transgenic animals modified to produce intact human antibodies or intact antibodies containing human variable regions in response to antigen challenge (see, for example, Lonberg, Nat Biotech 23, 1117-1125 (2005)). Human antibodies and human variable regions can also be generated by isolating selected Fv clone variable region sequences from human-derived phage display libraries (see, for example, Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phages typically present antibody fragments either as single-chain Fv (scFv) fragments or as Fab fragments.

[0223] In certain embodiments, the antigen-binding domains contained in the antibodies described herein are manipulated to enhance binding affinity, for example, according to the methods disclosed in PCT International Publication No. 2012 / 020006 (see examples relating to affinity maturation) or U.S. Patent Publication No. 2004 / 0132066. The binding ability of the antibodies of the present invention to a particular antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (Liljeblad, et al., Glyco J 17, 323-329 (2000)) and classical binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competitive assays can be used to identify antigen-binding molecules that compete with the reference antibody for binding to a particular antigen. In certain embodiments, such competitive antigen-binding molecules bind to the same epitope (e.g., a linear epitope or conformational epitope) bound by a reference antigen-binding molecule. Detailed exemplary methods for mapping the epitopes to which antigen-binding molecules bind are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In an exemplary competitive assay, an immobilized antigen is incubated in a solution containing a first labeled antigen-binding molecule that binds to the antigen and a second unlabeled antigen-binding molecule that is being tested for its ability to compete with the first antigen-binding molecule for binding to the antigen. The second antigen-binding molecule may be present in the hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution containing the first labeled antigen-binding molecule but not the second unlabeled antigen-binding molecule. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed, and the amount of label associated with the immobilized antigen is measured. If the amount of label bound to the immobilized antigen is substantially reduced in the test sample compared to the control sample, it indicates that the second antigen-binding molecule is competing with the first antigen-binding molecule for binding to the antigen.See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0224] The bispecific antibodies prepared herein can be purified by techniques known in the art, such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify specific proteins will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be obvious to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the antigen-binding molecule binds can be used. For example, a matrix containing protein A or protein G may be used to purify the antigen-binding molecule of the present invention by affinity chromatography. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate the antigen-binding molecule, essentially as described in the examples. The purity of the antibody or its fragments can be determined by any of a variety of well-known analytical methods, including gel electrophoresis and high-pressure liquid chromatography. For example, the bispecific antibodies expressed as described in the examples have been shown to be intact and well-assembled, as demonstrated by reduced and unreduced SDS-PAGE.

[0225] Assay The BCMA antibodies or antibodies that specifically bind to BCMA and CD28 provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0226] 1. Affinity assay The affinity of the antigen-binding molecules provided herein to the corresponding target can be determined by surface plasmon resonance (SPR) using standard instruments such as a Proteon instrument (Bio-rad) and receptor or target protein, such as those obtained by recombinant expression, according to the method described in the Examples. The affinity of the antigen-binding molecule to the target cell antigen can also be determined by surface plasmon resonance (SPR) using standard instruments such as a Proteon instrument (Bio-rad), and receptor or target protein, such as those obtained by recombinant expression. According to one embodiment, K D This is measured using a Proteon® instrument (Bio-Rad) by surface plasmon resonance at 25°C.

[0227] 2. Binding assays and other assays The binding of antibodies or bispecific antibodies provided herein to cells expressing the corresponding receptors can be evaluated, for example, by flow cytometry (FACS) using cell lines expressing specific receptors or target antigens. In one embodiment, human CD28-expressing CHO cells (a parent cell line CHO-k1 ATCC#CCL-61 modified to stably overexpress human CD28) are used in the binding assay.

[0228] In a further embodiment, the binding of a bispecific antigen-binding molecule to this target cell antigen was demonstrated using cancer cell lines expressing BCMA.

[0229] 3. Activity assay In one embodiment, an assay is provided for identifying the biological activity of antibodies that specifically bind to BCMA and CD28. Examples of biological activity include T cell proliferation and cytokine secretion, as measured by the method described in Example 4. Antigen-binding molecules having such biological activity in vivo and / or in vitro are also provided.

[0230] Pharmaceutical compositions, formulations, and routes of administration In further embodiments, the present invention provides a pharmaceutical composition comprising one of the antibodies specifically binding to BCMA and CD28 provided herein for use in, for example, any of the following therapeutic methods. In one embodiment, the pharmaceutical composition comprises the antibodies specifically binding to BCMA and CD28 provided herein and at least one pharmaceutically acceptable additive. In another embodiment, the pharmaceutical composition comprises the antibodies specifically binding to BCMA and CD28 provided herein and at least one additional therapeutic agent, for example, described below.

[0231] The pharmaceutical compositions disclosed herein comprise one or more antigen-binding molecules in a therapeutically effective amount dissolved or dispersed in pharmaceutically acceptable additives. The expression “pharmaceutically acceptable” means molecular entities and compositions that are generally non-toxic to the recipient at the doses and concentrations used, i.e., that do not cause adverse reactions, allergic reactions, or other adverse reactions when administered to animals, such as humans. The preparation of pharmaceutical compositions containing at least one antibody specifically binding to BCMA and CD28, and optionally additional active ingredients, will be known to those skilled in the art in the view of this disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. In particular, compositions are lyophilized formulations or aqueous solutions. As used herein, “pharmaceutically acceptable additives” include, as known to those skilled in the art, any solvent, buffer, dispersion medium, coating agent, surfactant, antioxidant, preservative (e.g., antimicrobial, antifungal), isotonic agent, salt, stabilizer, and combinations thereof.

[0232] Parenteral compositions include those designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the antibody or bispecific antibody may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulations such as suspensions, stabilizers, and / or dispersants. Alternatively, antibodies that specifically bind to BCMA and CD28 may be in powder form for formulation in a suitable vehicle (e.g., pyrogen-free sterile water) before use. Sterile injectable solutions are prepared by incorporating the required amount of the fusion protein of the present invention, along with various other components listed below if necessary, into a suitable solvent. Sterilization can be easily achieved, for example, by filtration with a sterile filtration membrane. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and / or other components. For sterile powders used to prepare sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying, which yields powders of the active ingredient and any further desired ingredients from an already sterile filtered liquid medium. The liquid medium should be appropriately buffered if necessary, and the liquid diluent should be first isotonicized with sufficient saline or glucose before injection. The composition must be stable under manufacturing and storage conditions and protected from microbial contamination such as bacteria and fungi. It will be recognized that endotoxin contamination should be kept to a safe level, for example, less than 0.5 ng / mg of protein.Appropriate pharmaceutically acceptable additives include, but are not limited to, buffering agents such as phosphates, citrates and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), and low molecular weight (less than approximately 10 residues) polypeptides. Examples of active compounds include proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). The aqueous injection suspension may also contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain appropriate stabilizers or agents that increase the solubility of the compounds to enable the preparation of highly concentrated solutions. Furthermore, the suspension of the active compound may be prepared as a suitable oil injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethylcrete or triglycerides, or liposomes.

[0233] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation technology or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), or encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such technologies are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. A suitable example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing a polypeptide, which may be in the form of a molded article, such as a film or microcapsule. In certain embodiments, sustained absorption of the injectable composition may be achieved by using an absorption-delaying agent in the composition, such as aluminum monostearate, gelatin, or a combination thereof. Exemplary pharmaceutically acceptable additives of the present invention further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Specific exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Applications Publications 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycans (e.g., chondroitinases). Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and International Publication No. 2006 / 044908, the latter of which includes histidine-acetate buffer. In addition to the aforementioned compositions, antibodies that specifically bind to BCMA and CD28 may be incorporated as depot formulations.Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Therefore, for example, antibodies that specifically bind to BCMA and CD28 can be formulated using suitable polymer or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as less soluble derivatives, such as less soluble salts.

[0234] Pharmaceutical compositions containing antibodies that specifically bind to BCMA and CD28 can be manufactured by conventional mixing, dissolution, emulsification, encapsulation, encapsulation, or lyophilization processes. The pharmaceutical compositions can be formulated in conventional ways using one or more physiologically acceptable carriers, diluents, additives, or auxiliaries that facilitate the processing of the protein into a pharmaceutically usable formulation. The appropriate formulation depends on the selected route of administration. Antibodies that specifically bind to BCMA and CD28 can be formulated in the composition in the form of free acids or free bases, neutral or salts. A pharmaceutically acceptable salt is one that substantially retains the biological activity of the free acid or free base. These include acid addition salts, such as those formed with free amino groups of a proteinaceous composition, or those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, and ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, and procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than their corresponding free base forms. The compositions herein may also contain multiple active ingredients necessary for the specific indication being treated, preferably having complementary activities that do not adversely affect each other. Such active ingredients are appropriately combined in amounts effective for the intended purpose. Formulations used for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtration with a sterile filtration membrane.

[0235] Treatment method and composition Any of the antibodies provided herein that specifically bind to BCMA and CD28 can be used alone or in combination in a therapeutic method.

[0236] In one embodiment, antibodies that specifically bind to BCMA and CD28 for use as pharmaceuticals are provided. In a further embodiment, antibodies that specifically bind to BCMA and CD28 for use in the treatment of cancer are provided. In a particular embodiment, antibodies that specifically bind to BCMA and CD28 for use in the treatment of hematological malignancies are provided. The term “hematological malignancies” includes diseases selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), and Hodgkin lymphoma, but also includes acute myeloid leukemia and acute lymphoblastic leukemia. In a particular embodiment, antibodies that specifically bind to BCMA and CD28 for use in the treatment of multiple myeloma (MM) are provided.

