Combination therapy with GPRC5D TCB and proteasome inhibitor

Abstract

The present invention relates to a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody with a proteasome inhibitor, which may further comprise a glucocorticosteroid.

Description

[Technical Field]

[0001] The present invention relates to a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody and a proteasome inhibitor, which may optionally include a glucocorticosteroid. [Background technology]

[0002] Multiple myeloma (MM), one of the most common hematological malignancies, accounts for approximately 75,000 new cases annually in the EU and the United States. It remains a disease with significant unmet medical needs. MM is characterized by terminally differentiated plasma cells secreting nonfunctional monoclonal immunoglobulins. In the short term, immunomodulatory drugs such as lenalidomide and pomalidomide and proteasome inhibitors such as carfilzomib or bortezomib may remain the backbone of first-line treatment for MM (Moreau, P. and S. V. Rajikumar, "Multiple Myeloma: Translation of Trial Results into Reality." Lancet, 2016, 388(10040):pp.111-3). However, these drugs do not specifically target pathological tumor cells, such as pathological plasma cells (PCs). Efforts to selectively deplete plasma cells have been ongoing in MM. The lack of surface proteins that specifically mark plasma cells has hindered the development of antibody or cell-based therapies for multiple myeloma. To date, biologics such as daratumumab (anti-CD38) and elotuzumab (anti-CD319) have had limited success. However, these two molecules are not uniquely expressed by plasma cells. Therefore, using RNA sequencing, novel targets from multiple myeloma plasma cells, such as G protein-coupled receptor class C group 5 member D (GPRC5D), which is differentially expressed by multiple myeloma plasma cells compared with plasma cells from healthy donors, have been identified.GPRC5D has been reported to be associated with the prognosis and tumor burden of multiple myeloma patients (Atamaniuk, J., et al., Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma. Eur J Clin Invest, 2012. 42(9): p. 953-60, and Cohen, Y., et al., GPRC5D is a promising marker for monitoring the tumor load and to target multiple myeloma cells. Hematology, 2013. 18(6): p. 348-51).

[0003] GPRC5D is an orphan receptor with no known ligand, and its biological properties in general and cancer in particular are largely unknown. The GPRC5D-encoding gene, located on chromosome 12p13.3, contains three exons and spans approximately 9.6 kb (Brauner-Osborne, H. et al., Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochim Biophys Acta, 2001, 1518(3):237-48). The first large exon encodes the seven transmembrane domain. GPRC5D has been shown to be involved in keratinogenesis in animal hair follicles (Gao, Y., et al., Comparative Transcriptome Analysis of Fetal Skin Reveals Key Genes Related to Hair Follicle Morphogenesis in Cashmere Goats. PLoS One, 2016. 11(3): p. e0151118, and Inoue, S., T. Nambu, and T. Shimomura, The RAIG family member, GPRC5D, is associated with hard-keratinized structures. J Invest Dermatol, 2004. 122(3): p. 565-73).

[0004] WO 2018 / 017786 and WO 2021 / 018859 disclose GPRC5D-specific antibodies or antigen-binding fragments that bind to GPRC5D on target cells and to an activating T cell antigen, such as CD3, on T cells. Simultaneous binding of such antibodies to both targets results in the formation of a T cell synapse and activation of (cytotoxic) T cells, which subsequently lyse the target cell.

[0005] The inability of standard therapies to cure patients with multiple myeloma highlights the need to develop novel, potent and specific therapeutic approaches. Accordingly, the present invention provides a combination of an anti-GPRC5D / anti-CD3 bispecific antibody with a proteasome inhibitor and, optionally, a glucocorticosteroid. Summary of the Invention

[0006] In a first aspect, the present invention provides an anti-GPRC5D / anti-CD3 bispecific antibody in combination with a proteasome inhibitor for use as a combination therapy in the treatment of cancer. In a further aspect, the present invention provides the use of an anti-GPRC5D / anti-CD3 bispecific antibody in combination with a proteasome inhibitor in the manufacture of a medicament for treating cancer. In another aspect, the present invention provides a method of treating cancer in an individual, comprising administering to the individual an anti-GPRC5D / anti-CD3 bispecific antibody in combination with a proteasome inhibitor. In yet another aspect, the present invention provides a kit comprising a first agent comprising an anti-GPRC5D / anti-CD3 bispecific antibody and a second agent comprising a proteasome inhibitor, optionally further comprising a package insert containing instructions for administering the first agent in combination with the second agent to treat cancer in an individual.

[0007] In one embodiment of any one of the above aspects, the anti-GPRC5D / anti-CD3 bispecific antibody comprises (i) a first antigen-binding portion that specifically binds to GPRC5D and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 12, an HCDR2 of SEQ ID NO: 13, and an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 15, an LCDR2 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO: 17, and (ii) a second antigen-binding portion that specifically binds to CD3 and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 18, an HCDR2 of SEQ ID NO: 19, and an HCDR3 of SEQ ID NO: 20, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 21, an LCDR2 of SEQ ID NO: 22, and an LCDR3 of SEQ ID NO: 23. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody comprises (i) a first antigen-binding portion that specifically binds GPRC5D comprising a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10 and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11, and (ii) a second antigen-binding portion that specifically binds CD3 comprising a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24 and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the first antigen-binding portion and / or the second antigen-binding portion of the anti-GPRC5D / anti-CD3 bispecific antibody is a Fab molecule. In a further embodiment, the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH of the Fab light chain and the Fab heavy chain, are replaced by each other.In one embodiment, the first antigen-binding moiety has in the constant domain: the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1: the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering). In another embodiment, the first antigen-binding moiety and the second antigen-binding moiety are fused to each other, optionally via a peptide linker. In further embodiments, the first and second antigen-binding moieties are each Fab molecules, and (i) the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety, or (ii) the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety.

[0008] In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody according to any of the above aspects comprises a third antigen-binding moiety. In a further embodiment, the third antigen-binding moiety is identical to the first antigen-binding moiety. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody comprises an Fc domain composed of a first and a second subunit. In one aspect, each of the first, second, and, if present, third antigen-binding moieties is a Fab molecule, wherein (i) the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and, if present, the third antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. In one embodiment, the Fc domain is an IgG Fc domain. In one embodiment, the Fc domain is an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain.

[0009] In one embodiment of any one of the above aspects, amino acid residues in the CH3 domain of a first subunit of the Fc domain are replaced with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of a second subunit, and amino acid residues in the CH3 domain of a second subunit of the Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit that can be positioned within the protrusion in the CH3 domain of the first subunit. In one embodiment, the Fc domain comprises one or more amino acid substitutions that reduce Fc receptor binding and / or effector function.

[0010] In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody of any one of this aspect comprises the polypeptide sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody of any one of this aspect is Formtamig.

[0011] In one embodiment, the proteasome inhibitor of any one of the above aspects belongs to the class of peptide boronates or peptide epoxyketones, hi one embodiment, the proteasome inhibitor is bortezomib or carfilzomib.

[0012] In another aspect, the combination as described in any one of the above aspects further comprises a glucocorticosteroid. In one embodiment, the glucocorticosteroid is dexamethasone. [Brief explanation of the drawings]

[0013] [Figure 1A-1B] Results of an efficacy experiment evaluating GPRC5D-TCB as a single agent and in combination with bortezomib with or without dexamethasone against KMS-12BM multiple myeloma tumors subcutaneously implanted in stem cell-humanized NSGs. (Figure 1A) GPRC5D-TCB was intravenously injected at 1 mg / kg once weekly and combined with twice-weekly intravenous administration of 0.25 mg / kg bortezomib with or without 2 mg / kg oral dexamethasone, and KMS-12BM tumor growth was monitored. (Figure 1B) KMS-12BM tumor burden in individual mice was assessed on day 35 (end of study). Statistical analysis, ordinary one-way ANOVA, Tukey's test: p = < 0.0001 (****), p = 0.0001–0.001 (***), p = 0.001–0.01 (**), p = 0.01–0.05 (*), p = ≥ 0.05 (ns). [Figures 2A-2D]Results of an efficacy experiment evaluating GPRC5D-TCB as a single agent and in combination with carfilzomib against NCI-H929 multiple myeloma tumors subcutaneously implanted in stem cell-humanized NSGs (Figure 2A). GPRC5D-TCB was intravenously injected weekly at 0.1 mg / kg and combined with twice-weekly intravenous administration of 3 mg / kg carfilzomib, and NCI-H929 tumor growth was monitored. IFN-γ (Figure 2B), IL-2 (Figure 2C), and TNF-α (Figure 2D) levels in mouse serum were measured 48 hours after the first GPRC5D-TCB injection and 24 hours after carfilzomib injection using multiplex technology. Statistical analysis, ordinary one-way ANOVA, Tukey's test: p = < 0.0001 (****), p = 0.0001–0.001 (***), p = 0.001–0.01 (**), p = 0.01–0.05 (*), p = ≥ 0.05 (ns). DETAILED DESCRIPTION OF THE INVENTION

[0014] definition Unless otherwise defined below, terms are used herein as commonly used in the art.

[0015] As used herein, the term "antigen-binding molecule" refers in the broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and derivatives (e.g., fragments) thereof.

[0016] The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each specific for a different antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigenic determinants (particularly, two antigenic determinants expressed on two distinct cells).

[0017] The term "valency," as used herein, refers to the presence of a specific number of antigen-binding sites within an antigen-binding molecule. In this context, the term "monovalent binding to an antigen" refers to the presence of one (and not more than one) antigen-binding site specific for an antigen within the antigen-binding molecule.

[0018] "Antigen-binding site" refers to the site (i.e., one or more amino acid residues) of an antigen-binding molecule that interacts with an antigen. For example, the antigen-binding site of an antibody comprises amino acid residues from the complementarity-determining regions (CDRs). A native immunoglobulin molecule typically contains two antigen-binding sites, and a Fab molecule typically has one antigen-binding site.

[0019] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding moiety can target a target site, e.g., a moiety (e.g., a second antigen-binding moiety) that binds to a specific type of tumor cell bearing the antigenic determinant. In another embodiment, an antigen-binding moiety can activate signaling through its target antigen, e.g., a T-cell receptor complex antigen. Antigen-binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen-binding moieties comprise the antigen-binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, an antigen-binding moiety may comprise an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of the two isotypes: κ and λ.

[0020] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a contiguous stretch of amino acids or a conformation composed of distinct regions of non-contiguous amino acids) to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Proteins (e.g., GPRC5D, CD3) referred to herein as antigens can be any naturally occurring form of the protein 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. In a specific embodiment, the antigen is a human protein. When a particular protein is referred to herein, the term encompasses not only the "full-length," unprocessed protein, but also any form of the protein resulting from processing within the cell. The term also encompasses naturally occurring variants of the protein, such as splice or allelic variants. Exemplary human proteins useful as antigens are CD3, particularly the epsilon subunit of CD3 (see UniProt No. P07766 (version 185), NCBI RefSeq No. NP_000724.1, SEQ ID NO: 4 for the human sequence, or UniProt No. Q95LI5 (version 69), NCBI GenBank No. BAB71849.1, SEQ ID NO: 5 for the cynomolgus monkey [Macaca fascicularis] sequence), or GPRC5D (see UniProt No. Q9NZD1 (version 115), NCBI RefSeq No. NP_061124.1, SEQ ID NO: 9 for the human sequence). In certain embodiments, the bispecific antigen-binding molecule binds to an epitope of CD3 or GPRC5D that is conserved among CD3 or GPRC5D antigens from different species. In a specific embodiment, the bispecific antigen-binding molecule binds to human GPRC5D.

[0021] "Specifically binds" means that the binding is antigen-selective and can be distinguished from unwanted or nonspecific interactions. The binding ability of an antigen-binding moiety to a specific 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 (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the antigen-binding moiety to an unrelated protein is less than about 10% of the binding of the antigen-binding moiety to the antigen, as measured, for example, by SPR. In certain embodiments, an antigen-binding portion that binds to an antigen, or an antigen-binding molecule comprising an antigen-binding portion, has a denaturing activity of ≦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, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Dissociation constant (K D )

[0022] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding moiety and an antigen, or a receptor and its ligand). The affinity of molecule X for its binding partner Y is typically measured by the dissociation constant (K D ), and the dissociation rate constant and association rate constant (k off and k on) is the ratio of the rate constants. Thus, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0023] "Decreased binding," e.g., decreased binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured, e.g., by SPR. For clarity, the term also includes a decrease in affinity to zero (or below the detection limit of the analytical method), i.e., a complete loss of interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0024] As used herein, a "T cell activation 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 antigen-binding molecule. Specifically, interaction of an antigen-binding molecule with an activating T cell antigen can induce T cell activation by triggering a signal transduction cascade in the T cell receptor complex. In a specific embodiment, the activating T cell antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt No. P07766 (version 144), NCBI RefSeq No. NP_000724.1, SEQ ID NO: 4 for the human sequence, or UniProt No. Q95LI5 (version 49), NCBI GenBank No. BAB71849.1, SEQ ID NO: 5 for the cynomolgus monkey [Macaca fascicularis] sequence).

[0025] 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 described herein.

[0026] As used herein, "target cell antigen" refers to an antigenic determinant displayed on the surface of a target cell, e.g., a cell within a tumor, such as a cancer cell or a cell of the tumor stroma. In a specific embodiment, the target cell antigen is GPRC5D, particularly human GPRC5D according to SEQ ID NO: 9.

[0027] As used herein, the terms "first," "second," or "third," with respect to Fab molecules, etc., are used for ease of distinction when more than one of each type of moiety is present. The use of these terms is not intended to confer a particular order or orientation of the bispecific antigen-binding molecules unless explicitly indicated as such.

[0028] By "fused" is meant that the components (eg, a Fab molecule and an Fc domain subunit) are linked by a peptide bond, either directly or via one or more peptide linkers.

[0029] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of a light chain (a "Fab light chain").

[0030] A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which the variable or constant domains of the Fab heavy and light chains have been exchanged (i.e., replaced with each other), i.e., the crossover Fab molecule comprises a peptide chain consisting of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1 from N-terminal to C-terminal), and a peptide chain consisting of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL from N-terminal to C-terminal). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0031] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native format, i.e., a Fab molecule comprising a heavy chain (VH-CH1 from N to C-terminus) composed of a heavy chain variable domain and a constant domain, and a light chain (VL-CL from N to C-terminus) composed of a light chain variable domain and a constant domain.

[0032] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light domain (CL), also called a light chain constant region. Immunoglobulin heavy chains can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further classified into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Immunoglobulins essentially consist of two Fab molecules and an Fc domain connected via an immunoglobulin hinge region.

[0033] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0034] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, each individual antibody in the population is identical and / or binds to the same epitope, excluding possible variant antibodies, including, for example, naturally occurring mutations or mutations that arise during production of the monoclonal antibody preparation. Such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0035] An "isolated" antibody is one that has been separated from a component of its natural environment (i.e., is not in its native environment). A specific level of purification is not required. For example, an isolated antibody can be removed from its native or natural environment. Recombinantly produced antibodies expressed in host cells are considered isolated for purposes of the present invention, as are native or recombinant antibodies that have been separated, fractionated, or partially or substantially purified by any suitable technique. Thus, the antibodies and bispecific antigen-binding molecules of the present invention are isolated. In some embodiments, antibodies are purified to greater than 95% or greater than 99% purity as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0036] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to that of a native antibody.