[0237] In certain embodiments, antibodies that specifically bind to BCMA and CD28 are provided for use in a therapeutic method. In certain embodiments, antibodies that specifically bind to BCMA and CD28 are provided for use in a method of treating an individual having cancer, comprising administering an effective amount of the antibody that specifically binds to BCMA and CD28 to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

[0238] In one embodiment, the antibodies described herein that specifically bind to BCMA and CD28 are intended for use in inhibiting the proliferation of cancer cells that express BCMA.

[0239] In certain embodiments, antibodies that specifically bind to BCMA and CD28 are provided for use in a therapeutic method. In certain embodiments, antibodies that specifically bind to BCMA and CD28 are provided for use in a method of treating an individual having cancer, comprising administering an effective amount of the antibody that specifically binds to BCMA and CD28 to the individual. In other embodiments, antibodies that specifically bind to BCMA and CD28 are provided for use in a method of treating an individual having cancer expressing BCMA, particularly hematological malignancies selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia, acute B lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, acute myeloid leukemia and acute lymphoblastic leukemia, comprising administering an effective amount of the antibody that specifically binds to BCMA and CD28 to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

[0240] In further embodiments, the use of antibodies specifically binding to BCMA and CD28 as described herein in the manufacture or preparation of a pharmaceutical is provided herein. In one embodiment, the pharmaceutical is for the treatment of cancer, in particular cancer expressing BCMA. In further embodiments, the pharmaceutical is for use in a method of treating cancer, comprising administering an effective amount of the pharmaceutical to an individual having cancer. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, e.g., as described below, to the individual. In another embodiment, the pharmaceutical is for the treatment of cancer expressing BCMA. In further embodiments, the pharmaceutical is for use in a method of treating cancer, in particular cancer expressing BCMA, comprising administering an effective amount of the pharmaceutical to an individual having cancer. In further embodiments, a method for treating cancer, in particular cancer expressing BCMA is provided herein. In one embodiment, the method comprises administering an effective amount of antibodies specifically binding to BCMA and CD28 to an individual having cancer. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, as described below. An "individual" in any of the above-described manner may be a human being.

[0241] In further embodiments, pharmaceutical formulations are provided herein that include, for example, one of the antibodies that specifically bind to BCMA and CD28 as reported herein, for use in any of the therapeutic methods described above. In one embodiment, the pharmaceutical formulation includes the antibodies that specifically bind to BCMA and CD28 as reported herein, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation includes the antibodies that specifically bind to BCMA and CD28 as reported herein, and at least one additional therapeutic agent.

[0242] The antibodies that specifically bind to BCMA and CD28 reported herein may be used in therapy either alone or in combination with other agents. For example, the antibodies that specifically bind to BCMA and CD28 reported herein may be co-administered with at least one additional therapeutic agent. Thus, antibodies that specifically bind to BCMA and CD28 described herein for use in cancer immunotherapy are provided. In certain embodiments, antibodies that specifically bind to BCMA and CD28 for use in a method of cancer immunotherapy are provided. The “individual” in any of the above embodiments is preferably a human.

[0243] Such combination therapies as described above include combined administration (containing two or more therapeutic agents in the same or separate formulations) and separate administrations, in which case, administration of the antibodies reported herein may occur before, concurrently with, and / or after the administration of the additional therapeutic agent(s). In one embodiment, the administration of antibodies that specifically bind to BCMA and CD28 and the administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days.

[0244] The antigen-binding molecules (and any additional therapeutic agents) reported herein may be administered by any preferred means, including parenteral, intrapulmonary, intranasal, and, if desired for topical treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any preferred route, e.g., by injection, such as intravenous or subcutaneous injection, depending in part whether the administration is short-term or chronic. Various dosing schedules, including but not limited to single or multiple doses at various time points, bolus administration, and pulse infusion, are envisioned herein.

[0245] Antibodies specifically binding to BCMA and CD28 as described herein may be formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the individual patient's clinical symptoms, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to the physician. Antibodies specifically binding to BCMA and CD28 may, but not necessarily, be formulated together with one or more other agents used concurrently to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors mentioned above. These are generally used by the same dosages and routes of administration as described herein, or at about 1–99% of the dosages described herein, or by any dosage and route as empirically / clinically deemed appropriate.

[0246] For the prevention or treatment of disease, the appropriate dosage of the antibodies specifically binding to BCMA and CD28 described herein (when used alone or in combination with one or more other additional therapeutic agents) will be determined by the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapies, the patient's medical history, response to the antibody, and the discretion of the attending physician. Antibodies specifically binding to BCMA and CD28 are preferably administered to the patient in a single dose or over a series of treatments. Depending on the type and severity of the disease, for example, whether administered as one or multiple separate doses or in a series of infusions, a bispecific agonist CD28 antigen-binding molecule in a dose of approximately 1 μg / kg to 15 mg / kg (e.g., 0.5 mg / kg to 10 mg / kg) may be the initial candidate dose for administration to the patient. A typical daily dose may range from approximately 1 μg / kg to 100 mg / kg, depending on the factors described above. In repeated administrations over several days or more, treatment is usually continued, depending on the symptoms, until the desired suppression of disease symptoms occurs. One exemplary dose of antibody may range from about 0.05 mg / kg to about 10 mg / kg. Therefore, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives about 2 to about 20 doses, or for example, about 6 doses of antibody). A higher loading dose may be administered first, followed by one or more lower doses. However, other drug regimens may be useful. The progress of this therapy is readily monitored by conventional techniques and assays.

[0247] Other drugs and treatments As described above, antibodies that specifically bind to BCMA and CD28 may be administered in combination with one or more other agents in therapy. For example, the antigen-binding molecules described herein may be co-administered with at least one additional therapeutic agent. The term “therapeutic agent” encompasses any agent that may be administered to treat a symptom or disease in an individual requiring such treatment. Such additional therapeutic agents may include active ingredients suitable for the specific indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is another anticancer agent, such as a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent. In certain embodiments, the additional therapeutic agent is an immunomodulator, a cell proliferation inhibitor, a cell adhesion inhibitor, a cytotoxic agent or cell proliferation inhibitor, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis-inducing factors.

[0248] Accordingly, the present invention provides for use in the treatment of cancer antibodies that specifically bind to BCMA and CD28, or pharmaceutical compositions containing them, which are administered in combination with chemotherapeutic agents, radiotherapy and / or other agents for use in cancer immunotherapy.

[0249] Such other agents are preferably present in combination in amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of fusion protein used, the type of disorder or treatment, and the other factors mentioned above. The bispecific antigen-binding molecules or antibodies of the present invention are typically used in the same doses and routes of administration described herein, or in 1-99% of the doses described herein, or in any dose and route that is experimentally / clinically deemed appropriate. Such combination therapies described above include combined administration (containing two or more therapeutic agents in the same or separate compositions) and separate administration (where the bispecific antigen-binding molecules or antibodies of the present invention may be administered before, concurrently with, and / or after the administration of additional therapeutic agents and / or adjuvants).

[0250] In a further embodiment, antibodies are provided that specifically bind to BCMA and CD28 as described herein for use in the treatment of cancer, particularly cancer expressing BCMA, wherein a bispecific antigen-binding molecule is administered in combination with another immunomodulator. The term “immunomodulator” refers to any substance, including monoclonal antibodies, that affects the immune system. The molecules of the present invention may be considered immunomodulators. Immunomodulators may be used as antitumor agents for the treatment of cancer. In one embodiment, immunomodulators include anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD1 antibodies (e.g., nivolumab or pembrolizumab), PD-L1 antibodies (e.g., atezolizumab, avelumab or durvalumab), OX-40 antibodies, 4-1BB antibodies, and GITR antibodies. Such combination therapies as described above include combined administration (where two or more therapeutic agents are contained in the same or separate compositions) and separate administration, in which case the administration of the bispecific antigen-binding molecule may be performed before, concurrently with, and / or after the administration of further therapeutic agents and / or adjuvants.

[0251] Combination with T cell bispecific antibody In one embodiment, an antibody that specifically binds to BCMA and CD28 may be administered in combination with a T-cell activating anti-CD3 bispecific antibody. The T-cell activating anti-CD3 bispecific antibody is specific to a tumor-associated antigen, such as GPRC5D, CD38, FcRH5, or BCMA. In one embodiment, the T-cell activating anti-CD3 bispecific antibody is an anti-GPRC5D / anti-CD3 bispecific antibody.

[0252] In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) containing (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 145, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 146, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 147. HGPRC5D) and a light chain variable region (V) containing (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 148, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO: 149, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO: 150. L The antibody comprises at least one antigen-binding domain capable of specific binding to GPRC5D, including GPRC5D. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 151, 153, 154, and 155. H GPRC5D) and a light chain variable region (V) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 152, 156, 157, 158, 159 and 160. L It includes at least one antigen-binding domain capable of specific binding to GPRC5D, including GPRC5D.

[0253] In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody has a heavy chain variable region (V) that contains an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 151. H GPRC5D) and a light chain variable region (V) containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 152. L It includes at least one antigen-binding domain capable of specific binding to GPRC5D, including GPRC5D. In particular, the antigen-binding domain capable of specific binding to GPRC5D is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 151. H Light chain variable region (V) containing the amino acid sequence of GPRC5D) and SEQ ID NO: 152 L Includes GPRC5D.

[0254] In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) containing (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 161, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 162, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 163.H GPRC5D) and a light chain variable region (V) containing (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 164, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO: 165, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO: 166. L The antibody comprises at least one antigen-binding domain capable of specific binding to GPRC5D, including GPRC5D. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 167, 168, 169, 170, 171, and 172. H GPRC5D) and a light chain variable region (V) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 173, 174, 175, 176, and 177. L The antibody comprises at least one antigen-binding domain capable of specific binding to GPRC5D, including GPRC5D. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody comprises at least one antigen-binding domain capable of specific binding to GPRC5D, wherein the antigen-binding domain is (a) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 151 (V H Light chain variable region (V) containing the amino acid sequence of GPRC5D) and SEQ ID NO: 152 L GPRC5D), or (b) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 155 (V H Light chain variable region (V) containing the amino acid sequence of GPRC5D) and SEQ ID NO: 158 L GPRC5D), or (c) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 151 (V H Light chain variable region (V) containing the amino acid sequence of GPRC5D) and SEQ ID NO: 158 L GPRC5D), or (d) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 167 (V H Light chain variable region (V) containing the amino acid sequence of GPRC5D) and SEQ ID NO: 175 L GPRC5D), or (e) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 169 (VH Light chain variable region (V) containing the amino acid sequence of GPRC5D) and SEQ ID NO: 174 L Includes GPRC5D.

[0255] In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) containing (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 178, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 179, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 180. H CD3) and a light chain variable region (V) containing (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 181, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO: 182, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO: 183. L It includes an antigen-binding domain capable of specific binding to CD3, which includes CD3.In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody has a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 184. H CD3) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 185 L It contains an antigen-binding domain capable of specific binding to CD3, including CD3.

[0256] In another embodiment, the provided anti-GPRC5D / anti-CD3 bispecific antibody comprises a heavy chain variable region (V) containing (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 186, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 187, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 188. H CD3) and a light chain variable region (V) containing (iv) CDR-L1 containing the amino acid sequence of SEQ ID NO: 189, (v) CDR-L2 containing the amino acid sequence of SEQ ID NO: 190, and (vi) CDR-L3 containing the amino acid sequence of SEQ ID NO: 191. L It includes an antigen-binding domain capable of specific binding to CD3, which includes CD3. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody includes a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 192. H CD3) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 193 LIt contains an antigen-binding domain capable of specific binding to CD3, including CD3.

[0257] In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody has a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 184. H CD3) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 185 L It includes an antigen-binding domain capable of specific binding to CD3 (including CD3) and two antigen-binding domains capable of specific binding to GPRC5D, and each antigen-binding domain capable of specific binding to GPRC5D contains a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 151 H GPRC5D) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 152 L The anti-GPRC5D / anti-CD3 bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 137, two polypeptides that are at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 138, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 139, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 140. In a further specific embodiment, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 137, the two polypeptide sequences of SEQ ID NO: 138, the polypeptide sequence of SEQ ID NO: 139, and the polypeptide sequence of SEQ ID NO: 140 (GPRC5D CD3 TCB). In a further specific embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody is forimtamig.

[0258] In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 141, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 142, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 143, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 144. In one embodiment, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 141, the polypeptide sequence of SEQ ID NO: 142, the polypeptide sequence of SEQ ID NO: 143, and the polypeptide sequence of SEQ ID NO: 144 (GPRC5D CD3 1+1 bispecific antibody).

[0259] In another embodiment, the T cell activating anti-CD3 bispecific antibody is an anti-BCMA / anti-CD3 bispecific antibody. In one embodiment, the anti-BCMA / anti-CD3 bispecific antibody includes the amino acid sequence of SEQ ID NO: 198, the two amino acid sequences of SEQ ID NO: 199, the amino acid sequence of SEQ ID NO: 200, and the amino acid sequence of SEQ ID NO: 201. In another embodiment, the anti-BCMA / anti-CD3 bispecific antibody includes the amino acid sequence of SEQ ID NO: 202, the amino acid sequence of SEQ ID NO: 203, the amino acid sequence of SEQ ID NO: 204, and the amino acid sequence of SEQ ID NO: 205. In yet another embodiment, the anti-BCMA / anti-CD3 bispecific antibody includes the amino acid sequence of SEQ ID NO: 206, the amino acid sequence of SEQ ID NO: 207, the amino acid sequence of SEQ ID NO: 208, and the amino acid sequence of SEQ ID NO: 209. In one embodiment, the anti-BCMA / anti-CD3 bispecific antibody is selected from the group consisting of Alnuctamab, Elranatamab, and Teclistamab.

[0260] In a further embodiment, the T-cell activating anti-CD3 bispecific antibody is an anti-FcRH5 / anti-CD3 bispecific antibody. Anti-FcRH5 / anti-CD3 bispecific antibodies are described, for example, in International Publication No. 2016 / 205520.

[0261] Such combination therapies described above include combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case the administration of a therapeutic agent may be performed before, simultaneously with, and / or after the administration of an additional therapeutic agent or drug. In one embodiment, the administration of a therapeutic agent and the administration of an additional therapeutic agent are performed within approximately one month of each other, or within approximately one, two, or three weeks, or within approximately one, two, three, four, five, or six days.

[0262] manufactured goods In another aspect of the present invention, a product is provided containing a substance useful for the treatment, prevention and / or diagnosis of the above-mentioned disorders. The product comprises a container and a label or accompanying information attached to or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, etc. The container may be formed from a variety of materials such as glass or plastic. The container holds a composition effective for the treatment, prevention and / or diagnosis of a symptom, either alone or in combination with another composition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured with a subcutaneous needle). At least one activator in the composition is an antibody that specifically binds to BCMA and CD28 as disclosed herein. The label or accompanying information indicates that the composition is used to treat a selected symptom. Furthermore, the product may comprise (a) a first container containing a composition comprising antibodies that specifically bind to BCMA and CD28; and (b) a second container containing a composition comprising further cytotoxic agents or other therapeutic agents. The product in this embodiment of the present invention may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition thereto, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. This may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. [Table 2] TIFF2026522235000004.tif249170TIFF2026522235000005.tif250170TIFF2026522235000006.tif249170TIFF2026522235000007.tif249170TIFF2026522235000008.tif252170TIFF2026522235000009.tif252170TIFF2026522235000010.tif252170TIFF2026522235000011.tif253170TIFF2026522235000012.tif249170TIFF2026522235000013.tif252170TIFF2026522235000014.tif253170TIFF2026522235000015.tif249170TIFF2026522235000016.tif252170TIFF2026522235000017.tif251170TIFF2026522235000018.tif252170TIFF2026522235000019.tif253170TIFF2026522235000020.tif250170TIFF2026522235000021.tif250170TIFF2026522235000022.tif252170TIFF2026522235000023.tif252170TIFF2026522235000024.tif252170TIFF2026522235000025.tif252170TIFF2026522235000026.tif156170

[0263] General information regarding the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of the antibody chain are numbered and referenced according to the numbering system by Kabat (Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) as defined above.

[0264] The following numbered sections describe aspects of the present invention.

[0265] 1. An antibody that specifically binds to B cell maturation antigen (BCMA), wherein the antibody includes a first antigen-binding domain, and the first antigen-binding domain is (i) Heavy chain variable region (V) including the heavy chain complementarity determination region of CDR-H1 (GYTFTNYWMH) of SEQ ID NO: 1, CDR-H2 (IIHPNSGSTNYNEKFQG) of SEQ ID NO: 2, and CDR-H3 (GIYDYPFAY) of SEQ ID NO: 3 H BCMA) and, (ii) Light chain variable region (V L BCMA) (a) A VL containing the light chain complementarity determination region of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASSLQS) of SEQ ID NO: 5, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6, or (b) A VL containing the light chain complementarity determination region of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASNLES) of SEQ ID NO: 7, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6, or (c) An antibody comprising a light chain variable region selected from the group consisting of VLs including the light chain complementarity determining regions of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASNLQS) of SEQ ID NO: 8, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6.

[0266] 2.V H BCMA contains an amino acid sequence selected from the group consisting of SEQ ID NO: 9 (VH1a) and SEQ ID NO: 10 (VH1b), and / or V L The antibody described in paragraph 1, wherein BCMA contains an amino acid sequence selected from the group consisting of SEQ ID NO: 11 (VL1f), SEQ ID NO: 12 (VL1a), SEQ ID NO: 13 (VL1b), SEQ ID NO: 14 (VL1c), SEQ ID NO: 15 (VL1d), and SEQ ID NO: 16 (VL1e).

[0267] 3. An antibody that specifically binds to BCMA, wherein the antibody includes a first antigen-binding domain, and the first antigen-binding domain is (a) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 11 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 9 H V containing the amino acid sequences of BCMA and SEQ ID NO: 12 L Antibodies containing BCMA.

[0268] 4. An antibody described in any one of paragraphs 1-3, wherein the first antigen-binding domain is a Fab molecule.

[0269] 5. An antibody described in any one of paragraphs 1 to 4, comprising an Fc domain composed of a first and a second subunit.

[0270] 6. An antibody described in any one of paragraphs 1 to 5, comprising a second antigen-binding domain that specifically binds to a second antigen.

[0271] 7. An antibody as described in any one of paragraphs 1 to 6, wherein the second antigen-binding domain that specifically binds to the second antigen is a Fab molecule, and the variable domains VL and VH or constant domains CL and CH1 of the Fab light chain and Fab heavy chain, particularly the variable domains VL and VH, are substituted for each other.