[0037] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. See U.S. Pat. No. 5,869,046 for a description of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having extended in vivo half-lives. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 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). Single domain antibodies are antibody fragments that contain 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, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, Massachusetts; see, e.g., U.S. Pat. No. 6,248,516).Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0038] The term "antigen-binding domain" refers to a part of an antibody that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). In particular, an antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0039] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. When used herein with respect to variable region sequences, "Kabat numbering" refers to the numbering system established by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0040] As used herein, amino acid positions in all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system, referred to herein as "Kabat numbering" or "Kabat numbering," as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Specifically, the Kabat numbering system (see Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) pp. 647-660) is used for the light chain constant domains CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pp. 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3), which is further clarified herein by referring to "Kabat EU index numbering."

[0041] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR", the CDRs of a heavy chain variable region / domain are abbreviated, e.g., as HCDR1, HCDR2, and HCDR3, and the CDRs of a light chain variable region / domain are abbreviated, e.g., as LCDR1, LCDR2, and LCDR3) and / or that forms structurally distinct loops ("hypervariable loops") and / or that contains antigen-contacting residues ("antigen contacts"). Generally, antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs of the invention include the following: (a) hypervariable loops occurring 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 located at 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)); (c) antigenic contacts 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)); and (d) A combination of (a), (b) and / or (c) comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0042] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0043] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences are usually designated in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0044] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. Such variable domains are referred to herein as "humanized variable regions." A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, some FR residues of a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant regions have been further modified or altered from those of the original antibody to create properties according to the invention, particularly in terms of C1q binding and / or Fc receptor (FcR) binding.

[0045] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or an antibody of non-human origin that utilizes human antibody-encoding sequences, such as the human antibody repertoire. This definition of human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. In certain embodiments, a human antibody is derived from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain embodiments, a human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from a human antibody library are also considered human antibodies or human antibody fragments of the invention.

[0046] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by the antibody or immunoglobulin heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0047] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to stretch from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, antibodies produced by host cells may contain a full-length heavy chain or a truncated variant of the full-length heavy chain (also referred to herein as a "truncated variant heavy chain"). This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. The amino acid sequence of a heavy chain comprising an Fc domain (or a subunit of an Fc domain as defined herein) is shown herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one embodiment of the invention, a heavy chain comprising an Fc domain subunit as specified herein contained in an antibody or bispecific antigen-binding molecule of the invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the EU index of Kabat). In one embodiment of the invention, a heavy chain comprising an Fc domain subunit as specified herein contained in an antibody or bispecific antigen-binding molecule of the invention comprises an additional C-terminal glycine residue (G446, numbered according to the EU index of Kabat). Compositions of the invention, e.g., pharmaceutical compositions described herein, comprise a population of antibodies or bispecific antigen-binding molecules of the invention. The population of antibodies or bispecific antigen-binding molecules may include molecules that contain full-length heavy chains and molecules that contain truncated variant heavy chains.A population of antibodies or bispecific antigen-binding molecules may consist of a mixture of molecules with full-length heavy chains and molecules with cleaved variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or bispecific antigen-binding molecules have cleaved variant heavy chains. In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antigen-binding molecules of the invention comprises antibodies or bispecific antigen-binding molecules comprising heavy chains comprising subunits of an Fc domain as specified herein, which comprise an additional C-terminal glycine-lysine dipeptide (G446 and K447, according to EU index of Kabat). In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antigen-binding molecules of the invention comprises antibodies or bispecific antigen-binding molecules comprising heavy chains comprising subunits of an Fc domain as specified herein, which comprise an additional C-terminal glycine residue (G446, according to EU index of Kabat). In one embodiment of the present invention, such compositions comprise a population of antibodies or bispecific antigen-binding molecules comprised of: molecules comprising heavy chains comprising subunits of an Fc domain as specified herein; molecules comprising heavy chains comprising subunits of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to the EU index of Kabat); molecules comprising heavy chains comprising subunits of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the EU index of Kabat). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also supra).As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0048] A "modification that promotes association of a first subunit and a second subunit of an Fc domain" refers to manipulation of the peptide backbone or post-translational modification of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. As used herein, a modification that promotes association specifically includes separate modifications made to each of the two Fc domain subunits (i.e., the first and second Fc domain subunits) that are desired to associate, which are complementary to each other to promote the association of the two Fc domain subunits. For example, a modification that promotes association can alter the structure or charge of one or both of the Fc domain subunits to sterically or electrostatically favor their association, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may not be identical in the sense that the additional components (e.g., antigen-binding moieties) fused to each of the subunits are not the same. In some embodiments, a modification that promotes association includes an amino acid mutation, specifically an amino acid substitution, within the Fc domain. In a specific embodiment, the association-promoting modifications comprise distinct amino acid mutations, particularly amino acid substitutions, in each of the two subunits of the Fc domain.

[0049] The term "effector function" refers to a 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 cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0050] As used herein, the terms "engineer, engineered, manipulating" are intended to include any manipulation or post-translational modification of the peptide backbone of a naturally occurring or recombinant polypeptide or fragment thereof. Manipulation includes modifying the amino acid sequence, modifying the glycosylation pattern, or modifying the side groups of individual amino acids, as well as combinations of these techniques.

[0051] The term "amino acid mutation" as used herein is intended to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., decreased binding to an Fc receptor or increased association with another peptide). Deletions and insertions in the amino acid sequence include amino- and / or carboxy-terminal deletions and insertions of amino acids. A particular amino acid mutation is an amino acid substitution. For example, to alter the binding characteristics of the Fc region, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods for modifying the side chain groups of amino acids other than genetic engineering, such as chemical modification, may also be useful. Various names may be used herein to refer to the same amino acid mutation. For example, a proline to glycine substitution at position 329 of the Fc domain is referred to as 329G, G329, G 329 , P329G or Pro329Gly.

[0052] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program in the FASTA package version 36.3.8c, followed by the BLOSUM50 comparison matrix. The FASTA program package was written by W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; W.R. Pearson (1996), "Effective protein sequence comparison," Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available at http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml.Alternatively, sequences can be compared using the public server accessible at http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch(globalprotein:protein) program and default options (BLOSUM50, open:-10, ext:-2, Ktup=2) to ensure a global, rather than local, alignment. The percent amino acid identity is shown in the output alignment header.

[0053] An "activating Fc receptor" is an Fc receptor that, following binding of the Fc domain of an antibody, triggers signaling events that stimulate the receptor-bearing cell to carry out an effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0054] Antibody-dependent cellular cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells to which an antibody or its derivative, including an Fc region, specifically binds via a protein portion generally N-terminal to the Fc region. As used herein, the term "reduced ADCC" is defined as either a decrease in the number of target cells lysed in a given time period at a given antibody concentration in the medium surrounding the target cells by the ADCC mechanism defined above, and / or an increase in the antibody concentration in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time period by the ADCC mechanism. The reduced ADCC is relative to ADCC mediated by the same antibody, but made using the same standard production, purification, formulation, and storage methods (known to those skilled in the art) and by the same type of host cell, but without engineering. For example, reduced ADCC mediated by an antibody containing an amino acid substitution in its Fc domain that reduces ADCC is compared to ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, WO 2006 / 082515 or WO 2012 / 130831).

[0055] An "effective amount" of a drug refers to the amount necessary to cause a physiological change in a cell or tissue to which the drug is administered.

[0056] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical composition) refers to an amount effective, at the necessary dosage and for the necessary period of time, to achieve a desired therapeutic or prophylactic result. For example, a therapeutically effective amount of an agent may eliminate, reduce, delay, minimize, or prevent the side effects of a disease.

[0057] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0058] The term "pharmaceutical composition" refers to a formulation in a form that allows for the biological activity of the active ingredient contained therein to be effective and does not contain additional components that are unacceptably toxic to the subject to which the composition is administered.

[0059] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0060] As used herein, "treatment" (and its grammatical variants, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of disease in the individual being treated, and can be carried out for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating the direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating disease symptoms, and achieving remission or improved prognosis. In some embodiments, an antibody or bispecific antigen-binding molecule of the invention is used to delay the onset of disease or to slow the progression of disease.

[0061] The term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings for use of such therapeutic product. Bispecific antigen-binding molecules that bind to GPRC5D and CD3

[0062] The anti-GPRC5D / anti-CD3 bispecific antigen-binding molecules (also referred to herein as "GPRC5D TCB") used in the combination therapies described herein comprise at least two antigen-binding moieties capable of specifically binding to two different antigenic determinants (first and second antigens). Suitable bispecific antigen-binding molecules that bind to GPRC5D and CD3 for use in the present invention are described, for example, in WO 2021 / 018859, WO 2019 / 154890, and WO 2018017786.

[0063] According to specific embodiments of the present invention, the antigen-binding moieties comprised in the bispecific antigen-binding molecule are Fab molecules (i.e., antigen-binding domains comprised of heavy and light chains, each comprising a variable domain and a constant domain). In one embodiment, the first and / or second antigen-binding moieties are Fab molecules. In one embodiment, the Fab molecules are human. In a specific embodiment, the Fab molecules are humanized. In yet another embodiment, the Fab molecules comprise human heavy and light chain constant domains.

[0064] Preferably, at least one of the antigen-binding portions is a crossover Fab molecule. Such modification reduces mismatches between heavy and light chains from different Fab molecules, thereby increasing the yield and purity of the bispecific antigen-binding molecules of the present invention during recombinant production. In certain crossover Fab molecules useful for the bispecific antigen-binding molecules of the present invention, the variable domains of the Fab light chain and the Fab heavy chain (VL and VH, respectively) are swapped. However, even with such domain swapping, the preparation of bispecific antigen-binding molecules may contain some by-products due to so-called Bence-Jones interactions between mismatched heavy and light chains (see Schaefer et al., PNAS, 108 (2011) 11187-11191). To further reduce mispairing of the heavy and light chains of different Fab molecules and thereby increase the purity and yield of the desired bispecific antigen-binding molecule, charged amino acids with opposite charges can be introduced into specific amino acid positions in the CH1 and CL domains of either the Fab molecule that binds to the first antigen (GPRC5D) or the Fab molecule that binds to the second antigen (CD3), as further described herein. The charge modifications are made in either the conventional Fab molecule contained in the bispecific antigen-binding molecule or the VH / VL crossover Fab molecule contained in the bispecific antigen-binding molecule (but not both). In a specific embodiment, the charge modifications are made in the conventional Fab molecule contained in the bispecific antigen-binding molecule (which in a specific embodiment binds to the first antigen, i.e., GPRC5D).

[0065] The bispecific antigen-binding molecule can simultaneously bind to a first antigen (i.e., GPRC5D) and a second antigen (i.e., CD3). By simultaneously binding GPRC5D and an activating T cell antigen, the bispecific antigen-binding molecule can crosslink T cells and target cells. Such simultaneous binding results in activation of T lymphocytes, particularly cytotoxic T lymphocytes, in a cellular response selected from the group consisting of lysis of target cells, particularly GPRC5D-expressing tumor cells, proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers.

[0066] In one embodiment, the bispecific antigen binding molecule is capable of redirecting the cytotoxic activity of a T cell to a target cell, hi a specific embodiment, said redirection being independent of MHC-mediated peptide antigen presentation by the target cell and / or the specificity of the T cell.

[0067] In particular, T cells according to embodiments of the present invention are cytotoxic T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells, especially CD8 + T cells.

[0068] First antigen-binding moiety A bispecific antigen-binding molecule comprises at least one antigen-binding portion, particularly a Fab molecule, that binds to GPRC5D (first antigen). In certain embodiments, a bispecific antigen-binding molecule comprises two antigen-binding portions, particularly Fab molecules, that bind to GPRC5D.

[0069] In certain such embodiments, each of these antigen-binding moieties binds to the same antigenic determinant. In yet further specific embodiments, all of these antigen-binding moieties are identical, i.e., they comprise the same amino acid sequence, including the same amino acid substitutions in the CH1 domain and CL domain (if present) described herein. In one embodiment, the bispecific antigen-binding molecule comprises no more than two antigen-binding moieties, particularly Fab molecules, that bind to GPRC5D.

[0070] In a specific embodiment, the antigen-binding moiety(ies) that bind to GPRC5D are conventional Fab molecules. In such embodiments, the antigen-binding moiety that binds to a second antigen is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains have been swapped / replaced.

[0071] In alternative embodiments, the antigen-binding moiety(s) that bind to GPRC5D are crossover Fab molecules (i.e., Fab molecules in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains have been swapped / replaced) as described herein. In such embodiments, the antigen-binding moiety that binds to the second antigen is a conventional Fab molecule.

[0072] The GPRC5D-binding moiety can direct the bispecific antigen-binding molecule to a target site, for example, a particular type of tumor cell that expresses GPRC5D.

[0073] The first antigen-binding portion of the bispecific antigen-binding molecule may incorporate any of the features described herein for antibodies that bind to GPRC5D, either alone or in combination, unless it is scientifically clearly unreasonable or impossible.

[0074] In one aspect, the bispecific antigen-binding molecule comprises: (a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding moiety comprises a heavy chain variable region (VH) comprising heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 12, HCDR2 of SEQ ID NO: 13, and HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 15, LCDR2 of SEQ ID NO: 16, and LCDR3 of SEQ ID NO: 17; and (b) a second antigen-binding moiety that binds to CD3.

[0075] In some embodiments, the first antigen-binding moiety is a humanized antibody (derived from a humanized antibody). In one embodiment, the VH is a humanized VH and / or the VL is a humanized VL. In one embodiment, the first antigen-binding moiety comprises the same CDRs as any of the above embodiments and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0076] In one embodiment, the VH of the first antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and the VL of the first antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0077] In one embodiment, the first antigen-binding portion comprises a VH sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a VL sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0078] In one embodiment, the first antigen-binding portion comprises a VH comprising the amino acid sequence of SEQ ID NO:10 and a VL comprising the amino acid sequence of SEQ ID NO:11.

[0079] In one embodiment, the first antigen-binding portion comprises the VH sequence of SEQ ID NO:10 and the VL sequence of SEQ ID NO:11.

[0080] In a specific embodiment, the first antigen-binding portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 53. In a specific embodiment, the first antigen-binding portion comprises the VH sequence of SEQ ID NO: 48 and the VL sequence of SEQ ID NO: 11.

[0081] In one embodiment, the first antigen-binding moiety comprises a human constant region. In one embodiment, the first antigen-binding moiety is a Fab molecule comprising a human constant region, in particular a human CH1 and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs: 1 and 2 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 3 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In some embodiments, the first antigen-binding moiety comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, in particular an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In particular, the light chain constant region may comprise the amino acid mutations described herein in a "charge-modified" state and / or, in the case of a crossover Fab molecule, may comprise deletions or substitutions of one or more (in particular two) N-terminal amino acids. In some embodiments, the first antigen-binding portion comprises a heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 3. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise an amino acid mutation described herein in a "charge-modified" state.