[0272] 8. The antibody described in any one of paragraphs 1 to 7, wherein the first antigen-binding domain is a Fab molecule, and in the constant domain CL, the amino acid at position 123 (numbered according to the Kabat EU index) is substituted with an amino acid selected from lysine (K), arginine (R), or histidine (H), and the amino acid at position 124 (numbered according to the Kabat EU index) is independently substituted with lysine (K), arginine (R), or histidine (H); and in the constant domain CH1, the amino acid at position 147 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D), and the amino acid at position 213 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index).

[0273] 9. The antibody described in any one of paragraphs 5-8, wherein the Fc domain is IgG, particularly the IgG1 Fc domain.

[0274] 10. An antibody described in any one of paragraphs 5-9, wherein the Fc domain is a human Fc domain.

[0275] 11. An antibody described in any one of paragraphs 5-10, wherein the Fc domain includes a modification that promotes the association of the first and second subunits of the Fc domain.

[0276] 12. The antibody described in paragraph 11, wherein the first subunit of the Fc domain contains amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain contains amino acid substitutions Y349C, T366S and Y407V (Kabat EU index numbering).

[0277] 13. An antibody as described in any one of paragraphs 5 to 12, wherein the Fc domain contains one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

[0278] 14. The Fc domain is the Fc domain of the human IgG1 subclass and includes amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index), as described in any one of paragraphs 5-13.

[0279] 15. An antibody described in any one of paragraphs 6-14, wherein the second antigen is CD28.

[0280] 16. The second antigen-binding domain that specifically binds to CD28 contains the heavy chain variable region (V) which includes the heavy chain complementarity determining regions of CDR-H1 of SEQ ID NO: 17, CDR-H2 of SEQ ID NO: 18, and CDR-H3 of SEQ ID NO: 19. H CD28) and the light chain variable region (V) including the light chain complementarity determination region of CDR-L1 of SEQ ID NO: 20, CDR-L2 of SEQ ID NO: 21, and CDR-L3 of SEQ ID NO: 22 L The antibodies described in paragraph 15, including CD28.

[0281] 17. The second antigen-binding domain that specifically binds to CD28 is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO: 23. H CD28) and the light chain variable region (V) containing the amino acid sequence of sequence number 24(v8). L The antibody described in paragraph 15 or 16, comprising CD28).

[0282] 18. The antibody contains the amino acid sequence of SEQ ID NO: 9. HV containing the amino acid sequences of BCMA and SEQ ID NO: 11 L The first antigen-binding domain contains BCMA, and V contains the amino acid sequence of SEQ ID NO: 23. H V containing the amino acid sequences of CD28 and SEQ ID NO: 24 L An antibody as described in any one of paragraphs 1 to 17, comprising a second antigen-binding domain containing CD28.

[0283] 19. An antibody according to any one of paragraphs 1 to 18, comprising a first light chain containing the amino acid sequence of SEQ ID NO: 25, a first heavy chain containing the amino acid sequence of SEQ ID NO: 26, a second heavy chain containing the amino acid sequence of SEQ ID NO: 27, and a second light chain containing the amino acid sequence of SEQ ID NO: 28.

[0284] 20. Antibodies that specifically bind to B cell maturation antigen (BCMA) and CD28, (A) The first antigen-binding domain is (i) Heavy chain variable region (V H BCMA) (a) VH including the heavy chain complementarity determining regions of CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYADSVKG) of SEQ ID NO: 30, and CDR-H3 (DGYH) of SEQ ID NO: 31, and (b) A heavy chain variable region selected from the group consisting of VH including the heavy chain complementarity determination regions of CDR-H1 (GFTFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYAAPVKG) of SEQ ID NO: 32, and CDR-H3 (DGYH) of SEQ ID NO: 31, (ii) Light chain variable region (V) including the light chain complementarity determination region of CDR-L1 (RASEDIRNGLA) of SEQ ID NO: 33, CDR-L2 (NANSLHT) of SEQ ID NO: 34, and CDR-L3 (EDTSKYPYT) of SEQ ID NO: 35 L A first antigen-binding domain including BCMA, (B) An antibody comprising a second antigen-binding domain that specifically binds to CD28.

[0285] 21.V HBCMA contains an amino acid sequence selected from the group consisting of SEQ ID NO: 36 (VH2a) and SEQ ID NO: 38 (VH1b), and / or V L The antibody described in paragraph 20, wherein BCMA contains an amino acid sequence selected from the group consisting of SEQ ID NO: 37 (VL2a) and SEQ ID NO: 39 (VL1a).

[0286] 22. An antibody that specifically binds to BCMA and CD28, wherein the antibody includes a first antigen-binding domain, and the first antigen-binding domain is (a) V containing the amino acid sequence of SEQ ID NO: 36 H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 38 H V containing the amino acid sequences of BCMA and SEQ ID NO: 39 L Antibodies containing BCMA.

[0287] 23. An antibody described in any one of paragraphs 20-22, wherein the first antigen-binding domain is a Fab molecule.

[0288] 24. An antibody described in any one of paragraphs 20-23, comprising an Fc domain composed of a first and a second subunit.

[0289] 25. An antibody described in any one of paragraphs 20-24, comprising a second antigen-binding domain that specifically binds to a second antigen.

[0290] 26. The antibody described in any one of paragraphs 20-25, wherein the second antigen-binding domain that specifically binds to the second antigen is a Fab molecule, and the variable domains VL and VH or constant domains CL and CH1 of the Fab light chain and Fab heavy chain, particularly the variable domains VL and VH, are substituted for each other.

[0291] 27. The antibody described in any one of paragraphs 20 to 26, wherein the first antigen-binding domain is a Fab molecule, and in the constant domain CL, the amino acid at position 123 (numbered according to the Kabat EU index) is substituted with an amino acid selected from lysine (K), arginine (R), or histidine (H), and the amino acid at position 124 (numbered according to the Kabat EU index) is independently substituted with lysine (K), arginine (R), or histidine (H); and in the constant domain CH1, the amino acid at position 147 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D), and the amino acid at position 213 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index).

[0292] 28. An antibody described in any one of paragraphs 20-27, wherein the Fc domain is IgG, particularly the IgG1 Fc domain.

[0293] 29. An antibody described in any one of paragraphs 24-28, wherein the Fc domain is a human Fc domain.

[0294] 30. An antibody described in any one of paragraphs 24-29, wherein the Fc domain includes a modification that promotes the association of the first and second subunits of the Fc domain.

[0295] 31. The antibody described in paragraph 30, wherein the first subunit of the Fc domain contains the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain contains the amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index).

[0296] 32. An antibody according to any one of paragraphs 24-31, wherein the Fc domain contains one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

[0297] 33. An antibody described in any one of paragraphs 24-32, wherein the Fc domain is the Fc domain of the human IgG1 subclass and contains amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index).

[0298] 34. The second antigen-binding domain that specifically binds to CD28 contains the heavy chain variable region (V) which includes the heavy chain complementarity determining regions of CDR-H1 of SEQ ID NO: 17, CDR-H2 of SEQ ID NO: 18, and CDR-H3 of SEQ ID NO: 19. H CD28) and the light chain variable region (V) including the light chain complementarity determination region of CDR-L1 of SEQ ID NO: 20, CDR-L2 of SEQ ID NO: 21, and CDR-L3 of SEQ ID NO: 22 L An antibody containing CD28, as described in any one of paragraphs 20-33.

[0299] 35. The second antigen-binding domain specifically binds to CD28, and the heavy chain variable region (V) contains the amino acid sequence of SEQ ID NO: 23. H CD28) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 24 L The antibody described in paragraph 34, which includes CD28.

[0300] 36. The antibody contains a first antigen-binding domain, and the first antigen-binding domain contains the amino acid sequence of SEQ ID NO: 36. H V containing the amino acid sequences of BCMA and SEQ ID NO: 37 L The first antigen-binding domain contains BCMA, and V contains the amino acid sequence of SEQ ID NO: 23. H V containing the amino acid sequences of CD28 and SEQ ID NO: 24 L An antibody as described in any one of paragraphs 20-35, comprising a second antigen-binding domain containing CD28.

[0301] 37. An antibody according to any one of paragraphs 20 to 36, comprising a first light chain containing the amino acid sequence of SEQ ID NO: 40, a first heavy chain containing the amino acid sequence of SEQ ID NO: 41, a second heavy chain containing the amino acid sequence of SEQ ID NO: 27, and a second light chain containing the amino acid sequence of SEQ ID NO: 28.

[0302] 38. One or more isolated polynucleotides encoding an antibody described in any one of paragraphs 1-37.

[0303] 39. One or more vectors, particularly expression vectors, containing the polynucleotides described in paragraph 38.

[0304] 40. A host cell containing the polynucleotide described in paragraph 38 or the vector described in paragraph 39.

[0305] 41. A method for producing an antibody that specifically binds to BCMA, comprising: a) culturing host cells described in paragraph 40 under conditions suitable for antibody expression; and optionally b) recovering the antibody.

[0306] 42. An antibody that specifically binds to BCMA, manufactured by the method described in paragraph 41.

[0307] 43. A pharmaceutical composition comprising an antibody described in any one of paragraphs 1 to 37 or 42, and at least one pharmaceutically acceptable additive.

[0308] 44. An antibody described in any one of paragraphs 1 to 37 or 42, or a pharmaceutical composition described in paragraph 43, for use as a pharmaceutical.