[0082] Second antigen-binding moiety The bispecific antigen-binding molecule comprises at least one antigen-binding moiety, specifically a Fab molecule, that binds to a second antigen (CD3).

[0083] In specific embodiments, the antigen-binding portion that binds to the second antigen is a crossover Fab molecule as described herein (i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains have been swapped / replaced). In such embodiments, the antigen-binding portion that binds to the first antigen (i.e., GPRC5D) is preferably a conventional Fab molecule. In embodiments in which there are two or more antigen-binding portions, particularly Fab molecules, that bind to GPRC5D contained in the bispecific antigen-binding molecule, the antigen-binding portion that binds to the second antigen is preferably a crossover Fab molecule, and the antigen-binding portion that binds to GPRC5D is a conventional Fab molecule.

[0084] In alternative embodiments, the antigen-binding portion that binds the second antigen is a conventional Fab molecule. In such embodiments, the antigen-binding portion(s) that bind the first antigen (i.e., GPRC5D) are crossover Fab molecules (i.e., Fab molecules in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains have been swapped / replaced) as described herein. In embodiments in which there are two or more antigen-binding portions, particularly Fab molecules, that bind the second antigen contained in the bispecific antigen-binding molecule, the antigen-binding portion that binds GPRC5D is preferably a crossover Fab molecule, and the antigen-binding portion that binds the second antigen is a conventional Fab molecule.

[0085] The second antigen, i.e., CD3, is an activating T cell antigen (also referred to herein as an "activating T cell antigen-binding portion, or an activating T cell antigen-binding Fab molecule"). In a specific embodiment, the bispecific antigen-binding molecule comprises no more than one antigen-binding portion capable of specifically binding to an activating T cell antigen. In one embodiment, the bispecific antigen-binding molecule provides monovalent binding to an activating T cell antigen.

[0086] The second antigen is CD3, specifically rabbit CD3 (SEQ ID NO: 4) or cynomolgus monkey CD3 (SEQ ID NO: 5), most specifically human CD3. In one embodiment, the second antigen-binding portion is cross-reactive with (i.e., specifically binds to) human and cynomolgus monkey CD3. In some embodiments, the second antigen is the epsilon subunit of CD3 (CD3 epsilon).

[0087] In one embodiment, the second antigen-binding moiety comprises an HCDR1 of SEQ ID NO: 18, an HCDR2 of SEQ ID NO: 19, an HCDR3 of SEQ ID NO: 20, an LCDR1 of SEQ ID NO: 21, an LCDR2 of SEQ ID NO: 22, and an LCDR3 of SEQ ID NO: 23. In one embodiment, the second antigen-binding moiety comprises a VH comprising an HCDR1 of SEQ ID NO: 18, an HCDR2 of SEQ ID NO: 19, and an HCDR3 of SEQ ID NO: 20, and a VL comprising an LCDR1 of SEQ ID NO: 21, an LCDR2 of SEQ ID NO: 22, and an LCDR3 of SEQ ID NO: 23. In some embodiments, the second antigen-binding moiety is a humanized antibody (derived from a humanized antibody). In one embodiment, the VH is a humanized VH and / or the VL is a humanized VL. In one embodiment, the second antigen-binding moiety comprises the same CDRs as any of the above embodiments and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In one embodiment, the second antigen-binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24. In one embodiment, the second antigen-binding portion comprises a VL sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the second antigen-binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24 and a VL sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the VH of the second antigen-binding portion comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24, and the VL of the second antigen-binding portion comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the second antigen-binding portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25. In one embodiment, the second antigen-binding portion comprises the VH sequence of SEQ ID NO: 24, and the VL sequence of SEQ ID NO: 25.

[0088] In one embodiment, the second antigen-binding moiety comprises a human constant region. In one embodiment, the second antigen-binding moiety is a Fab molecule comprising a human constant region, particularly a human CH1 and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs: 1 and 2 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 3 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In some embodiments, the second antigen-binding moiety comprises a light chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, particularly to the amino acid sequence of SEQ ID NO: 1. In particular, the light chain constant region may comprise amino acid mutations described herein in a "charge-modified" state and / or, in the case of a crossover Fab molecule, may comprise deletions or substitutions of one or more (particularly two) N-terminal amino acids. In some embodiments, the second antigen-binding portion comprises a heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 3. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise an amino acid mutation described herein in a "charge-modified" state.

[0089] In some embodiments, the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain, in particular the variable domains VL and VH, are replaced with each other (i.e., according to such embodiments, the second antigen-binding moiety is a crossover Fab molecule in which the variable or constant domains of the Fab light chain and the Fab heavy chain are exchanged). In one such embodiment, the first (and, if present, the third) antigen-binding moiety is a conventional Fab molecule.

[0090] In one embodiment, no more than one antigen-binding moiety that binds to the second antigen (i.e., CD3) is present in the bispecific antigen-binding molecule (i.e., the bispecific antigen-binding molecule provides monovalent binding to the second antigen).

[0091] charge modification Bispecific antigen-binding molecules may contain amino acid substitutions in the Fab molecules contained therein that are particularly effective in reducing mismatching of light chains with mismatched heavy chains (Bence-Jones by-products) that can occur in the production of bi / multispecific antigen-binding molecules derived from Fabs having a VH / VL exchange in one (or more in the case of molecules containing more than two antigen-binding Fab molecules) binding arm. (See also PCT Publication No. WO 2015 / 150447, which is incorporated herein by reference in its entirety, in particular the Examples therein.) The ratio of desired bispecific antigen-binding molecules compared to undesired by-products (particularly Bence-Jones by-products present in bispecific antigen-binding molecules with a VH / VL domain exchange in one of the binding arms) can be improved by introducing charged amino acids with opposite charges at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

[0092] Thus, in some embodiments, the first and second antigen-binding moieties of the bispecific antigen-binding molecule are both Fab molecules, and in one of the antigen-binding moieties (particularly the second antigen-binding moiety), the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other: i) in the constant domain CL of the first antigen-binding moiety the amino acid at position 124 is substituted by a positively charged amino acid (Kabat numbering) and in the constant domain CH1 of the first antigen-binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (Kabat EU index numbering), or ii) in the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is substituted by a positively charged amino acid (Kabat numbering), and in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (Kabat EU index numbering).

[0093] A bispecific antigen-binding molecule does not contain both modifications described in i) and ii): the constant domains CL and CH1 of the antigen-binding moiety having a VH / VL exchange are not replaced with each other (i.e., remain unexchanged).

[0094] In a more specific embodiment, i) in the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), or ii) in the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is substituted independently by lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0095] In one such embodiment, in the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat); and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0096] In a further embodiment, in the constant domain CL of the first antigen-binding moiety the amino acid at position 124 is independently substituted by a lysine (K), an arginine (R) or a histidine (H) (Kabat numbering) and in the constant domain CH1 of the first antigen-binding moiety the amino acid at position 147 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (Kabat EU index numbering).

[0097] In a specific embodiment, in the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (Kabat numbering), and the amino acid at position 123 is independently substituted by lysine (K), arginine (R), or histidine (H) (Kabat numbering); and in the constant domain CHI of the first antigen-binding moiety, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0098] In a more specific embodiment, in the constant domain CL of said first antigen-binding moiety the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), and in the constant domain CHI of said first antigen-binding moiety the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index), and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0099] In an even more specific embodiment, in the constant domain CL of said first antigen-binding moiety the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and in the constant domain CHI of said first antigen-binding moiety the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0100] In a specific embodiment, when the amino acid substitutions according to the above embodiments are made in the constant domain CL and the constant domain CH1 of the first antigen-binding moiety, the constant domain CL of the first antigen-binding moiety is of the kappa isotype.

[0101] Alternatively, the amino acid substitutions in the above embodiments may be made in the constant domains CL and CH1 of the second antigen-binding moiety, rather than in the constant domains CL and CH1 of the first antigen-binding moiety. In a specific such embodiment, the constant domain CL of the second antigen-binding moiety is of the kappa isotype.

[0102] Thus, in one embodiment, in the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0103] In a further embodiment, in the constant domain CL of the second antigen-binding moiety the amino acid at position 124 is substituted independently by a lysine (K), an arginine (R) or a histidine (H) (Kabat numbering) and in the constant domain CH1 of the second antigen-binding moiety the amino acid at position 147 is substituted independently by a glutamic acid (E) or an aspartic acid (D) (Kabat EU index numbering).

[0104] In yet another embodiment, in the constant domain CL of said second antigen-binding moiety, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering); and in the constant domain CHI of said second antigen-binding moiety, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0105] In one embodiment, in the constant domain CL of said second antigen-binding moiety the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), and in the constant domain CHI of said second antigen-binding moiety the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index), and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0106] In another embodiment, in the constant domain CL of said second antigen-binding moiety the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and in the constant domain CHI of said second antigen-binding moiety the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index), and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0107] In a specific embodiment, the bispecific antigen-binding molecule comprises: (a) a first antigen-binding portion that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion is a Fab molecule comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 12, an HCDR2 of SEQ ID NO: 13, and an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 15, an LCDR2 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO: 17; (b) a second antigen-binding portion that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding portion is a Fab molecule comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 18, an HCDR2 of SEQ ID NO: 19, and an HCDR3 of SEQ ID NO: 20, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 21, an LCDR2 of SEQ ID NO: 22, and an LCDR3 of SEQ ID NO: 23, wherein the variable domains VL and VH of the Fab heavy chain are substituted for each other; In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted by lysine (K), arginine (R), or histidine (H) (in particular embodiments, independently by lysine (K) or arginine (R)) (Kabat numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R), or histidine (H) (in specific embodiments, independently by lysine (K) or arginine (R)) (Kabat numbering), and in the constant domain CHI of the first antigen-binding moiety, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0108] Bispecific antigen-binding molecule format In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, multispecific antibodies have three or more binding specificities. In certain embodiments, bispecific antibodies can bind to two (or more) different epitopes of GPRC5D. Multispecific (e.g., bispecific) antibodies can also be used to localize cytotoxic agents to cells expressing GPRC5D. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0109] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (see, e.g., WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605), using conventional light chain technology to circumvent light chain mispairing problems (see, e.g., WO 98 / 50431), using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605), al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and by the preparation of triabodies as described, for example, in Tutorial. J. Immunol. 147:60 (1991).

[0110] Also included herein are engineered antibodies, or DVD-Igs, having three or more antigen-binding sites, including, for example, "octopus antibodies" (see, e.g., WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual acting FAbs" or "DAFs" that contain antigen-binding sites that bind to GPRC5D and CD3 (see, e.g., U.S. Patent Application Publication Nos. 2008 / 0069820 and WO 2015 / 095539).

[0111] Multispecific antibodies can also be provided in asymmetric form with domain crossovers in one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253), or complete Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299; see also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, for example, WO 2016 / 172485.

[0112] A variety of additional molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0113] Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, so-called "BiTE" (bispecific T cell engager) molecules in which two scFv molecules are fused by a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567; Nagorsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)), diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies ("TandAb", Kipriyanov et al., J Mol Biol 293, 41-56 (1999)), "DART" (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for further stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), as well as the so-called triomabs, which are whole mouse / rat IgG hybrid molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Particular T cell bispecific antibody formats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309, and Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0114] The components of the bispecific antigen-binding molecule may be fused to each other in a variety of configurations.

[0115] In specific embodiments, the antigen-binding moieties comprised in the bispecific antigen-binding molecule are Fab molecules. In such embodiments, the first, second, third, etc. antigen-binding moieties may be referred to herein as first, second, third, etc. Fab molecules, respectively.

[0116] In one embodiment, the first and second antigen-binding portions of the bispecific antigen-binding molecule are fused to each other, optionally via a peptide linker. In a specific embodiment, the first and second antigen-binding portions are each Fab molecules. In one such embodiment, the second antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion at the C-terminus of the Fab heavy chain. In another such embodiment, the first antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the second antigen-binding portion at the C-terminus of the Fab heavy chain. In embodiments in which (i) the second antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion at the C-terminus of the Fab heavy chain, or (ii) the first antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the second antigen-binding portion at the C-terminus of the Fab heavy chain, the Fab light chain of the first antigen-binding portion and the Fab light chain of the second antigen-binding portion may further be fused to each other, optionally via a peptide linker.

[0117] Bispecific antigen-binding molecules having a single antigen-binding moiety (e.g., a Fab molecule) capable of specifically binding to a target cell antigen such as GPRC5D are useful, particularly when internalization of the target cell antigen is expected after binding of the high-affinity antigen-binding moiety. In such cases, the presence of more than one antigen-binding moiety specific for the target cell antigen can enhance internalization of the target cell antigen, thereby reducing its availability.

[0118] However, in other cases, it may be advantageous to have a bispecific antigen-binding molecule that contains two or more antigen-binding moieties (e.g., Fab molecules) specific for target cell antigens, for example, to optimize targeting to the target site or to enable cross-linking of target cell antigens.

[0119] Thus, in a specific embodiment, the bispecific antigen-binding molecule comprises a third antigen-binding moiety.

[0120] In one embodiment, the third antigen-binding moiety binds to the first antigen, i.e., GPRC5D. In one embodiment, the third antigen-binding moiety is a Fab molecule.

[0121] In one embodiment, the third antigen-binding moiety is identical to the first antigen-binding moiety.

[0122] The third antigen-binding portion of the bispecific antigen-binding molecule may incorporate any of the features described herein for the first antigen-binding portion and / or antibodies that bind to GPRC5D, either alone or in combination, unless it is scientifically clearly unreasonable or impossible.

[0123] In one embodiment, the third antigen-binding portion comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 12, an HCDR2 of SEQ ID NO: 13, and an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 15, an LCDR2 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO: 17.

[0124] In some embodiments, the third antigen-binding moiety is a humanized antibody (derived from a humanized antibody). In one embodiment, the VH is a humanized VH and / or the VL is a humanized VL. In one embodiment, the third antigen-binding moiety comprises the same CDRs as any of the above embodiments and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0125] In one embodiment, the VH of the third antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and the VL of the third antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0126] In one embodiment, the third antigen-binding portion comprises a VH sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a VL sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0127] In one embodiment, the third antigen-binding portion comprises a VH comprising the amino acid sequence of SEQ ID NO:10 and a VL comprising the amino acid sequence of SEQ ID NO:11.

[0128] In one embodiment, the third antigen-binding portion comprises the VH sequence of SEQ ID NO:10 and the VL sequence of SEQ ID NO:11.

[0129] In a specific embodiment, the third antigen-binding portion comprises a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 53. In a specific embodiment, the third antigen-binding portion comprises a VH sequence of SEQ ID NO: 48 and a VL sequence of SEQ ID NO: 11.

[0130] In one embodiment, the third antigen-binding moiety comprises a human constant region. In one embodiment, the third antigen-binding moiety is a Fab molecule comprising a human constant region, particularly a human CH1 and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs: 1 and 2 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 3 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In some embodiments, the third antigen-binding moiety comprises a light chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, particularly to the amino acid sequence of SEQ ID NO: 1. In particular, the light chain constant region may comprise the amino acid mutations described herein in a "charge-modified" state and / or, in the case of a crossover Fab molecule, may comprise deletions or substitutions of one or more (particularly two) N-terminal amino acids. In some embodiments, the third antigen-binding portion comprises a heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 3. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise an amino acid mutation described herein in a "charge-modified" state.