[0309] 45. An antibody according to any one of paragraphs 1 to 37 or 42, or a pharmaceutical composition according to paragraph 43, for use in (a) T cell activation or (b) enhancement of T cell effector function.

[0310] 46. ​​An antibody described in any one of paragraphs 1 to 37 or 42, or a pharmaceutical composition described in paragraph 43, for use in the treatment of a disease.

[0311] 47. An antibody or pharmaceutical composition for use as described in paragraph 46, wherein the disease is cancer, particularly multiple myeloma.

[0312] 48. An antibody as described in any one of paragraphs 1 to 37 or 42, or a pharmaceutical composition as described in paragraph 43, for use in the treatment of cancer, which is intended to be administered in combination with other agents for use in chemotherapy, radiotherapy and / or cancer immunotherapy.

[0313] 49. An antibody according to any one of paragraphs 1 to 37 or 42, or a pharmaceutical composition according to paragraph 43, for use in the treatment of cancer, which is intended to be administered in combination with a T cell-activating anti-CD3 bispecific antibody.

[0314] 50. An antibody or pharmaceutical composition for use in paragraph 49, wherein the T cell activating anti-CD3 bispecific antibody is an anti-GPRC5D / anti-CD3 antibody.

[0315] 52. Use of an antibody described in any one of paragraphs 1 to 37 or 42, or use of a pharmaceutical composition described in paragraph 43, in the manufacture of a pharmaceutical for the treatment of a disease, particularly cancer.

[0316] 53. A method for treating a disease in an individual, particularly cancer, comprising administering to the individual an effective amount of an antibody described in any one of paragraphs 1 to 37 or 42, or a pharmaceutical composition described in paragraph 43.

[0317] 54. The method according to paragraph 53, further comprising administering in combination with other agents for use in chemotherapy, radiotherapy and / or cancer immunotherapy, in particular in combination with a T cell-activating anti-CD3 bispecific antibody. [Examples]

[0318] The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments may be practiced, based on the general description provided above.

[0319] Recombinant DNA Techniques: DNA was manipulated using standard methods as described in Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. General information regarding the nucleotide sequences of the light and heavy chains of human immunoglobulins is given below: Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242.

[0320] DNA sequencing: The DNA sequence was determined by double-strand sequencing.

[0321] Gene Synthesis: Where necessary, desired gene segments were generated by PCR using appropriate templates, or synthesized by automated gene synthesis from synthetic oligonucleotides and PCR products at Geneart AG (Regensburg, Germany) or Genscript (New Jersey, USA). Gene segments adjacent to a single restriction endonuclease cleavage site were cloned into standard cloning / sequencing vectors. Plasmid DNA was purified from transformed bacteria and its concentration was measured by UV spectroscopy. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene segments were designed using appropriate restriction sites to enable subcloning into their respective expression vectors. All constructs were designed using 5' terminal DNA sequences encoding leader peptides targeting proteins for secretion in eukaryotic cells.

[0322] Production of IgG and bispecific antibodies: DNA sequences encoding the variable heavy and light chain regions of BCMA antibodies (and, optionally, CD28 antibodies) were cloned into mammalian expression vectors using conventional cloning techniques. The antibodies described herein were produced using a shaking flask in FedBatch mode. Recombinant production was performed by transient transfection of Expi293® cells in defined serum-free medium. ExpiFectamine® 293 Transfection Kit (Gibco) was used for transfection. Cell culture supernatant was collected 7–12 days after transfection.

[0323] Quantification of protein titer: The protein titer of the supernatant sample was determined by affinity chromatography using a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Scientific, Waltham, Massachusetts, USA) with a POROS A 20 μm column, 2.1 × 30 mm (Life Technologies, Carlsbad, California, USA). The supernatant was loaded onto a column equilibrated with 0.2 M Na2HPO4, pH 7.4, and subsequently eluted with 0.1 M citrate, 0.2 M NaCl, pH 2.5. Titer was quantified by measuring the absorbance at 280 nm, and the protein concentration was then calculated by comparing the elution peak area (under the curve) of the analyte with a reference standard curve.

[0324] Alternatively, the Fc-containing construct in the supernatant was quantified by Protein A-HPLC on an Agilent HPLC system equipped with a UV detector. The supernatant was injected into POROS A 20 μm (Applied Biosystems), washed with 10 mM Tris, 50 mM Glycine, 100 mM NaCl, pH 8.0, and eluted with the same buffer at pH 2.0. Titer was quantified by measuring absorbance at 280 nm, and the protein concentration was subsequently calculated by comparing the elution peak area (below the curve) of the analyte with a reference standard curve.

[0325] Purification of IgG and bispecific antibodies: Proteins were purified from cell culture supernatant following standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatant by protein A affinity chromatography (equilibrium buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5 or PBS; elution buffer: 20 mM, 25 mM or 50 mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0, followed immediately by neutralization of the sample pH. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15, #UFC903096), and aggregated proteins were separated from monomeric proteins by size exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0326] Analysis of IgG and bispecific antibodies: The concentration of purified protein was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423. Protein purity and molecular weight were analyzed by CE-SDS in the presence and absence of a reducing agent using LabChip GXII or LabChip GX Touch (Perkin Elmer). Aggregate content was determined by HPLC chromatography at 25°C using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000, Tosoh Bioscience) equilibrated in running buffer (200 mM KH2PO4, 250 mM KCl pH 6.2, 0.02% NaN3). For all molecules, the monomer content in CE-SDS varied from approximately 70% to nearly 100%, and the final quality was good for all molecules with a purity of >90%. In conclusion, all IgGs and bispecific antibodies were produced with good quality.

[0327] Mass Spectrometry: This section describes the characterization of multispecific antibodies (VH / VL CrossMab), including VH / VL exchange, with an emphasis on accurate assembly. The expected primary structure of deglycosylated intact CrossMab and deglycosylated / plasmin-digested CrossMab or deglycosylated / LysC-limited digested CrossMab was analyzed by electrospray ionization mass spectrometry (ESI-MS). VH / VL CrossMab was deglycosylated with N-glycosidase F in phosphate buffer or Tris buffer at 37°C for up to 17 hours at a protein concentration of 1 mg / ml. Plasmin digestion or LysC (Roche)-limited digestion was performed using 100 μg of deglycosylated VH / VL CrossMab in Tris buffer (pH 8) at room temperature for 120 hours and at 37°C for 40 minutes, respectively. Prior to mass spectrometry, the samples were desalted by HPLC using a Sephadex G25 column (GE Healthcare). The total mass was determined by ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

[0328] Determination of binding and binding affinity of multispecific antibodies to each antigen using surface plasmon resonance (SPR) (BIACORE): The binding of the generated antibodies to each antigen was observed by surface plasmon resonance using a BIACORE instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurement, goat anti-human IgG and JIR 109-005-098 antibodies were immobilized on a CM5 chip via amine coupling for antibody presentation against each antigen. Binding was measured in HBS buffer (HBS-P (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, pH 7.4) at 25°C (or alternatively, 37°C). Antigens (R&D Systems or in-house purified) were added to the solution at various concentrations. Association was measured by antigen injection for 80 seconds to 3 minutes, dissociation was measured by washing the tip surface with HBS buffer for 3 to 10 minutes, and the KD value was estimated using a 1:1 Langmuir binding model. Negative control data (e.g., buffer curve) was subtracted from the sample curve to correct for system-specific baseline drift and reduce noise signals. Each Biacore Evaluation Software was used for sensorgram analysis and affinity data calculation.

[0329] Example 1 Production of optimal anti-BCMA antibodies and manufacturing 1.1 Creation of a humanized variant of the anti-BCMA antibody E04 1.1.1 Methodology The anti-BCMA antibody E04 is disclosed in International Publication No. 2012 / 163805 and possesses the VH domain of SEQ ID NO: 42 and the VL domain of SEQ ID NO: 43. Its optimized variant was prepared as described below. A combination of two methodologies was used to identify a suitable human acceptor framework during humanization. Firstly, the mouse input sequence (excised into the variable region) was queried against the BLASTp database of human V and J region sequences using conventional methods. The selection criteria for human acceptor frameworks were sequence homology, same or similar CDR length, and estimated frequency of human germline, as well as conservation of specific amino acids at the VH-VL domain interface. Following the germline identification step, the CDRs of the mouse input sequence were transplanted into the human acceptor framework regions. The differences in each amino acid between these initial CDR grafts and the parental antibody were evaluated for their potential impact on the structural integrity of their respective variable regions, and “reverse mutations” against the parental sequence were introduced whenever deemed appropriate. Structural evaluation was performed using an in-house antibody structural homology modeling protocol implemented with BIOVIA Discovery Studio Environment, version 17R2, based on Fv region homology models of both the parental antibody and the humanized variant. Several humanized variants included "forward mutations," i.e., amino acid exchanges that change the original amino acid occurring at a given CDR position in the parental conjugate to an amino acid found at an equivalent position in the human acceptor germline. The objective is to enhance the overall humaneness of the humanized variants (beyond the framework region) to further reduce the risk of immunogenicity.

[0330] Meanwhile, we used an in-house developed in silico tool to predict the VH-VL domain orientation of paired VH and VL humanized variants (International Publication No. 2016 / 062734). By comparing the results with the predicted VH-VL domain orientation of the parent conjugate, we selected a framework combination that closely resembled the shape of the original antibody. The rationale is to detect the possibility of amino acid exchange in the VH-VL interface region, which could lead to disruptive changes in the pairing of the two domains that could negatively affect binding properties.