[0131] In specific embodiments, the third antigen-binding moiety and the first antigen-binding moiety are each Fab molecules, and the third antigen-binding moiety is identical to the first antigen-binding moiety. Thus, in these embodiments, the first and third antigen-binding moieties comprise the same heavy and light chain amino acid sequences and have the same domain configuration (i.e., conventional or crossover). Furthermore, in these embodiments, the third antigen-binding moiety, if present, comprises the same amino acid substitutions as the first antigen-binding moiety. For example, the amino acid substitutions described herein as "charge-modifying" are made in the constant domains CL and CHI of the first and third antigen-binding moieties, respectively. Alternatively, the amino acid substitutions may be made in the constant domains CL and CHI of the second antigen-binding moiety (which in specific embodiments is also a Fab molecule), rather than in the constant domains CL and CHI of the first and third antigen-binding moieties.

[0132] Like the first antigen-binding portion, the third antigen-binding portion is particularly a conventional Fab molecule. However, embodiments in which the first and third antigen-binding portions are crossover Fab molecules (and the second antigen-binding portion is a conventional Fab molecule) are also contemplated. Thus, in specific embodiments, the first antigen-binding portion and the third antigen-binding portion are each conventional Fab molecules, and the second antigen-binding portion is a crossover Fab molecule as described herein (i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are swapped / replaced with each other). In other embodiments, the first antigen-binding portion and the third antigen-binding portion are each crossover Fab molecules, and the second antigen-binding portion is a conventional Fab molecule.

[0133] When a third antigen-binding moiety is present, in a specific embodiment, the first and third antigen-binding moieties bind to GPRC5D and the second antigen-binding moiety binds to CD3, particularly CD3 epsilon.

[0134] In a specific embodiment, the bispecific antigen-binding molecule comprises an Fc domain composed of a first and a second subunit, wherein the first and second subunits of the Fc domain are capable of stable association.

[0135] Bispecific antigen-binding molecules may have different configurations, i.e., the first, second (and optionally third) antigen-binding moieties may be fused to each other and to the Fc domain in different ways. The components may be fused to each other directly or, preferably, via one or more suitable peptide linkers. When fusion of a Fab molecule is to the N-terminus of a subunit of the Fc domain, the fusion is typically via the immunoglobulin hinge region.

[0136] In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety are each a Fab molecule, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second Fc domain subunit. In such embodiments, the first antigen-binding moiety may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety or to the N-terminus of the other Fc domain subunit. In certain such embodiments, the first antigen-binding moiety is a conventional Fab molecule, and the second antigen-binding moiety is a crossover Fab molecule as described herein (i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are swapped / replaced). In other such embodiments, the first Fab molecule is a crossover Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0137] In one embodiment, the first antigen-binding moiety and the second antigen-binding moiety are each Fab molecules, and the second antigen-binding moiety is fused at the C-terminus of its Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the first antigen-binding moiety is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety. In a specific embodiment, the bispecific antigen-binding molecule consists essentially of first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, and the first Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fc domain of the first or second subunit. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.

[0138] In another embodiment, the first antigen-binding portion and the second antigen-binding portion are each Fab molecules, and each of the first antigen-binding portion and the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits. In a specific embodiment, the bispecific antigen-binding molecule consists essentially of first and second Fab molecules, and the Fc domain is composed of the first and second subunits and, optionally, one or more peptide linkers, and each of the first and second Fab molecules is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits. The first Fab molecule and the second Fab molecule may be fused to the Fc domain directly or via a peptide linker. In a specific embodiment, the first and second Fab molecules are each fused to the Fc domain by an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1 Fc domain.

[0139] In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety are each Fab molecules, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In such embodiments, the second antigen-binding moiety may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety or to the N-terminus of the other subunit of the Fc domain (as described above). In certain such embodiments, the first antigen-binding moiety is a conventional Fab molecule, and the second antigen-binding moiety is a crossover Fab molecule as described herein (i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are swapped / replaced). In other such embodiments, the first Fab molecule is a crossover Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0140] In one embodiment, the first antigen-binding moiety and the second antigen-binding moiety are each Fab molecules, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety. In a specific embodiment, the bispecific antigen-binding molecule consists essentially of first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.

[0141] In some embodiments, a third antigen-binding moiety, particularly a third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In certain such embodiments, the first and third Fab molecules are each conventional Fab molecules, and the second Fab molecule is a crossover Fab molecule as described herein (i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains have been swapped / replaced). In other such embodiments, the first and third Fab molecules are each crossover Fab molecules, and the second Fab molecule is a conventional Fab molecule.

[0142] In certain such embodiments, the second and third antigen-binding moieties are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In a specific embodiment, the bispecific antigen-binding molecule consists essentially of first, second, and third Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The second and third Fab molecules may be fused to the Fc domain directly or via peptide linkers. In a specific embodiment, the second and third Fab molecules are each fused to an Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.

[0143] In another such embodiment, the first and third antigen-binding moieties are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety. In a specific embodiment, the bispecific antigen-binding molecule consists essentially of first, second, and third Fab molecules, an Fc domain composed of the first and second subunits, and, optionally, one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first Fc domain subunit, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit. The first and third Fab molecules may be fused to the Fc domain directly or via peptide linkers. In a specific embodiment, the first and third Fab molecules are each fused to an Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.

[0144] In the construction of a bispecific antigen-binding molecule in which a Fab molecule is fused via an immunoglobulin hinge region from the C-terminus of the Fab heavy chain to the N-terminus of each subunit of the Fc domain, the two Fab molecules, the hinge region, and the Fc domain essentially form an immunoglobulin molecule. In a specific embodiment, the immunoglobulin molecule is an immunoglobulin of the IgG class. In a more specific embodiment, the immunoglobulin is an immunoglobulin of the IgG1 subclass. In another embodiment, the immunoglobulin is an immunoglobulin of the IgG4 subclass. In a more specific embodiment, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the immunoglobulin comprises a human constant region, particularly a human Fc region.

[0145] In some bispecific antigen-binding molecules, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are fused to each other, optionally via a peptide linker. Depending on the structure of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. Fusing the Fab light chains of the first and second Fab molecules further reduces mispairing of incompatible Fab heavy and light chains and also reduces the number of plasmids required to express a portion of the bispecific antigen-binding molecule.

[0146] The antigen-binding portions may be fused to the Fc domain or to each other, either directly or via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or G4 (SG4) n Peptide linkers are included. "n" is generally an integer from 1 to 10, typically from 2 to 4. In one embodiment, the peptide linker has a length of at least 5 amino acids, in one embodiment from 5 to 100 amino acids, and in a further embodiment from 10 to 50 amino acids. In one embodiment, the peptide linker has a length of (GxS) n or (GxS) n G mwhere G=glycine, S=serine, (x=3, n=3, 4, 5 or 6, m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5, m=0, 1, 2 or 3), in certain embodiments x=4, n=2 or 3, and in further embodiments x=4, n=2. In one embodiment, the peptide linker is (G4S)2. A peptide linker particularly suitable for fusing the Fab light chains of the first and second Fab molecules to each other is (G4S)2. An exemplary peptide linker suitable for linking the Fab heavy chains of the first and second Fab fragments comprises the sequence (D)-(G4S)2 (SEQ ID NOs: 7 and 8). Another suitable such linker comprises the sequence (G4S)4. In addition, the linker may comprise (part of) an immunoglobulin hinge region. In particular, when a Fab molecule is fused to the N-terminus of an Fc domain subunit, the fusion may be via an immunoglobulin hinge region or part thereof, with or without an additional peptide linker.

[0147] In certain embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), thereby causing the Fab heavy chain constant region of the second Fab molecule to share a carboxy-terminal peptide bond with an Fc domain subunit (VL (2) -CH1 (2) -CH2-CH3(-CH4)) and a polypeptide in which the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (1) -CH1 (1) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1)In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0148] In certain embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), whereby the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CL (2) -CH2-CH3(-CH4)) and a polypeptide in which the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (1) -CH1 (1) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) )) and a Fab light chain polypeptide (VL) of the first Fab molecule. (1) -CL (1) In certain embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0149] In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, where the heavy chain variable region is replaced by a light chain variable region), whereby the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, whereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. (2) -CH1 (2)-VH (1) -CH1 (1) In other embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VH) in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain variable region of a second Fab molecule, whereby the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, whereby the heavy chain variable region is replaced by a light chain variable region), whereby the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. (1) -CH1 (1) -VL (2) -CH1 (2) -CH2-CH3(-CH4)).

[0150] In some of these embodiments, the bispecific antigen-binding molecule comprises a crossover Fab light chain polypeptide (VH) of a second Fab molecule, in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) In some other of these embodiments, the bispecific antigen-binding molecule further comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, whereby the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule. (2) -CL (2) -VL (1) -CL (1) ), or a polypeptide in which the Fab light chain polypeptide of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, whereby the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VL(1) -CL (1) -VH (2) -CL (2) ) as needed.

[0151] The bispecific antigen-binding molecule according to these embodiments comprises (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL (3) -CL (3) In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0152] In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, where the heavy chain constant region is replaced by a light chain constant region), whereby the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, whereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. (2) -CL (2) -VH (1) -CH1 (1)In other embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VH) in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, whereby the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), whereby the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. (1) -CH1 (1) -VH (2) -CL (2) -CH2-CH3(-CH4)).

[0153] In some of these embodiments, the bispecific antigen-binding molecule comprises a crossover Fab light chain polypeptide (VL) of a second Fab molecule, in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule. (2) -CH1 (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) In some other of these embodiments, the bispecific antigen-binding molecule further comprises a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, whereby the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule. (2) -CH1 (2) -VL (1) -CL (1) ), or a polypeptide in which the Fab light chain polypeptide of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, whereby the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VL (1) -CL (1) -VL(2) -CH1 (2) ) as needed.

[0154] The bispecific antigen-binding molecule according to these embodiments comprises (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL (3) -CL (3) In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0155] In certain embodiments, the bispecific antigen-binding molecule does not comprise an Fc domain. In certain such embodiments, the first and, if present, third Fab molecules are each conventional Fab molecules, and the second Fab molecule is a crossover Fab molecule as described herein (i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains have been swapped / replaced). In other such embodiments, the first and, if present, third Fab molecules are each crossover Fab molecules, and the second Fab molecule is a conventional Fab molecule.

[0156] In one such embodiment, the bispecific antigen-binding molecule consists essentially of a first and a second antigen-binding moiety, and optionally one or more peptide linkers, wherein the first and second antigen-binding moieties are both Fab molecules, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety.

[0157] In another such embodiment, the bispecific antigen-binding molecule consists essentially of a first and a second antigen-binding moiety and, optionally, one or more peptide linkers, wherein the first and second antigen-binding moieties are both Fab molecules and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety.

[0158] In some embodiments, the first Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the second Fab molecule, and the bispecific antigen-binding molecule further comprises a third antigen-binding moiety, particularly a third Fab molecule, said third Fab molecule fused at its C-terminus to the N-terminus of the Fab heavy chain of the first Fab molecule. In certain such embodiments, the bispecific antigen-binding molecule consists essentially of first, second, and third Fab molecules and optionally one or more peptide linkers, wherein the first Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the second Fab molecule, and the third Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the first Fab molecule.

[0159] In some embodiments, the second Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the first Fab molecule, and the bispecific antigen-binding molecule further comprises a third antigen-binding moiety, particularly a third Fab molecule, said third Fab molecule fused at its N-terminus to the C-terminus of the Fab heavy chain of the first Fab molecule. In certain such embodiments, the bispecific antigen-binding molecule consists essentially of first, second, and third Fab molecules and optionally one or more peptide linkers, wherein the second Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the first Fab molecule, and the third Fab molecule is fused at its N-terminus to the C-terminus of the Fab heavy chain of the first Fab molecule.

[0160] In certain embodiments, a bispecific antigen-binding molecule comprises a polypeptide (VH) in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, whereby the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by the light chain variable region). (1) -CH1 (1) -VL (2) -CH1 (2) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) and further.

[0161] In certain embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VH) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), thereby causing the Fab heavy chain constant region of the second Fab molecule to share a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. (2) -CH1 (2) -VL (1) -CH1 (1) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) and further.

[0162] In certain embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), whereby the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. (2) -CL (2) -VH (1) -CH1 (1) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) )) and a Fab light chain polypeptide (VL) of the first Fab molecule. (1) -CL (1) ) and further.

[0163] In certain embodiments, a bispecific antigen-binding molecule according to the invention comprises a polypeptide (VL) in which the Fab light chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), thereby causing the Fab heavy chain constant region of the second Fab molecule to share a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. (2) -CH1 (2) -VH (1) -CH1 (1) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) and further.

[0164] In certain embodiments, the bispecific antigen-binding molecule comprises a Fab heavy chain of a third Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a first Fab molecule, whereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain variable region of a second Fab molecule, whereby the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, whereby the heavy chain variable region is replaced by a light chain variable region), and a polypeptide (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VL (2) -CH1 (2) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) In some embodiments, the bispecific antigen-binding molecule further comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0165] In certain embodiments, bispecific antigen binding occurs when the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, whereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, whereby the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, whereby the heavy chain constant region is replaced by the light chain constant region), and the polypeptide (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VH(2) -CL (2) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) )) and a Fab light chain polypeptide (VL) of the first Fab molecule. (1) -CL (1) In some embodiments, the bispecific antigen-binding molecule further comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0166] In certain embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VL) in which the Fab light chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, where the heavy chain variable region is replaced by a light chain variable region), whereby the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and whereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule. (2) -CH1 (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) In some embodiments, the bispecific antigen-binding molecule further comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0167] In certain embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VH) in which the Fab heavy chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, where the heavy chain constant region is replaced by a light chain constant region), whereby the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, and whereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule. (2) -CL (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) )) and a Fab light chain polypeptide (VL) of the first Fab molecule. (1) -CL (1) In some embodiments, the bispecific antigen-binding molecule further comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0168] In certain embodiments, the bispecific antigen-binding molecule comprises a Fab heavy chain of a second Fab molecule that shares a carboxy-terminal peptide bond with a Fab light chain variable region of a first Fab molecule, whereby the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises crossover Fab heavy chains, whereby the heavy chain variable region is replaced by a light chain variable region), whereby the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain variable region of a third Fab molecule, whereby the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of a third Fab molecule (i.e., the third Fab molecule comprises crossover Fab heavy chains, whereby the heavy chain variable region is replaced by a light chain variable region), and a polypeptide (VH (2) -CH1 (2) -VL (1) -CH1 (1) -VL (3) -CH1 (3) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab heavy chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a first Fab molecule (VH (1) -CL (1) ) and the Fab light chain polypeptide (VL) of the second Fab molecule (2) -CL (2) In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. (3) -CL (3) ) further includes.

[0169] In certain embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VH) in which the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, whereby the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises crossover Fab heavy chains, whereby the heavy chain constant region is replaced by a light chain constant region), and whereby the Fab light chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a third Fab molecule, whereby the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises crossover Fab heavy chains, whereby the heavy chain constant region is replaced by a light chain constant region). (2) -CH1 (2) -VH (1) -CL (1) -VH (3) -CL (3) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab light chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a first Fab molecule (VL (1) -CH1 (1) ) and a Fab light chain polypeptide (VL) of a second Fab molecule. (2) -CL (2) In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL (3) -CH1 (3) ) further includes.