[0331] 1.1.2 Selection and Adaptation of Acceptor Frameworks The acceptor framework was selected as shown in Table 1 below. [Table 3]

[0332] The framework region after CDR3 is human IGHJ germline IGHJ4*01(YFDY WGQGTLVTVSS , Sequence ID No. 194) and Human IGKJ Germline IGKJ4*01(LT FGGGTKVEIK, Adapted from Sequence ID No. 195). Sections related to the acceptor framework are underlined.

[0333] Based on structural considerations, reverse mutations from the human acceptor framework to amino acids in the parental clone were introduced at specific locations in the E04 humanized variant. Furthermore, several locations were identified as promising candidates for forward mutations, in which amino acids in the parental conjugate CDR are replaced with amino acids found in the human acceptor germline. The changes are detailed in Table 2 below. [Table 4]

[0334] 1.1.3 T cell epitope prediction To evaluate the potential T cell epitope development in humanized sequences, NetMHCIIpan 4.0 predictions were used (Reynisson B et al: NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data, Nucl. Acids Res., 48(W1):W449-W454 (2020)). Predictions were made for the following human MHC class II alleles: DRB1*01:01, DRB1*03:01, DRB1*04:01, DRB1*07:01, DRB1*08:01, DRB1*09:01, DRB1*11:01, DRB1*13:01, and DRB1*15:01.

[0335] The thresholds for strongly bound and weakly bound 15-mer peptides were set to percentile ranks 1 and 5, respectively. Binding 15-mer peptides with percentile ranks greater than 5 were not considered. Similarly, all binding 15-mer peptides with 9-mer core peptides occurring in 10 or more human V-region germlines were not considered. Germline sequences were obtained from the IMGT database (Giudicelli, V. et al.: IMGT / LIGM-DB, the IMGT® comprehensive database of immunoglobulin and T cell receptor nucleotide sequences. Nucl. Acids Res., 34(S1):D781-D784 (2006)). Since many of the predicted 15-mer conjugates share the same 9-mer core peptide, Table 3 below also details the number of unique 9-mer cores present in each sequence that are predicted to bind within the percentile rank range of ≤5. [Table 5]

[0336] 1.1.4 VH and VL domains of the obtained humanized BCMA antibody The VH domains from which humanized BCMA antibodies were obtained can be found in Table 4 below, and the VL domains from which humanized BCMA antibodies were obtained are listed in Table 5 below. [Table 6] [Table 7]

[0337] The humanized amino acid sequences of the heavy-chain and light-chain variable domains of the E04 humanized variant were fused to a single-armed human IgG1 skeleton / human CH1-hinge-CH2-CH3 containing effector silent Fc domains (P329G; L234A, L235A) that inhibit binding to the Fcγ receptor, and also fused to the light chain, according to the method described in International Publication No. 2012 / 130831A1. Human Fc containing the effector silent Fc domain was used for the correct assembly of single-armed IgG1. The amino acid sequences were back-translated into DNA, and the resulting cDNA was synthesized (GeneArt or Twist Biosciences). It was then cloned into a heavy-chain expression vector as a fusion protein with the human IgG1 skeleton and into an expression vector as a fusion protein with human C-kappa. The light-chain (LC) and heavy-chain (HC) plasmids were then co-transfected in HEK293 cells, and the supernatant was purified after 7 days using standard methods for antibody purification.

[0338] 1.2 Creation of humanized variants of anti-BCMA antibody 54 1.2.1 Methodology Anti-BCMA antibody 54 is disclosed in International Publication No. 2013 / 072415 and has the VH domain of SEQ ID NO: 44 and the VL domain of SEQ ID NO: 45. BCMA-54 is a humanized antibody with 84.8% identity to the most similar human HV germline (IGHV3-15*01) and 83.2% identity to the most similar human KV germline (IGKV1-6*01). The variable region of BCMA-54 is based on a human-derived framework region, but these regions have several positions that do not correspond to human germline amino acids. Examples of these positions include VH-16 (Ala), VH-44 (Arg), VH-84 (Lys), VL-22 (Ala), VL-83 (Glu), and VL-95 (Ile). Furthermore, there is a possibility of humanizing a portion of the CDR to reduce the immunogenicity of this conjugate and decrease the number of potential T cell epitopes.

[0339] To realize this potential for optimization, appropriate human acceptor frameworks were identified by querying the BLASTp database of human V and J region sequences for the original BCMA-54 sequence. Selection criteria for the human acceptor framework included sequence homology, same or similar CDR length, and estimated human germline frequency, as well as conservation of specific amino acids at the VH-VL domain interface. Following the germline identification step, the CDRs of the BCMA-54 input sequence were transplanted into the human acceptor framework regions. Each amino acid difference between these initial CDR grafts and the parental antibody was evaluated for its potential impact on the structural integrity of the respective variable regions, and “reverse mutations” to the parental sequence were introduced whenever deemed appropriate. Structural evaluation was performed using an in-house antibody structural homology modeling protocol implemented with BIOVIA Discovery Studio Environment, version 17R2, based on Fv region homology models for both BCMA-54 and the optimization candidate variants. Some humanized variants involved "forward mutations," i.e., amino acid exchanges that replaced the original amino acid occurring at a given CDR position in the parent conjugate with an amino acid found at an equivalent position in the human acceptor germline.

[0340] Using an in-house developed in silico tool, we predicted the VH-VL domain orientation of paired VH and VL humanized variants (International Publication No. 2016 / 062734). By comparing the results with the predicted VH-VL domain orientation of the parent conjugate, we selected a framework combination that closely resembled the shape of the original antibody. The rationale is to detect the possibility of amino acid exchange in the VH-VL interface region, which could lead to disruptive changes in the pairing of the two domains, potentially negatively impacting the binding properties.

[0341] 1.2.2 Selection and Adaptation of Acceptor Frameworks The subsequent acceptor frameworks were selected as shown in Table 6 below. [Table 8]

[0342] The framework region after CDR3 is human IGHJ germline IGHJ1*01(AEYFQH WGQGTLVTVSS , Sequence ID No. 196), and Human IGKJ Germline IGKJ2*01(YT FGQGTKLEIK Adapted from sequence number 197). Sections related to the acceptor framework are underlined.

[0343] Based on structural considerations, reverse mutations to amino acids in the original BCMA-54 sequence were introduced from the human acceptor framework at specific locations in the optimized variants. Furthermore, several locations were identified as promising candidates for forward mutations, in which amino acids in the parent conjugate CDR are replaced with amino acids found in the human acceptor germline. In addition to the basic variants (VH1a, VH1b, VH2a, VL1a, and VL2a), additional sequence variants were defined that typically introduce further forward mutations ("germlineization") at either individual locations or regions of their respective sequences. One variant aimed to improve the predicted hydrophobic surface patch, thereby potentially improving the biophysical properties of the VH region (VH1a_W197Y). The changes are detailed in Table 7 below. [Table 9]

[0344] 1.2.3 T cell epitope prediction To evaluate the potential T cell epitope development in humanized sequences, NetMHCIIPan 4.0 predictions were used (Reynisson B et al: NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data, Nucl. Acids Res., 48(W1):W449-W454 (2020)). Predictions were made for the following human MHC class II alleles: DRB1*01:01, DRB1*03:01, DRB1*04:01, DRB1*07:01, DRB1*08:01, DRB1*09:01, DRB1*11:01, DRB1*13:01, and DRB1*15:01.

[0345] The thresholds for strongly bound and weakly bound 15-mer peptides were set to percentile ranks 1 and 5, respectively. Binding 15-mer peptides with percentile ranks greater than 5 were not considered. Similarly, all binding 15-mer peptides with 9-mer core peptides occurring in 10 or more human V-region germlines were not considered. Germline sequences were obtained from the IMGT database (Giudicelli, V. et al.: IMGT / LIGM-DB, the IMGT® comprehensive database of immunoglobulin and T cell receptor nucleotide sequences. Nucl. Acids Res., 34(S1):D781-D784 (2006)). Since many of the predicted 15-mer conjugates share the same 9-mer core peptide, Table 8 below also details the number of unique 9-mer cores present in each sequence that are predicted to bind within the percentile rank range of ≤5. [Table 10]

[0346] 1.2.4 VH and VL domains of the obtained humanized BCMA antibody The VH domains from which humanized BCMA antibodies were obtained can be found in Table 9 below, and the VL domains from which humanized BCMA antibodies were obtained are listed in Table 10 below. [Table 11] [Table 12]

[0347] The humanized amino acid sequences of the heavy-chain and light-chain variable domains of the E04 humanized variant were fused to a single-armed human IgG1 skeleton / human CH1-hinge-CH2-CH3 containing effector silent Fc domains (P329G; L234A, L235A) with knob mutations, according to the method described in International Publication No. 2012 / 130831A1, to inhibit binding to the Fcγ receptor, and then fused to the light chain, following the knob-into-hole technique. Human Fc containing the effector silent Fc domain was used for the correct assembly of single-armed IgG1. The amino acid sequences were back-translated into DNA, and the resulting cDNA was synthesized (GeneArt or Twist Biosciences). It was then cloned into a heavy-chain expression vector as a fusion protein with the human IgG1 skeleton and into an expression vector as a fusion protein with human C-kappa. Next, light chain (LC) and heavy chain (HC) plasmids were co-transfected into HEK293 cells, and the supernatant was purified 7 days later using standard antibody purification methods.

[0348] 1.3 Characterization of humanized anti-BCMA variants To characterize the humanized anti-BCMA antibody variants, all clones were expressed as monovalent, single-arm IgG-like constructs (Figure 1A). This format was chosen to characterize binding to BCMA in a 1:1 model.

[0349] Seventy-six variants were generated with the intention of selecting two preferred humanized variants of anti-BCMA antibody E04 and two preferred humanized variants of anti-BCMA antibody 54. From these 76 variants, 29 variants with the highest affinity for huBCMA (15 BCMA 54 variants and 14 BCMA E04 variants) were pre-selected and further characterized by measuring their affinity for cyBCMA.

[0350] The 29 variants and their corresponding VH / VL pairs for the parent antibody, construct IDs (TaPIR IDs), and corresponding sequence numbers are listed in Table 11 below. [Table 13] TIFF2026522235000038.tif42170

[0351] The protein concentration of the purified construct was determined by measuring the optical density (OD) at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence, according to Pace, et al, Protein Science, 1995, 4, 2411-1423. Protein purity and molecular weight were analyzed by CE-SDS in and out of the presence of a reducing agent using LabChipGXII (Perkin Elmer). Aggregate contents were measured by HPLC chromatography at 25°C using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffer (25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, pH 6.7, or 200 mM KH2PO4, 250 mM KCl, pH 6.2, respectively). A summary of the product yield and purification parameters for all monovalent anti-BCMA variants is shown in Table 12.

[0352] The binding kinetics of monovalent humanized BCMA antibody variants to human BCMA and cynomolgus monkey BCMA were investigated by surface plasmon resonance (SPR) using a BIACORE T200 instrument (GE Healthcare). All experiments were performed at 25°C using HBS-P buffer (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) as the running buffer and dilution buffer. Anti-Fc IgG capture antibodies specific to the PGLALA mutant Fc region were immobilized on series S Sensor Chip CM5 (Cytiva) using standard amine coupling chemistry, obtaining a surface density of approximately 15,000 resonance units (RU). Anti-BCMA antibodies were captured on the surface at a flow rate of 5 μl / min for 30 seconds, resulting in a capture response of 50–200 RU. A dilution series of antigens (human BCMA Fc homodimer (R&D Systems) or cynomolgus monkey BCMA Fc homodimer (R&D Systems)) was injected into the surface at concentrations from 3 to 300 nM at a rate of 30 μl / min for 120 seconds (association phase). The dissociation phase was monitored for 300–600 seconds by washing with running buffer. The surface was regenerated by injecting (freshly prepared) 5 mM NaOH for 2 x 30 seconds. Differences in bulk refractive index were corrected by subtracting blank injections and responses obtained from reference flow cells that did not capture antibody. The derived curves were fitted to a 1:1 Langmuir binding model using BIAevaluation software.

[0353] K of monovalent constructs containing 29 preferred BCMA variants D The values ​​are shown in Table 12 below. [Table 14]

[0354] Based on in silico evaluation results regarding the potential T cell epitope generation in humanized sequences using NetMHCIIpan4.0 prediction (see Example 1.2.3), antibodies P1AG5080, P1AG5072, P1AG5028, P1AG5031, P1AG5063, and P1AG5036 (four BCMA 54 variants and two BCMA E04 variants) were selected as molecules with the lowest potential T cell epitopes. In combination with their binding behavior, P1AG5072 and P1AG5031 were selected as BCMA 54 variants, and P1AG5063 and P1AG5036 were selected as BCMA E04 variant antibodies to be included in bispecific antibodies.

[0355] Example 2 Design and production of bispecific antigen-binding molecules targeting BCMA 2.1 Cloning of bispecific antigen-binding molecules targeting BCMA and CD28 For the construction of expression plasmids, the sequences of each variable domain were used and subcloned in frame with the respective constant regions pre-inserted into each recipient mammalian expression vector. In the Fc domain, Pro329Gly, Leu234Ala, and Leu235Ala mutations (PG-LALA) were introduced into the constant region of the human IgG1 heavy chain to suppress binding to the Fcγ receptor, according to the method described in International Publication No. 2012 / 130831 of the international patent application. For the production of bispecific antibodies, the Fc fragments contained either a "knob" mutation (S354C / T366W mutation, numbered by the Kabat EU index) or a "hole" mutation (Y349C / T366S / L368A / Y407V mutation by the Kabat EU index) to avoid mispairing of the heavy chain. To avoid mispairing of light chains in bispecific antigen-binding molecules, an exchange of VH / VL or CH1 / C kappa domains was introduced at one binding site (CrossFab technology). As described in International Publication No. 2015 / 150447 of the international patent application, a charge was introduced to the CH1 domain and C kappa domain at another binding site.

[0356] The preparation and manufacturing of the anti-CD28 antibodies CD28 v.8 and CD28 v.15 used herein are described in International Publication No. 2020 / 127618A1 of the International Patent Application. CD28 v.8 has the VH of SEQ ID NO: 23 and the VL of SEQ ID NO: 24. CD28 v.15 has the VH of SEQ ID NO: 90 and the VL of SEQ ID NO: 91.

[0357] A schematic diagram of the bispecific antibody structure is shown in Figure 1B or Figure 1C. Table 13 summarizes the specific anti-BCMA / anti-CD28 bispecific antibodies prepared, their identifiers, and the sequences of the heavy chain (HC1 knob and HC2 hole) and light chain (LC1 and LC2). [Table 15]

[0358] 2.2 Production and Purification of Bispecific Antigen-Binding Molecules Targeting BCMA and CD28 DNA sequences encoding the variable heavy chain and variable light chain regions of the BCMA and CD28 antigen-binding domains were cloned into mammalian expression vectors using conventional cloning techniques. The bispecific antibodies described herein were prepared using a shaking flask in FedBatch mode. Recombinant production was performed by transient transfection of Expi293® cells in defined serum-free medium. The ExpiFectamine® 293 Transfection Kit (Gibco) was used for transfection. Cell culture supernatant was collected 7–12 days after transfection.

[0359] Quantification of protein titer: The protein titer of the supernatant sample was determined by affinity chromatography using a high-performance liquid chromatography system (Ultimate 3000 HPLC system, Thermo Scientific, Waltham, Massachusetts, USA) with a POROS A 20 μm column, 2.1 × 30 mm (Life Technologies, Carlsbad, California, USA). The supernatant was loaded onto a column equilibrated with 0.2 M Na2HPO4, pH 7.4, and subsequently eluted with 0.1 M citrate, 0.2 M NaCl, pH 2.5. Titer was quantified by measuring the absorbance at 280 nm, and the protein concentration was then calculated by comparing the elution peak area (under the curve) of the analyte with a reference standard curve.

[0360] Purification of bispecific antibodies: Proteins were purified from cell culture supernatants following a standard protocol. Briefly, Fc-containing proteins were purified from cell culture supernatants by protein A affinity chromatography (equilibrium buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5 or PBS; elution buffer: 20 mM, 25 mM or 50 mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0, followed immediately by neutralization of the sample pH. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15, #UFC903096), and aggregated proteins were separated from monomeric proteins by size exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0361] Analysis of bispecific antibodies: The concentration of purified proteins was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423. Protein purity and molecular weight were analyzed by CE-SDS in the presence and absence of a reducing agent using LabChip GXII or LabChip GX Touch (Perkin Elmer). Aggregate content was determined by HPLC chromatography at 25°C using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000, Tosoh Bioscience) equilibrated in running buffer (200 mM KH2PO4, 250 mM KCl pH 6.2, 0.02% NaN3).

[0362] Table 14 shows an overview of the purification parameters for the selected molecules. [Table 16]

[0363] 2.3 Biophysical and biochemical characterization of bispecific antibodies targeting BCMA and CD28 To predict the potential for developing bispecific antibodies, their biophysical and biochemical properties were evaluated using computational methods and assays.

[0364] Predictions based on the sequence of protein net charge as a function of pH, and therefore the isoelectric point (calculated pI), obtained using standard software (e.g., EMBOSS tools), helped estimate whether bispecific antibodies were suitable for binding to and unbinding to cation exchange and anion exchange chromatography media, and thus suitable for common purification methods during production. All BCMA-CD28 bsAbs produced had favorable pI values ​​in the range of >8 (see Table 15 below).

[0365] The thermal stability of the prepared BCMA-CD28 bsAb was monitored using an Optim2 instrument (Avacta Analytical, UK) by dynamic light scattering (DLS) and by monitoring temperature-dependent intrinsic protein fluorescence by applying a temperature gradient. A 10 μg filtered protein sample with a protein concentration of 1 mg / ml was applied in two parallel units to the Optim2 instrument. The temperature was inc...

Claims

1. An antibody that specifically binds to a B cell maturation antigen (BCMA), wherein the antibody comprises a first antigen-binding domain, and the first antigen-binding domain is (i) Heavy chain variable region (V) including the heavy chain complementarity determination region of CDR-H1 (GYTFTNYWMH) of SEQ ID NO: 1, CDR-H2 (IIHPNSGSSTNYNEKFQG) of SEQ ID NO: 2, and CDR-H3 (GIYDYPFAY) of SEQ ID NO: 3 Ｈ BCMA) and, (ii) Light chain variable region (V Ｌ BCMA) (a) A VL containing the light chain complementarity determination region of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASSLQS) of SEQ ID NO: 5, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6, or (b) A VL containing the light chain complementarity determination region of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASNLES) of SEQ ID NO: 7, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6, or (c) VL containing the light chain complementarity determination regions of CDR-L1 (RASESVSIHGTHLMH) of SEQ ID NO: 4, CDR-L2 (AASNLQS) of SEQ ID NO: 8, and CDR-L3 (QQSIEDPYT) of SEQ ID NO: 6 A light chain variable region selected from the group consisting of, Antibodies containing antibodies.