[0170] In certain embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VL) in which the Fab light chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain, where the heavy chain variable region is replaced by a light chain variable region), whereby the Fab heavy chain constant region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a first Fab molecule, and whereby the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain, where the heavy chain variable region is replaced by a light chain variable region), whereby the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule. (3) -CH1 (3) -VL (1) -CH1 (1) -VH (2) -CH1 (2) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab heavy chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a first Fab molecule (VH (1) -CL (1) ) and the Fab light chain polypeptide (VL) of the second Fab molecule (2) -CL (2) In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. (3) -CL (3) ) further includes.

[0171] In certain embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VH) in which the Fab heavy chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain, where the heavy chain constant region is replaced by a light chain constant region), whereby the Fab heavy chain constant region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a first Fab molecule, and whereby the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain, where the heavy chain constant region is replaced by a light chain constant region), whereby the Fab light chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule. (3) -CL (3) -VH (1) -CL (1) -VH (2) -CH1 (2) In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab light chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a first Fab molecule (VL (1) -CH1 (1) ) and a Fab light chain polypeptide (VL) of a second Fab molecule. (2) -CL (2) In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL (3) -CH1 (3) ) further includes.

[0172] In a specific embodiment, the present invention provides a first antigen-binding portion that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion is a Fab molecule comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 12, an HCDR2 of SEQ ID NO: 13, and an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 15, an LCDR2 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO: 17. a) a first binding portion that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH of a Fab light chain and the constant domains CL and CH1 of a Fab heavy chain are replaced with each other, and the Fab molecule has a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 18, an HCDR2 of SEQ ID NO: 19, and an HCDR3 of SEQ ID NO: 20, a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 21, an HCDR2 of SEQ ID NO: 22, and a light chain complementarity determining region (LCDR) 2 of SEQ ID NO: 23. a) a second antigen-binding portion comprising a light chain variable region (VL) comprising LCDR2 and LCDR3 of SEQ ID NO: 23; c) a third antigen-binding portion that binds to a first antigen and is identical to the first antigen-binding portion; and d) an Fc domain composed of first and second subunits, wherein (i) the first antigen-binding portion of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion of b); and or (ii) the second antigen-binding portion of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion of a), and the first antigen-binding portion of a) and the third antigen-binding portion of c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d).

[0173] In another embodiment, the present invention provides a antibody comprising: a) a first antigen-binding portion that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 12, HCDR2 of SEQ ID NO: 13, and HCDR3 of SEQ ID NO: 14; a) a first antigen-binding portion that is a Fab molecule comprising a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 15, an LCDR2 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO: 17; b) a second antigen-binding portion that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain are replaced with each other, and the Fab molecule comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 18, an HCDR2 of SEQ ID NO: 19, and an LCDR3 of SEQ ID NO: 20. and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 21, an LCDR2 of SEQ ID NO: 22, and an LCDR3 of SEQ ID NO: 23; and c) an Fc domain composed of first and second subunits, wherein (i) the first antigen-binding portion of a) and the second antigen-binding portion of b) are each fused to the N-terminus of one of the subunits of the Fc domain of c) at the C-terminus of the Fab heavy chain.

[0174] In all of the different configurations of the bispecific antigen-binding molecules, the amino acid substitutions described herein, if present, may be in either the CH1 and CL domains of the first and (if present) third antigen-binding moiety / Fab molecule, or the CH1 or CL domain of the second antigen-binding moiety / Fab molecule. Preferably, they are in the CH1 and CL domains of the first and (if present) third antigen-binding moiety / Fab molecule. In accordance with the concept of the present invention, when an amino acid substitution described herein is made in the first (and, if present, third) antigen-binding moiety / Fab molecule, such an amino acid substitution is not made in the second antigen-binding moiety / Fab molecule. Conversely, when an amino acid substitution described herein is made in the second antigen-binding moiety / Fab molecule, such an amino acid substitution is not made in the first (and, if present, third) antigen-binding moiety / Fab molecule. The amino acid substitutions are particularly made in bispecific antigen-binding molecules comprising Fab molecules, in which the variable domains VL and VH1 of the Fab light chain and Fab heavy chain are replaced with each other.

[0175] In specific embodiments, the constant domain CL of the first (and, if present, the third) Fab molecule of the bispecific antigen-binding molecule is of the kappa isotype, particularly when the amino acid substitutions as described herein are made in the first (and, if present, the third) antigen-binding moiety / Fab molecule. In other embodiments of bispecific antigen-binding molecules according to the invention, particularly in embodiments where the amino acid substitutions described herein are made in the second antigen-binding moiety / Fab molecule, the constant domain CL of the second antigen-binding moiety / Fab molecule is of the kappa isotype. In some embodiments, the constant domain CL of the first (and, if present, the third) antigen-binding moiety / Fab molecule and the constant domain CL of the second antigen-binding moiety / Fab molecule are of the kappa isotype.

[0176] In one embodiment, the bispecific antigen-binding molecule comprises: a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 12, an HCDR2 of SEQ ID NO: 13, and an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 15, an LCDR2 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO: 17; and b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are interchanged, and the Fab molecule comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 18, an HCDR2 of SEQ ID NO: 19, and an HCDR3 of SEQ ID NO: 20. a) a second antigen-binding portion comprising a variable region (VH) and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 21, a LCDR2 of SEQ ID NO: 22, and a LCDR3 of SEQ ID NO: 23; c) a third antigen-binding portion that binds to a first antigen and is identical to the first antigen-binding portion; and d) an Fc domain composed of a first and a second subunit, wherein in the constant domain CL of the first antigen-binding portion of a) and the third antigen-binding portion of c), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat); and in the constant domain CH1 of the first antigen-binding portion of a) and the third antigen-binding portion of c), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat). (numbering according to the EU index), and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat(numbered according to the EU index), (i) the first antigen-binding portion of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion of b), and the second antigen-binding portion of b) and the third antigen-binding portion of c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of d), or (ii) the second antigen-binding portion of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion of a), and the first antigen-binding portion of a) and the third antigen-binding portion of c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of d).

[0177] In a specific embodiment, the bispecific antigen-binding molecule comprises: a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 12, an HCDR2 of SEQ ID NO: 13, and an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 15, an LCDR2 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO: 17; and b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are interchanged, and the Fab molecule comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 18, an HCDR2 of SEQ ID NO: 19, and an HCDR3 of SEQ ID NO: 20. a) a second antigen-binding portion comprising a chain variable region (VH) and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 21, a LCDR2 of SEQ ID NO: 22, and a LCDR3 of SEQ ID NO: 23; c) a third antigen-binding portion that binds to a first antigen and is identical to the first antigen-binding portion; and d) an Fc domain composed of a first and a second subunit, wherein in the constant domain CL of the first antigen-binding portion of a) and the third antigen-binding portion of c), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat); and in the constant domain CH1 of the first antigen-binding portion of a) and the third antigen-binding portion of c), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat). (numbering according to the EU index), and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat(numbered according to the EU index), (i) the first antigen-binding portion of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion of b), and the second antigen-binding portion of b) and the third antigen-binding portion of c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of d), or (ii) the second antigen-binding portion of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion of a), and the first antigen-binding portion of a) and the third antigen-binding portion of c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of d).

[0178] In another embodiment, the bispecific antigen-binding molecule comprises: a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 12, an HCDR2 of SEQ ID NO: 13, and an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 15, an LCDR2 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO: 17; and b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are interchanged, and the Fab molecule comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 18, an HCDR2 of SEQ ID NO: 19, and an HCDR3 of SEQ ID NO: 20. a) a second antigen-binding portion comprising a variable region (VH) and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 21, a LCDR2 of SEQ ID NO: 22, and a LCDR3 of SEQ ID NO: 23; c) a third antigen-binding portion that binds to a first antigen and is identical to the first antigen-binding portion; and d) an Fc domain composed of a first and a second subunit, wherein in the constant domain CL of the first antigen-binding portion of a) and the third antigen-binding portion of c), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding portion of a) and the third antigen-binding portion of c), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat). a) a first antigen-binding moiety and b) a second antigen-binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0179] According to any of the above embodiments, the components of the bispecific antigen-binding molecule (e.g., Fab molecule, Fc domain) may be fused directly or via various linkers, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, as described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or G4 (SG4) n A peptide linker is included, where n is an integer usually from 1 to 10, typically from 2 to 4.

[0180] In a particular aspect, the bispecific antigen-binding molecule comprises: a) a first and a third antigen-binding moiety that bind to a first antigen, wherein the first antigen is GPRC5D, and the first and second antigen-binding moieties are each (conventional) Fab molecules comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are interchanged, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24. and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25; and c) an Fc domain composed of first and second subunits, wherein in the constant domain CL of the first and third antigen-binding portions of a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the first and third antigen-binding portions of a) the amino acid at position 147 is substituted by glutamic acid (E) (Kabat numbering). and wherein the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to EU index, Kabat); and wherein the first antigen-binding portion of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion of b), and the second antigen-binding portion of b) and the third antigen-binding portion of a) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0181] In one embodiment, in the first subunit of the Fc domain of the bispecific antigen-binding molecule, the threonine residue at position 366 is substituted with a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is substituted with a valine residue (Y407V), and optionally, the threonine residue at position 366 is substituted with a serine residue (T366S), and the leucine residue at position 368 is substituted with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0182] In a further embodiment, the first subunit of the Fc domain of the bispecific antigen-binding molecule further comprises a replacement of the serine residue at position 354 by a cysteine ​​residue (S354C) or a replacement of the glutamic acid residue at position 356 by a cysteine ​​residue (E356C) (particularly, a replacement of the serine residue at position 354 by a cysteine ​​residue), and a replacement of the tyrosine residue at position 349 by a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index).

[0183] In still further embodiments, in each of the first and second subunits of the Fc domain of the bispecific antigen-binding molecule, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to the Kabat EU index).

[0184] In an even further embodiment, the Fc domain is an IgG1 Fc domain.

[0185] In another specific embodiment, the bispecific antigen-binding molecule comprises a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 26, a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 27, a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 28, and a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 29. In a further specific embodiment, the bispecific antigen-binding molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 26, a polypeptide comprising the amino acid sequence of SEQ ID NO: 27, a polypeptide comprising the amino acid sequence of SEQ ID NO: 28, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 29. In one embodiment, the bispecific antigen-binding molecule is a formatamig.

[0186] Fc domain In a specific embodiment, the bispecific antigen-binding molecule comprises an Fc domain composed of a first and a second subunit.

[0187] The Fc domain of a bispecific antigen-binding molecule consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment, a bispecific antigen-binding molecule of the present invention comprises no more than one Fc molecule.

[0188] In one embodiment, the Fc domain of the bispecific antigen-binding molecule is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228, in particular the amino acid substitution S228P (Kabat EU index numbering). This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG1 Fc domain. An exemplary sequence of a human IgG1 Fc region is shown in SEQ ID NO: 6.

[0189] Fc domain modifications that promote heterodimerization Bispecific antigen-binding molecules contain different antigen-binding moieties that can be fused to one or the other of the two subunits of the Fc domain; therefore, the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides and subsequent dimerization generates several possible combinations of the two polypeptides. To increase the yield and purity of bispecific antigen-binding molecules in recombinant production, it is advantageous to introduce modifications to the Fc domain of the bispecific antigen-binding molecule that promote the association of the desired polypeptides.

[0190] Thus, in a specific embodiment, the Fc domain of the bispecific antigen-binding molecule comprises a modification that promotes the association of the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is within the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0191] There are several approaches to modifications in the CH3 domain of an Fc domain to enhance heterodimerization, which are fully described in, for example, WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches, the CH3 domain of the first Fc domain subunit and the CH3 domain of the second Fc domain subunit are both engineered in a complementary manner, such that each CH3 domain (or the heavy chain containing it) is directed not to homodimerize with itself but to heterodimerize with another complementary engineered CH3 domain (so that the first CH3 domain and the second CH3 domain heterodimerize, and homodimers between the two first CH3 domains or the two second CH3 domains do not form). These different approaches for improved heavy chain heterodimerization are envisioned as different alternatives in combination with heavy chain-light chain modifications (e.g., swapping / replacing VH and VL in one binding arm and introducing oppositely charged amino acid substitutions at the CH1 / CL interface) in bispecific antigen-binding molecules with reduced heavy chain / light chain mispairing and Bence Jones-type by-products.

[0192] In a specific embodiment, the modification that promotes association of the first and second subunits of the Fc domain is a so-called "knob-in-hole" modification, which comprises a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0193] Knob-into-hole technology 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 protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion can locate within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Complementary cavities of identical or similar size to the protrusions are created on the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0194] Thus, in a specific embodiment, in the CH3 domain of a first subunit of the Fc domain of the bispecific antigen-binding molecule, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be repositioned within a cavity in the CH3 domain of the second subunit, and in the CH3 domain of a 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 in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be repositioned.

[0195] Preferably, said amino acid residues having larger side chain volumes are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0196] Preferably, said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

[0197] 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.

[0198] In a specific embodiment, in the (CH3 domain of) the first subunit of the Fc domain (the "knob" subunit), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the (CH3 domain of) the second subunit of the Fc domain (the "hole" subunit), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, the second subunit of the Fc domain further replaces the threonine residue at position 366 with a serine residue (T366S) and the leucine residue at position 368 with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0199] In yet a further embodiment, the first subunit of the Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C) or a replacement of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (particularly, the replacement of the serine residue at position 354 with a cysteine ​​residue), and the second subunit of the Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). The introduction of these two cysteine ​​residues creates a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0200] In a specific embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0201] In a specific embodiment, an antigen-binding moiety that binds a second antigen (e.g., an activating T cell antigen) is fused (optionally via a first antigen-binding moiety that binds GPRC5D and / or a peptide linker) to a first subunit of an Fc domain (comprising a "knob" modification). Without wishing to be bound by theory, fusing an antigen-binding moiety that binds a second antigen (e.g., an activating T cell antigen) to a knob-containing subunit of an Fc domain (further) minimizes the generation of antigen-binding molecules comprising two antigen-binding moieties that bind to an activating T cell antigen (steric clashes between the two knob-containing polypeptides).

[0202] Other techniques for CH3 modifications that enhance heterodimerization are contemplated as alternatives in accordance with the present invention and are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, and WO 2013 / 096291.

[0203] In one embodiment, the heterodimerization approach described in EP 1870459 is used instead. This approach is based on the introduction of oppositely charged amino acids at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. One preferred embodiment of the bispecific antigen-binding molecule of the present invention has the amino acid mutations R409D, K370E in one of the two CH3 domains (of the Fc domain), and D399K, E357K in the other CH3 domain of the Fc domain (numbering according to the Kabat EU index).

[0204] In another embodiment, the bispecific antigen binding molecule comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain, the amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and further amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain, and amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain (numbering according to Kabat EU index).