2. The aforementioned V Ｈ BCMA comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 (VH1a) and SEQ ID NO: 10 (VH1b), and / or the V Ｌ The antibody according to claim 1, wherein BCMA comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11 (VL1f), SEQ ID NO: 12 (VL1a), SEQ ID NO: 13 (VL1b), SEQ ID NO: 14 (VL1c), SEQ ID NO: 15 (VL1d), and SEQ ID NO: 16 (VL1e).

3. An antibody that specifically binds to BCMA, wherein the antibody includes a first antigen-binding domain, and the first antigen-binding domain is (a) V containing the amino acid sequence of SEQ ID NO: 9 Ｈ V containing the amino acid sequences of BCMA and SEQ ID NO: 11 Ｌ BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 9 Ｈ V containing the amino acid sequences of BCMA and SEQ ID NO: 12 Ｌ BCMA Antibodies containing antibodies.

4. The antibody according to any one of claims 1 to 3, wherein the first antigen-binding domain is a Fab molecule.

5. The antibody according to any one of claims 1 to 4, comprising an Fc domain composed of a first and a second subunit.

6. The antibody according to any one of claims 1 to 5, comprising a second antigen-binding domain that specifically binds to a second antigen.

7. The antibody according to any one of claims 1 to 6, wherein the second antigen-binding domain that specifically binds to the second antigen is a Fab molecule, and the variable domains VL and VH or constant domains CL and CH1 of the Fab light chain and the Fab heavy chain, particularly the variable domains VL and VH, are substituted for each other.

8. The antibody according to claim 6 or 7, wherein the second antigen is CD28.

9. The second antigen-binding domain that specifically binds to CD28 comprises a heavy-chain variable region (V Ｈ CD28) and a light-chain variable region (V Ｌ CD28) containing the heavy-chain complementarity-determining regions of CDR-H1 of SEQ ID NO: 17, CDR-H2 of SEQ ID NO: 18, and CDR-H3 of SEQ ID NO: 19, and the light-chain complementarity-determining regions of CDR-L1 of SEQ ID NO: 20, CDR-L2 of SEQ ID NO: 21, and CDR-L3 of SEQ ID NO: 22, the antibody according to claim 8.

10. The second antigen-binding domain that specifically binds to CD28 is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO:

23. Ｈ CD28) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 24 (v8) Ｌ The antibody according to claim 8 or 9, comprising CD28.

11. The antibody contains the amino acid sequence of SEQ ID NO:

9. Ｈ V containing the amino acid sequences of BCMA and SEQ ID NO: 11 Ｌ The first antigen-binding domain contains BCMA and V contains the amino acid sequence of SEQ ID NO:

23. Ｈ V containing the amino acid sequences of CD28 and SEQ ID NO: 24 Ｌ An antibody according to any one of claims 1 to 10, comprising a second antigen-binding domain containing CD28.

12. The antibody according to any one of claims 1 to 11, comprising a first light chain containing the amino acid sequence of SEQ ID NO: 25, a first heavy chain containing the amino acid sequence of SEQ ID NO: 26, a second heavy chain containing the amino acid sequence of SEQ ID NO: 27, and a second light chain containing the amino acid sequence of SEQ ID NO:

28.

13. An antibody that specifically binds to B cell maturation antigen (BCMA) and CD28, (A) The first antigen-binding domain is (i) Heavy chain variable region (V Ｈ BCMA) (a) VH including the heavy chain complementarity determination region of CDR-H1 (GFTFFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYADSVKG) of SEQ ID NO: 30, and CDR-H3 (DGYH) of SEQ ID NO: 31, and (b) VH including the heavy chain complementarity determination region of CDR-H1 (GFTFFSNAWMD) of SEQ ID NO: 29, CDR-H2 (QITAKSNNYATYYAAPVKG) of SEQ ID NO: 32, and CDR-H3 (DGYH) of SEQ ID NO: 31 A heavy chain variable region selected from the group consisting of, (ii) Light chain variable region (V) including the light chain complementarity determination region of CDR-L1 (RASEDIRNGLA) of SEQ ID NO: 33, CDR-L2 (NANSLHT) of SEQ ID NO: 34, and CDR-L3 (EDTSKYPYT) of SEQ ID NO: 35 Ｌ BCMA) and, The first antigen-binding domain, (B) A second antigen-binding domain that specifically binds to CD28, Antibodies containing antibodies.

14. The aforementioned V Ｈ BCMA comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36 (VH2a) and SEQ ID NO: 38 (VH1b), and / or the V Ｌ The antibody according to claim 13, wherein BCMA comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37 (VL2a) and SEQ ID NO: 39 (VL1a).

15. An antibody that specifically binds to BCMA and CD28, wherein the antibody comprises a first antigen-binding domain, and the first antigen-binding domain is (a) V containing the amino acid sequence of SEQ ID NO: 36 Ｈ V containing the amino acid sequences of BCMA and SEQ ID NO: 37 Ｌ BCMA, or (b) V containing the amino acid sequence of SEQ ID NO: 38 Ｈ V containing the amino acid sequences of BCMA and SEQ ID NO: 39 Ｌ BCMA Antibodies containing antibodies.

16. The antibody according to any one of claims 13 to 15, wherein the first antigen-binding domain is a Fab molecule.

17. The antibody according to any one of claims 13 to 16, wherein the second antigen-binding domain that specifically binds to the second antigen is a Fab molecule, and the variable domains VL and VH or constant domains CL and CH1 of the Fab light chain and the Fab heavy chain, particularly the variable domains VL and VH, are substituted for each other.

18. The second antigen-binding domain that specifically binds to CD28 includes a heavy chain variable region (V) which contains the heavy chain complementarity determining regions of CDR-H1 of SEQ ID NO: 17, CDR-H2 of SEQ ID NO: 18, and CDR-H3 of SEQ ID NO:

19. Ｈ CD28) and the light chain variable region (V) which includes the light chain complementarity determination region of CDR-L1 of SEQ ID NO: 20, CDR-L2 of SEQ ID NO: 21, and CDR-L3 of SEQ ID NO: 22 Ｌ The antibody according to any one of claims 13 to 17, comprising CD28.

19. The second antigen-binding domain that specifically binds to CD28 is a heavy chain variable region (V) containing the amino acid sequence of SEQ ID NO:

23. Ｈ CD28) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 24 Ｌ The antibody according to claim 18, comprising CD28.

20. The antibody contains the amino acid sequence of SEQ ID NO:

36. Ｈ V containing the amino acid sequences of BCMA and SEQ ID NO: 37 Ｌ The first antigen-binding domain contains BCMA and V contains the amino acid sequence of SEQ ID NO:

23. Ｈ V containing the amino acid sequences of CD28 and SEQ ID NO: 24 Ｌ An antibody according to any one of claims 13 to 19, comprising a second antigen-binding domain containing CD28.

21. The antibody according to any one of claims 13 to 20, comprising a first light chain containing the amino acid sequence of SEQ ID NO: 40, a first heavy chain containing the amino acid sequence of SEQ ID NO: 41, a second heavy chain containing the amino acid sequence of SEQ ID NO: 27, and a second light chain containing the amino acid sequence of SEQ ID NO:

28.

22. One or more isolated polynucleotides encoding an antibody according to any one of claims 1 to 21.

23. One or more vectors, particularly expression vectors, comprising the polynucleotide described in claim 22.

24. A host cell comprising the polynucleotide described in claim 22 or the vector described in claim 23.

25. A method for producing an antibody that specifically binds to BCMA, comprising: a) culturing host cells described in claim 24 under conditions suitable for the expression of the antibody; and optionally b) recovering the antibody.

26. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 21 and at least one pharmaceutically acceptable additive.

27. An antibody according to any one of claims 1 to 21, or a pharmaceutical composition according to claim 26, for use as a pharmaceutical.

28. An antibody according to any one of claims 1 to 21, or a pharmaceutical composition according to claim 26, for use in (a) T cell activation or (b) enhancement of T cell effector function.

29. An antibody according to any one of claims 1 to 21, or a pharmaceutical composition according to claim 26, for use in the treatment of cancer, particularly multiple myeloma.

30. An antibody according to any one of claims 1 to 21, or a pharmaceutical composition according to claim 26, for use according to claim 29, wherein the use is for administration in combination with other agents for use in chemotherapy, radiotherapy and / or cancer immunotherapy.

31. The antibody according to any one of claims 1 to 21, or the pharmaceutical composition according to claim 26, for use according to claim 29, wherein the use is for administration in combination with a T cell-activating anti-CD3 bispecific antibody.

32. The antibody or pharmaceutical composition for use according to claim 31, wherein the T-cell activating anti-CD3 bispecific antibody is an anti-GPRC5D / anti-CD3 antibody.

33. Use of an antibody according to any one of claims 1 to 21, or use of a pharmaceutical composition according to claim 26, in the manufacture of a pharmaceutical for the treatment of a disease, particularly cancer.

34. A method for treating a disease in an individual, particularly cancer, comprising administering to the individual an effective amount of an antibody according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 26.

35. The method according to claim 34, further comprising administering in combination with other agents for use in chemotherapy, radiotherapy and / or cancer immunotherapy, in particular in combination with a T-cell activating anti-CD3 bispecific antibody.

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