[0205] In another embodiment, the bispecific antigen-binding molecule comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain; or said bispecific antigen-binding molecule comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain and further comprises the amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain (all numbered according to Kabat EU index).

[0206] In one embodiment, the heterodimerization approach described in WO 2013 / 157953 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbering according to the Kabat EU index). In a further embodiment, the first CH3 domain comprises the additional amino acid mutation L351K. In a further embodiment, the second CH3 domain further comprises an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numbering according to the Kabat EU index).

[0207] In one embodiment, the heterodimerization approach described in WO 2012 / 058768 is used instead. In one embodiment, the heterodimerization approach described in WO 2012 / 058768 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain comprises an additional amino acid mutation at positions T411, D399, S400, F405, N390 or K392, such as (a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W; (b) D399R, D399W, D399Y or (c) S400E, S400D, S400R or S400K, (d) F405I, F405M, F405T, F405S, F405V or F405W, (e) N390R, N390K or N390D, or (f) K392V, K392M, K392R, K392L, K392F or K392E (numbering according to the Kabat EU index). In a further embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366V, K409F. In a further embodiment, the first CH3 domain comprises the amino acid mutation Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the Kabat EU index).

[0208] In one embodiment, the heterodimerization approach described in WO 2011 / 143545 is used instead, e.g., with an amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to the Kabat EU index).

[0209] In one embodiment, the heterodimerization approach described in WO 2011 / 090762, which also uses the knob-in-hole technique described above, is used instead. In one embodiment, the first CH3 domain contains the amino acid mutation T366W and the second CH3 domain contains the amino acid mutation Y407A. In one embodiment, the first CH3 domain contains the amino acid mutation T366Y and the second CH3 domain contains the amino acid mutation Y407T (numbering according to the Kabat EU index).

[0210] In one embodiment, the bispecific antigen-binding molecule or its Fc domain is of the IgG2 subclass and the heterodimerization approach described in WO 2010 / 129304 is used instead.

[0211] In an alternative embodiment, the modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates electrostatic steering effects, e.g., as described in WO 2009 / 089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues, such that homodimer formation is electrostatically unfavorable, but heterodimerization is electrostatically favorable. In one such embodiment, the first CH3 domain comprises an amino acid substitution at K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain comprises an amino acid substitution at D399, E356, D356, or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K, or E357K, more preferably D399K and E356K). In a further embodiment, the first CH3 domain further comprises an amino acid substitution at K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In a further embodiment, the first CH3 domain also or alternatively comprises an amino acid substitution at K439 and / or K370 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) (all numbered according to the Kabat EU index).

[0212] In yet further embodiments, the heterodimerization approach described in WO 2007 / 147901 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K, and K322D, and the second CH3 domain comprises the amino acid mutations D239K, E240K, and K292D (numbering according to the Kabat EU index).

[0213] In yet another embodiment, the heterodimerization approach described in WO 2007 / 110205 may be used instead.

[0214] In one embodiment, the first subunit of the Fc domain comprises the amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises the amino acid substitutions D356K and D399K (numbering according to the Kabat EU index).

[0215] Fc domain modifications that reduce Fc receptor binding and / or effector function The Fc domain confers desirable pharmacokinetic properties to bispecific antigen-binding molecules, including a long serum half-life and a desirable tissue-to-blood distribution ratio, which contribute to favorable accumulation in target tissues. However, at the same time, it may result in undesirable targeting of the bispecific antigen-binding molecule to cells expressing Fc receptors rather than to preferred antigen-bearing cells. Furthermore, coactivation of the Fc receptor signaling pathway, combined with T cell activation properties (e.g., in embodiments of bispecific antigen-binding molecules in which the second antigen-binding moiety binds to an activating T cell antigen) and the long half-life of the bispecific antigen-binding molecule, may result in excessive activation of cytokine receptors and cytokine release, which leads to severe side effects upon systemic administration. Activation of immune cells (bearing Fc receptors) other than T cells may even reduce the efficacy of bispecific antigen-binding molecules (particularly bispecific antigen-binding molecules in which the second antigen-binding moiety binds to an activating T cell antigen), due to potential destruction of T cells by, for example, NK cells.

[0216] Thus, in a specific embodiment, the Fc domain of the bispecific antigen-binding molecule exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain. In one such embodiment, the Fc domain (or a bispecific antigen-binding molecule comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to Fc receptors compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule comprising a native IgG1 Fc domain), and / or exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or a bispecific antigen-binding molecule comprising said Fc domain) does not substantially bind to an Fc receptor and / or does not induce effector function. In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a specific embodiment, the effector function is ADCC. In certain embodiments, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or a bispecific antigen-binding molecule comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, and more particularly greater than about 90% of the binding affinity to FcRn compared to a native IgG1 Fc domain (or a bispecific antigen-binding molecule comprising a native IgG1 Fc domain).

[0217] In certain embodiments, the Fc domain is engineered to reduce its binding affinity to an Fc receptor and / or its effector function compared to an unengineered Fc domain. In a specific embodiment, the Fc domain of the bispecific antigen-binding molecule comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain for an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain for an Fc receptor. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain for an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there are more than one amino acid mutations that reduce the binding affinity of the Fc domain for an Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain for an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, a bispecific antigen-binding molecule comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity of an Fc receptor compared to a bispecific antigen-binding molecule comprising a non-engineered Fc domain. In a specific embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, and most particularly human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complementary components (particularly binding affinity to C1q) is also reduced. In one embodiment, binding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn (i.e., preservation of the binding affinity of the Fc domain to the receptor) is achieved when the Fc domain (or a bispecific antigen-binding molecule comprising the Fc domain) exhibits a binding affinity for FcRn that is greater than about 70% of the binding affinity of an unengineered form of the Fc domain (or a bispecific antigen-binding molecule comprising this unengineered form of Fc). The Fc domain, or a bispecific antigen-binding molecule of the invention comprising the Fc domain, may exhibit greater than about 80%, or even greater than about 90%, of such affinity. In certain embodiments, the Fc domain of the bispecific antigen-binding molecule is engineered to have reduced effector function compared to the unengineered Fc domain. Reduced effector function can include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular 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 to induce apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group consisting of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a specific embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a bispecific antigen-binding molecule comprising a non-engineered Fc domain).

[0218] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or the effector function is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329 (numbering according to Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235, and P329 (numbering according to Kabat EU index). In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to Kabat EU index). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and an additional amino acid substitution at a position selected from E233, L234, L235, N297, and P331 (numbering according to the Kabat EU index). In a more specific embodiment, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a specific embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA," "PGLALA," or "LALAPG").In particular, in a specific embodiment, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) in each of the first and second subunits of the Fc domain (Kabat EU index numbering).

[0219] In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor (and complement) binding of the human IgG1 Fc domain, as described in WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and for determining their properties, such as Fc receptor binding or effector function.

[0220] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions compared to IgG1 antibodies. Accordingly, in some embodiments, the Fc domain of the bispecific antigen-binding molecule is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numbering according to the Kabat EU index). To further reduce binding affinity to Fc receptors and / or their effector functions, in one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numbering according to the Kabat EU index). In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numbering according to the Kabat EU index). In a specific embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically the amino acid substitutions S228P, L235E and P329G (numbering according to the Kabat EU index). Such IgG4 Fc domain variants and their Fcγ receptor binding properties are described in WO 2012 / 130831, which is incorporated herein by reference in its entirety.

[0221] In specific embodiments, the Fc domain that exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain is a human IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numbering according to the Kabat EU index).

[0222] In certain embodiments, N-glycosylation of the Fc domain is ablated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, specifically an asparagine to alanine (N297A) or an aspartic acid to aspartic acid (N297D) (numbering according to the Kabat EU index).

[0223] In addition to the Fc domains described herein and in WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also comprise substitutions of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbering according to the Kabat EU index). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variants with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0224] Variant 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 the encoding DNA sequence, PCR, gene synthesis, etc. The correct nucleotide changes can be confirmed, for example, by screening.

[0225] Binding to an Fc receptor can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare) and an Fc receptor that can be obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain or a bispecific antigen-binding molecule containing an Fc domain for an Fc receptor may be assessed using a cell line known to express a particular Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor).

[0226] The effector function of an Fc domain or a bispecific antigen-binding molecule containing an Fc domain can be measured by methods known in the art. Examples of in vitro assays for assessing the ADCC activity of a 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, and Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (see, e.g., the ACTI® Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0227] In some embodiments, binding of the Fc domain to complement components, particularly C1q, is reduced. Thus, in some embodiments in which the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. To determine whether an Fc domain, or a bispecific antigen-binding molecule comprising an Fc domain, is capable of binding to C1q and therefore has CDC activity, a C1q binding assay may be performed. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., 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)).

[0228] Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929).

[0229] Compositions, Formulations, and Routes of Administration In a further aspect, the present invention provides pharmaceutical compositions comprising any of the antibodies or bispecific antigen-binding molecules provided herein, e.g., for use in any of the following therapeutic methods. In one embodiment, the pharmaceutical composition comprises any of the antibodies or bispecific antigen-binding molecules provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the antibodies or bispecific antigen-binding molecules provided herein and at least one additional therapeutic agent, e.g., as described below.

[0230] Furthermore, there is provided a method for producing an antibody or bispecific antigen-binding molecule of the present invention in a form suitable for in vivo administration, the method comprising: (a) obtaining an antibody or bispecific antigen-binding molecule according to the present invention; and (b) combining the antibody or bispecific antigen-binding molecule with at least one pharmaceutically acceptable carrier, thereby formulating a preparation of the antibody or bispecific antigen-binding molecule for in vivo administration.

[0231] Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of an antibody or bispecific antigen-binding molecule dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic, or other untoward reactions when administered to animals, e.g., humans, as needed. The preparation of pharmaceutical compositions containing an antibody or bispecific antigen-binding molecule and, optionally, additional active ingredients will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, incorporated herein by reference. Furthermore, it will be understood that for animal (e.g., human) administration, the formulation must meet sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards or corresponding national agencies. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0232] The immunoconjugates of the invention (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if desired, intralesional administration for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, e.g., by injection, e.g., intravenous or subcutaneous, depending in part on whether administration is temporary or chronic.

[0233] 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 antigen-binding molecule of the present invention may be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline buffer. Solutions may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the antibody or bispecific antigen-binding molecule may be in powder form for constitution with a suitable vehicle, e.g., pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the antibody or bispecific antigen-binding molecule of the present invention in the required amount in an appropriate solvent, with various other ingredients, as required, as listed below. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. Typically, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and / or other ingredients. In the case of sterile powders for preparing sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered liquid medium. The liquid medium should be appropriately buffered, if necessary, and the liquid diluent is first rendered isotonic with sufficient saline or glucose prior to injection. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept at a minimum safe level, e.g., below 0.5 ng / mg protein.Suitable pharmaceutically acceptable carriers include, but are not limited to, buffers, e.g., phosphate, citrate 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, alkyl parabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides. , proteins such as serum albumin, gelatin, or immunoglobulins; 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may also contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, and the like. Optionally, suspensions may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl acetate or triglycerides, or liposomes.

[0234] The active ingredient can also be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions, for example, by microcapsules prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may also be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, for example, films or microcapsules. In specific embodiments, sustained absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption (e.g., aluminum monostearate, gelatin, or combinations thereof).

[0235] In addition to the compositions described above, antibodies or bispecific antigen-binding molecules can also be formulated as depot preparations. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or intramuscular injection. Thus, for example, antibodies or bispecific antigen-binding molecules can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as sparingly soluble salts.

[0236] Pharmaceutical compositions containing the antibodies or bispecific antigen-binding molecules of the present invention can be produced by conventional mixing, dissolving, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, additives, or adjuvants that facilitate the processing of proteins into pharmaceutically usable preparations. The appropriate formulation depends on the selected route of administration.

[0237] Antibodies or bispecific antigen-binding molecules can be formulated in the composition in free acid or base, neutral, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include acid addition salts, such as those formed with free amino groups of proteinaceous compositions, or those formed with organic acids, such as hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0238] Proteasome inhibitors The term "proteasome inhibitor" refers to compounds that block the action of the proteasome, a cellular complex that breaks down proteins. Proteasome inhibitors include classes of peptide boronates (e.g., bortezomib or CEP-188770), peptide aldehydes (e.g., MG132), peptide vinyl sulfones, peptide epoxyketones (e.g., epoxomicin, carfilzomib), beta-lactone inhibitors (e.g., lactacystin, MLN 519, marizomib, NPI-0052, salinosporamide A), compounds that form dithiocarbamate complexes with metals (e.g., disulfiram), and certain antioxidants (e.g., epigallocatechin-3-gallate).

[0239] The term "bortezomib" refers to a compound having the following chemical structure: [ka]

[0240] The empirical formula of bortezomib is C 19 H 25 BN4O4, CAS Registry Number 179324-69-7, with a gram molecular weight of 384.24.

[0241] The term "carfilzomib" refers to a compound having the following chemical structure: [ka]

[0242] The empirical formula of carfilzomib is C 40 H 57 N5O7, CAS Registry Number 868540-17-4, with a gram molecular weight of 719.9.

[0243] The present invention provides a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody with a proteasome inhibitor (PI). The proteasome inhibitor used in the combination therapy described herein may be selected from the group consisting of peptide boronates, peptide aldehydes, peptide vinyl sulfones, peptide epoxyketones, and beta-lactone inhibitors. In one embodiment, the proteasome inhibitor belongs to the class of peptide boronates or peptide epoxyketones. In one embodiment, the proteasome inhibitor is bortezomib or carfilzomib.

[0244] Glucocorticosteroids The present invention further provides a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody with a proteasome inhibitor and a glucocorticosteroid.

[0245] As used herein, "glucocorticosteroids" or "glucocorticoids" refer to the corticosteroid class of compounds that are involved in carbohydrate, protein, and fat metabolism and have anti-inflammatory activity. Glucocorticosteroids are primarily used therapeutically for their anti-inflammatory and immunosuppressive effects. Glucocorticosteroids include, but are not limited to, dexamethasone, prednisone, prednisolone, methylprednisolone, and alternatives.

[0246] The term "dexamethasone" refers to a compound having the following chemical structure: [ka]

[0247] The empirical formula for dexamethasone is C 22 H 29 FO5, CAS Registry Number 50-02-2, gram molecular weight 392.46.

[0248] The present invention further provides a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody with a proteasome inhibitor and a glucocorticoid, hi one embodiment, the glucocorticosteroid is dexamethasone.

[0249] Therapeutic methods and compositions The present invention includes combination therapies comprising an anti-GPRC5D / anti-CD3 bispecific antibody in combination with a proteasome inhibitor. Optionally, the combination therapies described herein may further comprise a glucocorticosteroid.

[0250] The present invention includes a method of treating a patient in need of treatment, comprising administering to the patient a therapeutically effective amount of a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody and a proteasome inhibitor.The present invention includes a method of treating a patient in need of treatment, comprising administering to the patient a therapeutically effective amount of a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody, a proteasome inhibitor and a glucocorticosteroid.

[0251] One preferred embodiment of the present invention is a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody with a proteasome inhibitor for use in the treatment of cancer or tumors. Another preferred embodiment of the present invention is a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody with a proteasome inhibitor and a glucocorticosteroid for use in the treatment of cancer or tumors.

[0252] One embodiment of the present invention is an anti-GPRC5D / anti-CD3 bispecific antibody described herein for use in the treatment of cancer or tumor in combination with a proteasome inhibitor described herein.One embodiment of the present invention is an anti-GPRC5D / anti-CD3 bispecific antibody described herein for use in the treatment of cancer or tumor in combination with a proteasome inhibitor described herein and a glucocorticosteroid described herein.

[0253] Another embodiment of the present invention is a proteasome inhibitor as described herein for use in the treatment of cancer or tumor in combination with an anti-GPRC5D / anti-CD3 bispecific antibody as described herein. Another embodiment of the present invention is a proteasome inhibitor as described herein for use in the treatment of cancer or tumor in combination with an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and a glucocorticosteroid as described herein.

[0254] A further embodiment is a glucocorticosteroid as described herein for use in the treatment of cancer or tumors in combination with an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and a proteasome inhibitor as described herein.

[0255] Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferation disorders that can be treated using the combination therapy of the present invention include, but are not limited to, tumors located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and genitourinary system. Precancerous conditions or lesions and cancer metastasis are also included. In certain embodiments, the cancer is selected from the group consisting of kidney cancer, bladder cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, and prostate cancer. In one embodiment, the cancer is a GPRC5D-expressing cancer. In one embodiment, the cancer is multiple myeloma.

[0256] One embodiment of the present invention is an anti-GPRC5D / anti-CD3 bispecific antibody as described herein in combination with a proteasome inhibitor as described herein, for use in treating any of the above cancers or tumors. One embodiment of the present invention is an anti-GPRC5D / anti-CD3 bispecific antibody as described herein in combination with a proteasome inhibitor as described herein and a glucocorticoid, for use in treating any of the above cancers or tumors.

[0257] One embodiment of the present invention is an anti-GPRC5D / anti-CD3 bispecific antibody as described herein in combination with a proteasome inhibitor as described herein for use in the treatment of multiple myeloma.One embodiment of the present invention is an anti-GPRC5D / anti-CD3 bispecific antibody as described herein in combination with a proteasome inhibitor as described herein and a glucocorticoid for use in the treatment of multiple myeloma.

[0258] The invention includes a method of treating a patient in need of treatment, comprising administering to the patient a therapeutically effective amount of a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody described herein and a proteasome inhibitor described herein. The invention further includes a method of treating a patient in need of treatment, comprising administering to the patient a therapeutically effective amount of a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody described herein, a proteasome inhibitor described herein, and a glucocorticosteroid described herein.

[0259] The present invention includes a method of treating cancer in an individual, comprising administering to the individual an anti-GPRC5D / anti-CD3 bispecific antibody described herein in combination with a proteasome inhibitor described herein. The present invention further includes a method of treating cancer in an individual, comprising administering to the individual an anti-GPRC5D / anti-CD3 bispecific antibody described herein in combination with a proteasome inhibitor described herein and a glucocorticosteroid described herein.

[0260] The invention includes a method for preventing or treating metastasis in a patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody described herein with a proteasome inhibitor described herein. The invention further includes a method for preventing or treating metastasis in a patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody described herein with a proteasome inhibitor described herein and a glucocorticosteroid described herein.

[0261] The present invention includes the use of a proteasome inhibitor according to the present invention and an anti-GPRC5D / anti-CD3 bispecific antibody for the combination therapy described.The present invention includes the use of a proteasome inhibitor according to the present invention and a glucocorticosteroid and an anti-GPRC5D / anti-CD3 bispecific antibody for the combination therapy described.

[0262] In a preferred embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the described combination treatments and medical uses is Forimtamig. In a further embodiment, the proteasome inhibitor used in the above-described combination treatments and medical uses is bortezomib or carfilzomib. In a further embodiment, the glucocorticoid used in the above-described combination treatments and medical uses is dexamethasone. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, and the proteasome inhibitor used in the above-described combination treatments and medical uses is bortezomib or carfilzomib. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the described combination treatments and medical applications comprises the polypeptide sequences of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, and the proteasome inhibitor used in the described combination treatments and medical applications is bortezomib. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the described combination treatments and medical applications comprises the polypeptide sequences of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, and the proteasome inhibitor used in the described combination treatments and medical applications is carfilzomib. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the described combination treatments and medical applications is forimtamig, and the proteasome inhibitor used in the described combination treatments and medical applications is bortezomib or carfilzomib. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the described combination treatments and medical applications is forimtamig and the proteasome inhibitor used in the described combination treatments and medical applications is bortezomib.In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the described combination treatments and medical applications is forimtamig, and the proteasome inhibitor used in the described combination treatments and medical applications is carfilzomib. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above combination treatments and medical applications comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, the proteasome inhibitor is bortezomib or carfilzomib, and the glucocorticosteroid is dexamethasone. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the described combination treatments and medical applications is forimtamig, the proteasome inhibitor is bortezomib or carfilzomib, and the glucocorticosteroid is dexamethasone. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above combination treatments and medical applications comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, the proteasome inhibitor is bortezomib and the glucocorticosteroid is dexamethasone. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the described combination treatments and medical applications is forimtamig, the proteasome inhibitor is bortezomib and the glucocorticosteroid is dexamethasone. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the described combination treatments and medical applications is forimtamig, the proteasome inhibitor is carfilzomib and the glucocorticosteroid is dexamethasone.

[0263] The present invention includes an anti-GPRC5D / anti-CD3 bispecific antibody described herein in combination with a proteasome inhibitor described herein for use in the manufacture of a medicament for treating cancer.The present invention includes an anti-GPRC5D / anti-CD3 bispecific antibody described herein in combination with a proteasome inhibitor described herein and a glucocorticoid described herein for use in the manufacture of a medicament for treating cancer.

[0264] In another aspect, the present invention provides compositions, e.g., pharmaceutical compositions, comprising an anti-GPRC5D / anti-CD3 bispecific antibody described herein and a proteasome inhibitor described herein, formulated together with a pharmaceutically acceptable carrier. In another aspect, the present invention provides compositions, e.g., pharmaceutical compositions, comprising an anti-GPRC5D / anti-CD3 bispecific antibody described herein, a proteasome inhibitor described herein, and a glucocorticosteroid described herein, formulated together with a pharmaceutically acceptable carrier.

[0265] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible antibodies, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption / resorption delaying agents, etc. Preferably, the carrier is suitable for injection or infusion.

[0266] The compositions of the present invention can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and mode of administration will vary depending on the desired results.

[0267] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.In addition to water, carriers can be, for example, isotonic buffered saline.

[0268] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0269] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of active ingredient (an effective amount) effective to achieve a desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention, or its ester, salt, or amide, employed, the mode of administration, the time of administration, the excretion rate of the particular compound employed, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, overall health, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0270] The present invention includes a proteasome inhibitor described herein in combination with an anti-GPRC5D / anti-CD3 bispecific antibody described herein for use in the manufacture of a medicament for treating cancer. The present invention includes a proteasome inhibitor described herein in combination with an anti-GPRC5D / anti-CD3 bispecific antibody described herein and a glucocorticoid described herein for use in the manufacture of a medicament for treating cancer. The present invention includes a glucocorticoid described herein in combination with an anti-GPRC5D / anti-CD3 bispecific antibody described herein and a proteasome inhibitor described herein. In one embodiment, an anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29. In one embodiment, an anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention is Forimtamig. In one embodiment, a proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is bortezomib or carfilzomib. In one embodiment, the glucocorticoid for use in the manufacture of a medicament for treating cancer according to the present invention is dexamethasone. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, and the proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is bortezomib or carfilzomib. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, and the proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is bortezomib.In one embodiment, an anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, and a proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is carfilzomib. In one embodiment, an anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention is forimtamig, and a proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is bortezomib or carfilzomib. In one embodiment, an anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention is forimtamig, and a proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is bortezomib. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention is forimtamig, and the proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is carfilzomib. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, the proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is bortezomib or carfilzomib, and the glucocorticoid for use in the manufacture of a medicament for treating cancer according to the present invention is dexamethasone. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention is forimtamig, the proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is bortezomib or carfilzomib, and the glucocorticoid for use in the manufacture of a medicament for treating cancer according to the present invention is dexamethasone.In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, the proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is bortezomib and the glucocorticoid for use in the manufacture of a medicament for treating cancer according to the present invention is dexamethasone. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention is forimtamig, the proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is bortezomib and the glucocorticoid for use in the manufacture of a medicament for treating cancer according to the present invention is dexamethasone. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for treating cancer according to the present invention is forimtamig, the proteasome inhibitor for use in the manufacture of a medicament for treating cancer according to the present invention is carfilzomib, and the glucocorticoid for use in the manufacture of a medicament for treating cancer according to the present invention is dexamethasone.

[0271] The present invention further provides the use of an anti-GPRC5D / anti-CD3 bispecific antibody according to the invention described herein and a proteasome inhibitor according to the invention described herein for the manufacture of a medicament, preferably together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.The present invention further provides the use of an anti-GPRC5D / anti-CD3 bispecific antibody according to the invention described herein and a proteasome inhibitor according to the invention described herein for the manufacture of a medicament, preferably together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.

[0272] In one aspect, the invention provides a kit intended for the treatment of a disease, comprising, in the same or separate containers, (a) an anti-GPRC5D / anti-CD3 bispecific antibody described herein, and (b) a proteasome inhibitor described herein, and optionally further comprising (c) a package insert comprising printed instructions directing the use of the combination treatment as a method for treating the disease. In one aspect, the invention provides a kit intended for the treatment of a disease, comprising, in the same or separate containers, (a) an anti-GPRC5D / anti-CD3 bispecific antibody described herein, and (b) a proteasome inhibitor described herein, and (c) a glucocorticosteroid described herein, and optionally further comprising (d) a package insert comprising printed instructions directing the use of the combination treatment as a method for treating the disease.

[0273] Furthermore, the kit may include (a) a first container containing a composition comprising an anti-GPRC5D / anti-CD3 bispecific antibody described herein, (b) a second container containing a composition comprising a proteasome inhibitor described herein, and optionally (c) a third container containing a composition contained therein, wherein the composition comprises an additional cytotoxic or other therapeutic agent. The kit in this embodiment may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the kit may further include a fourth container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0274] The kit may further include (a) a first container containing a composition, the composition comprising an anti-GPRC5D / anti-CD3 bispecific antibody described herein; (b) a second container containing a composition, the composition comprising a proteasome inhibitor described herein; (c) a third container containing a composition, the composition comprising a glucocorticosteroid described herein; and, optionally, (d) a fourth container containing a composition, the composition comprising an additional cytotoxic therapeutic agent or another therapeutic agent. The kit in this embodiment may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the kit may further include a fifth container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0275] In one aspect, the invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising an anti-GPRC5D / anti-CD3 bispecific antibody described herein, and (b) a package insert directing the use of the anti-GPRC5D / anti-CD3 bispecific antibody in combination therapy with a proteasome inhibitor described herein as a method for treating the disease. In one aspect, the invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising an anti-GPRC5D / anti-CD3 bispecific antibody described herein, and (b) a package insert directing the use of the anti-GPRC5D / anti-CD3 bispecific antibody in combination therapy with a proteasome inhibitor and a glucocorticosteroid described herein as a method for treating the disease.

[0276] In another aspect, the present invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising a proteasome inhibitor described herein, and (b) a package insert comprising instructions directing the use of the proteasome inhibitor in combination therapy with an anti-GPRC5D / anti-CD3 bispecific antibody described herein as a method of treating the disease. In another aspect, the present invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising a proteasome inhibitor described herein, and (b) a package insert comprising instructions directing the use of the proteasome inhibitor in combination therapy with an anti-GPRC5D / anti-CD3 bispecific antibody described herein and a glucocorticoid as a method of treating the disease.

[0277] In another aspect, the present invention provides a kit intended for the treatment of a disease, comprising (a) a container containing a glucocorticoid as described herein, and (b) a package insert containing instructions directing the use of the glucocorticoid in combination therapy with an anti-GPRC5D / anti-CD3 bispecific antibody and a proteasome inhibitor as described herein as a method of treating the disease.

[0278] In a further aspect, the present invention provides a medicament intended for the treatment of a disease comprising an anti-GPRC5D / anti-CD3 bispecific antibody as described herein, which medicament is for use in combination therapy with a proteasome inhibitor as described herein, and optionally comprising a package insert comprising printed instructions directing the use of the combination therapy as a method for treating the disease. In a further aspect, the present invention provides a medicament intended for the treatment of a disease comprising an anti-GPRC5D / anti-CD3 bispecific antibody as described herein, which medicament is for use in combination therapy with a proteasome inhibitor as described herein and a glucocorticosteroid, and optionally comprising a package insert comprising printed instructions directing the use of the combination therapy as a method for treating the disease.

[0279] The term "method of treating," or its equivalents, when applied to cancer, for example, refers to a treatment or course of action designed to reduce or eliminate the number of cancer cells in a patient or to alleviate the symptoms of cancer. A "method of treating" cancer or another proliferative disease does not necessarily mean that cancer cells or other disorders are actually eliminated, that the number of cells or disorders is actually reduced, or that the symptoms of the cancer or other disorder are actually alleviated. In many cases, a method of treating cancer is undertaken even when the likelihood of success is low, but is still deemed to induce an overall beneficial course of action given the patient's medical history and projected survival potential.

[0280] The terms "administered in combination with" or "co-administration," "concurrent administration," "combination therapy," or "combined treatment" refer to the administration of an anti-GPRC5D / anti-CD3 bispecific antibody described herein and a proteasome inhibitor described herein, and optionally a glucocorticosteroid, for example, as separate formulations / applications (or a single formulation / application). Co-administration may be simultaneous or sequential in any order, desirably with a period during which all active agents simultaneously exert their biological activity. The active agents are co-administered simultaneously or sequentially (e.g., intravenously (iv)) by continuous infusion, or orally. When all therapeutic agents are co-administered sequentially, the doses are administered on the same day in two separate administrations, or one of the agents is administered on day 1 and the second agent is co-administered from days 2 through 7, preferably from days 2 through 4. Thus, in one embodiment, the term "sequentially" refers to within 7 days after administration of the first component, preferably within 4 days after administration of the first component, and the term "concurrently" refers to administration at the same time. The term "co-administration" with respect to maintenance doses of anti-GPRC5D / anti-CD3 bispecific antibody and / or proteasome inhibitor and / or proteasome inhibitor and / or glucocorticosteroid, if applicable, means that maintenance doses can be co-administered simultaneously, e.g., weekly, if treatment cycles are appropriate for all drugs.

[0281] It will be apparent that antibodies are administered to a patient in a "therapeutically effective amount" (or simply "effective amount"), that is the amount of each compound or combination that elicits the biological or medical response in a tissue, system, animal, or human that is being sought by the researcher, veterinarian, physician, or other clinician.

[0282] The amount of co-administration and timing of co-administration will depend on the type (species, sex, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated. The anti-GPRC5D / anti-CD3 bispecific antibody and / or proteasome inhibitor and / or glucocorticosteroid, if applicable, are suitably co-administered to the patient at once or over a series of treatments, e.g., on the same day, or on subsequent days, or at weekly intervals.

[0283] Those skilled in the art will readily recognize that in many cases, a combination therapy will not provide a cure and may provide only partial benefit. In some embodiments, any physiological change that has any benefit is also considered therapeutically beneficial. Thus, in some embodiments, the amount of combination therapy that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount."

[0284] Aspects of the invention 1. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with a proteasome inhibitor for use as a combination therapy in the treatment of cancer.

[0285] 2. Use of an anti-GPRC5D / anti-CD3 bispecific antibody in combination with a proteasome inhibitor in the manufacture of a medicament for treating cancer.

[0286] 3. A method of treating cancer in an individual, comprising administering to said individual an anti-GPRC5D / anti-CD3 bispecific antibody in combination with a proteasome inhibitor.

[0287] 4. A kit comprising a first agent comprising an anti-GPRC5D / anti-CD3 bispecific antibody and a second agent comprising a proteasome inhibitor, optionally further comprising a package insert containing instructions for administering the first agent in combination with the second agent to treat cancer in an individual. Aspects of the invention 5. Anti-GPRC5D / anti-CD3 bispecific antibodies (i) a first antigen-binding portion that specifically binds to GPRC5D and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 12, an HCDR2 of SEQ ID NO: 13, and an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 15, an LCDR2 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO: 17; and (ii) An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 1 to 4, comprising a second antigen-binding portion that specifically binds to CD3 and comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 18, HCDR2 of SEQ ID NO: 19, and HCDR3 of SEQ ID NO: 20, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 21, LCDR2 of SEQ ID NO: 22, and LCDR3 of SEQ ID NO: 23.

[0288] 6. Anti-GPRC5D / anti-CD3 bispecific antibodies (i) a first antigen-binding portion that specifically binds to GPRC5D, comprising a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11; and (ii) an anti-GPRC5D / anti-CD3 bispecific antibody, use, method, or kit for use in combination with a proteasome inhibitor according to any one of aspects 1 to 5, comprising a second antigen-binding portion that specifically binds CD3, the second antigen-binding portion comprising a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24, and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25.

[0289] 7. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to aspect 5 or 6, wherein the first antigen-binding portion and / or the second antigen-binding portion of the anti-GPRC5D / anti-CD3 bispecific antibody is a Fab molecule.

[0290] 8. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 5 to 7, wherein the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain, in particular the variable domains VL and VH, have been replaced by one another.

[0291] 9. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 5 to 8, wherein the first antigen-binding portion is a Fab molecule in which in the constant domain the amino acid at position 124 (numbering according to Kabat) is independently substituted by lysine (K), arginine (R) or histidine (H), and the amino acid at position 123 (numbering according to Kabat) is independently substituted by lysine (K), arginine (R) or histidine (H), and in the constant domain CH1 the amino acid at position 147 (numbering according to Kabat EU index) is independently substituted by glutamic acid (E) or aspartic acid (D), and the amino acid at position 213 (numbering according to Kabat EU index) is independently substituted by glutamic acid (E) or aspartic acid (D).

[0292] 10. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 5 to 9, wherein the first antigen-binding portion and the second antigen-binding portion are fused to each other, optionally via a peptide linker.

[0293] 11. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 5 to 10, wherein the first antigen-binding moiety and the second antigen-binding moiety are each Fab molecules, and either (i) the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety, or (ii) the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety.

[0294] 12. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 1 to 11, wherein the anti-GPRC5D / anti-CD3 bispecific antibody comprises a third antigen-binding moiety.

[0295] 13. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to aspect 12, wherein the third antigen-binding portion is identical to the first antigen-binding portion.

[0296] 14. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 1 to 13, wherein the anti-GPRC5D / anti-CD3 bispecific antibody comprises an Fc domain composed of a first and a second subunit.

[0297] 15. The first antigen-binding portion, the second antigen-binding portion, and, if present, the third antigen-binding portion are each Fab molecules; (i) the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion, and the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion, and the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, 15. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with an anti-CD38 antibody according to any one of aspects 5 to 14, wherein the third antigen-binding moiety, if present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0298] 16. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to aspect 14 or 15, wherein the Fc domain is an IgG Fc domain.

[0299] 17. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 14 to 16, wherein the Fc domain is an IgG1 Fc domain.

[0300] 18. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 14 to 17, wherein the Fc domain is a human Fc domain.

[0301] 19. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 14 to 18, wherein an amino acid residue in the CH3 domain of a first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of the second subunit, and wherein an amino acid residue in the CH3 domain of a second subunit of the Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit that can be positioned within the protrusion in the CH3 domain of the first subunit.

[0302] 20. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 14 to 19, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0303] 21. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 1 to 20, characterized in that the anti-GPRC5D / anti-CD3 bispecific antibody comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29.

[0304] 22. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 1 to 21, wherein the proteasome inhibitor belongs to the class of peptide boronates or peptide epoxyketones.

[0305] 23. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 1 to 22, wherein the proteasome inhibitor is bortezomib or carfilzomib.

[0306] 24. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 1 to 23, wherein the combination further comprises a glucocorticosteroid.

[0307] 25. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of aspects 24, wherein the glucocorticosteroid is dexamethasone. Amino acid sequence TIFF2025533183000004.tif198170TIFF2025533183000005.tif235170TIFF20255331830 00006.tif206170TIFF2025533183000007.tif169170TIFF2025533183000008.tif125170 [Example]

[0308] The following are examples of methods and compositions of the present invention. Given the general description provided above, it is understood that various embodiments may be practiced.

[0309] Materials and Methods All in vivo efficacy and PD experiments were performed in humanized NSG mice bearing subcutaneous multiple myeloma xenograft tumors. Female humanized NSG mice were purchased from Jackson Laboratories and transfected with human CD34 +After 14–20 weeks of hematopoietic stem cell engraftment, the mice were sent to the animal facility at the Roche Innovation Center Munich. After arrival, the animals were maintained for one week for acclimatization and observation. In accordance with relevant guidelines (GV-Solas, Felasa, TierschG), mice were maintained under specific pathogen-free conditions with a 12-hour light / 12-hour dark cycle daily. Continuous health monitoring was performed regularly. The experimental study protocol was reviewed and approved by the local authorities (ROB-55.2-2532.Vet_03-16-10 or ROB-55.2-2532.Vet_03-20-170). To evaluate the therapeutic effects on established multiple myeloma tumors, humanized NSG mice were subcutaneously implanted with human tumor cell lines. Tumor cell lines were obtained from different donors and, after expansion, deposited in the Roche Munich internal cell bank (Table 1). All tumor cells were cultured at 37 °C in a water-saturated atmosphere of 5% CO2, and 50 μl of Matrigel was co-injected into the flank of anesthetized humanized NSG mice at different cell numbers and with a survival rate of over 90% in the right flank of the animals (Table 1). Subcutaneous tumors were 200–300 mm 3When the tumor reached an average volume of 1000 mg / kg, the humanized mice were randomized into different treatment groups based on tumor volume and body weight. Upon randomization, animals were treated with GPRC5D-TCB (SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29; RO7425781, forimtamig, as disclosed in WO 2021 / 018859) as monotherapy or in combination with a proteasome inhibitor, a standard of care (SoC) agent for the treatment of multiple myeloma. The treatment schedule, dose, and route of administration for each treatment are summarized in Table 2. All treatments were freshly prepared before injection. Animals were monitored daily for clinical symptoms and adverse effects. Animal termination criteria were visible illness (shabby fur, hunched back, respiratory distress, impaired movement), weight loss greater than 20%, or tumor size. Tumor growth was monitored twice weekly using caliper measurements. To quantify tumor-infiltrating lymphocytes, in some experiments, tumors from scout animals were harvested and single-cell suspensions were subjected to flow cytometry using a FACSFortessa instrument and FlowJo software. Cytokines in the serum of treated mice were analyzed using BioRad's Bio-Plex Multiplex Immunoassay System in combination with the Bio-Plex Pro Human Cytokine 27 plex Assay. Statistical analysis was performed using GraphPad Prism software. To compare results for ImmunoPD, tumor volume, or cytokine levels between different treatment groups, data were subjected to one-way ANOVA analysis corrected for multiple comparisons (Tukey's test). [Table 1] [Table 2]

[0310] result Proteasome inhibitors (PIs) inhibit T cell responses 1、2 It has been reported that it weakens the immune system and induces lymphopenia in patients. 3This may limit the potential for combination with T cell-engaging therapies such as T cell bispecific antibodies. Surprisingly, the efficacy of GPRC5D-TCB against KM12-BM tumors was not impaired by combination with bortezomib and was further enhanced by triple combination with dexamethasone (Figure 1A and Figure 1B). Combination of GPRC5D-TCB with carfilzomib, a second-generation protease inhibitor, in NCI-H929 tumor-bearing humanized mice resulted in a faster onset of tumor growth inhibition (Figure 2A) and delayed tumor recurrence compared with GPRC5D-TCB monotherapy, despite a slight reduction in cytokine release (Figure 2B, 2C, and 2D). In summary, combination therapy of GPRC5D-TCB with a proteasome inhibitor improved antitumor responses and reduced the number of escapers, but had minimal impact on T cell activation.

[0311] References: 1.Berges C,Haberstock H,Fuchs D,Miltz M,Sadeghi M,Opelz G,Daniel V,Naujokat C.Proteasome inhibition suppresses essential immune functions of human CD4+T cells.Immunology.2008 Jun;124(2):234-46.doi:10.1111 / j.1365-2567.2007.02761.x.Epub 2008 Jan 23.PMID:18217957;PMCID:PMC2566628. 2.Yanaba K, Yoshizaki A, Muroi E, Hara T, Ogawa F, Shimizu K, Sato S.The proteasome inhibitor bortezomib inhibits T cell-dependent inflammatory responses.J Leukoc Biol.2010 Jul;88(1):117-22.doi:10.1189 / jlb.1009666.Epub 2010 Apr 23.PMID:20418448. 3.Jung SH,Bae SY,Ahn JS,Kang SJ,Yang DH,Kim YK,Kim HJ,Lee JJ.Lymphocytopenia is associated with an increased risk of severe infections in patients with multiple myeloma treated with bortezomib-based regimens.Int J Hematol.2013 Mar;97(3):382-7.doi:10.1007 / s12185-013-1270-7.Epub 2013 Jan 25.PMID:23355264.

[0312] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but the illustrations and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with a proteasome inhibitor for use as a combination therapy in the treatment of cancer.

2. Use of an anti-GPRC5D / anti-CD3 bispecific antibody in combination with a proteasome inhibitor in the manufacture of a medicament for treating cancer.

3. 1. A method of treating cancer in an individual, comprising administering to said individual an anti-GPRC5D / anti-CD3 bispecific antibody in combination with a proteasome inhibitor.

4. 1. A kit comprising a first agent comprising an anti-GPRC5D / anti-CD3 bispecific antibody and a second agent comprising a proteasome inhibitor, optionally further comprising a package insert containing instructions for administering the first agent in combination with the second agent to treat cancer in an individual.

5. the anti-GPRC5D / anti-CD3 bispecific antibody (i) a first antigen-binding portion that specifically binds to GPRC5D and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 12, a HCDR2 of SEQ ID NO: 13, and a HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 15, a LCDR2 of SEQ ID NO: 16, and a LCDR3 of SEQ ID NO: 17; and (ii) a second antigen-binding portion that specifically binds to CD3 and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 18, a HCDR2 of SEQ ID NO: 19, and a HCDR3 of SEQ ID NO: 20, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 21, a LCDR2 of SEQ ID NO: 22, and a LCDR3 of SEQ ID NO:

23.

6. the anti-GPRC5D / anti-CD3 bispecific antibody (i) a first antigen-binding portion that specifically binds to GPRC5D, comprising a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11; and 6. The anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit of any one of claims 1 to 5 for use in combination with a proteasome inhibitor, comprising: (ii) a second antigen-binding portion that specifically binds CD3 comprising a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24, and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

25.

7. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to claim 5 or 6, wherein the first antigen-binding portion and / or the second antigen-binding portion of the anti-GPRC5D / anti-CD3 bispecific antibody is a Fab molecule.

8. 8. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 5 to 7, wherein the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain, in particular the variable domains VL and VH, are replaced by each other.

9. 9. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 5 to 8, wherein the first antigen-binding moiety is a Fab molecule in which in the constant domain the amino acid at position 124 (numbering according to Kabat) is independently substituted by lysine (K), arginine (R) or histidine (H) and the amino acid at position 123 (numbering according to Kabat) is independently substituted by lysine (K), arginine (R) or histidine (H); and in the constant domain CH1 the amino acid at position 147 (numbering according to Kabat EU index) is independently substituted by glutamic acid (E) or aspartic acid (D) and the amino acid at position 213 (numbering according to Kabat EU index) is independently substituted by glutamic acid (E) or aspartic acid (D).

10. 10. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 5 to 9, wherein the first antigen-binding moiety and the second antigen-binding moiety are fused to each other, optionally fused to each other via a peptide linker.

11. 11. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 5 to 10, wherein the first antigen-binding moiety and the second antigen-binding moiety are each Fab molecules, and either (i) the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety, or (ii) the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety.

12. 12. The anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 1 to 11, wherein the anti-GPRC5D / anti-CD3 bispecific antibody comprises a third antigen-binding moiety.

13. 13. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to claim 12, wherein the third antigen-binding portion is identical to the first antigen-binding portion.

14. The anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 1 to 13, wherein the anti-GPRC5D / anti-CD3 bispecific antibody comprises an Fc domain composed of a first and a second subunit.

15. the first antigen-binding portion, the second antigen-binding portion, and, if present, the third antigen-binding portion, are each a Fab molecule; (i) the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, 15. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 5 to 14, wherein the third antigen-binding moiety, if present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

16. 16. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to claim 14 or 15, wherein the Fc domain is an IgG Fc domain.

17. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 14 to 16, wherein the Fc domain is an IgG1 Fc domain.

18. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 14 to 17, wherein the Fc domain is a human Fc domain.

19. 19. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 14 to 18, wherein an amino acid residue in the CH3 domain of a first subunit of the Fc domain is substituted with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is substituted with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit that can be positioned within the protrusion in the CH3 domain of the first subunit.

20. 20. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 14 to 19, wherein the Fc domain comprises one or more amino acid substitutions that reduce Fc receptor binding and / or effector function.

21. 21. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 1 to 20, characterized in that the anti-GPRC5D / anti-CD3 bispecific antibody comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO:

29.

22. 21. The anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 1 to 20, wherein the anti-GPRC5D / anti-CD3 bispecific antibody is formtamig.

23. 23. An anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 1 to 22, wherein said proteasome inhibitor belongs to the class of peptide boronates or peptide epoxyketones.

24. 24. The anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 1 to 23, wherein said proteasome inhibitor is bortezomib or carfilzomib.

25. 25. The anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to any one of claims 1 to 24, wherein said combination further comprises a glucocorticosteroid.

26. 26. The anti-GPRC5D / anti-CD3 bispecific antibody, use, method or kit for use in combination with a proteasome inhibitor according to claim 25, wherein the glucocorticosteroid is dexamethasone.

Images