Combination therapy of GPRC5D TCB and IMID

Abstract

The present invention relates to a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody with an IMiD, 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 an immunomodulatory imid drug (IMiD), which may include glucocorticosteroids. [Background technology]

[0002] Multiple myeloma (MM), with approximately 75,000 new cases diagnosed annually in Europe and the United States, is one of the most common hematological malignancies and remains a significant unmet medical need. MM is characterized by well-differentiated plasma cells secreting nonfunctional monoclonal immunoglobulins. In the short term, immunomodulatory drugs such as lenalidomide and pomalidomide, as well as proteasome inhibitors such as carfilzomib or bortezomib, may remain the mainstay 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 affected tumor cells, such as affected plasma cells (PCs). Efforts have been made to selectively deplete plasma cells 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, including daratumumab (anti-CD38) and elotuzumab (anti-CD319), have had little success. It is important to note that these two molecules are not exclusively expressed by plasma cells. Therefore, novel targets from multiple myeloma plasma cells have been identified using RNA-sequencing, such as G protein-coupled receptor class C group 5 member D (GPRC5D), which is differentially expressed between multiple myeloma plasma cells and plasma cells from healthy donors.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 biology in men in general and cancer in particular is largely unknown. The gene encoding GPRC5D is 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;48)). The large first exon encodes seven transmembrane domains. 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;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 activate T cell antigens, such as CD3 on T cells. Simultaneous binding of such antibodies to both of their targets results in the formation of a T cell synapse, activating cytotoxic T cells and subsequently lysing the target cell. T cell bispecific antibodies (TCBs) have become a novel treatment option for patients with relapsed and refractory multiple myeloma (RRMM) based on their promising objective response rates (ORR), favorable safety profile, and off-the-shelf availability compared to CAR-T cell therapy (van de Donk, NWCJ et al., T-cell redirecting bispecific and trispecific antibodies in multiple myeloma beyond BCMA. Curr Opin Oncol, 2023. 35:000-000). BCMA- and GPRC5D-targeted CAR T Cells have been reported to induce profound clinical responses, but antigenic drift represents a tumor-intrinsic resistance mechanism that limits the durability of responses (Mailankody, S. et al., GPRC5D-Targeted CAR T Cells for Myeloma. N Engl J Med. 2022;387(13):1196-1206). Given that all standard-of-care treatments are unable to cure multiple myeloma patients, there is a clear need to develop novel, effective and specific therapies. Accordingly, the present invention provides a combination of an anti-GPRC5D / anti-CD3 bispecific antibody with an IMiD and, optionally, a glucocorticosteroid. Summary of the Invention

[0005] In a first aspect, the present invention provides an anti-GPRC5D / anti-CD3 bispecific antibody in combination with an immunomodulatory imid drug (IMiD) 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 an immunomodulatory imid drug (IMiD) in the manufacture of a medicament for the treatment of cancer. In a still further 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 an immunomodulatory imid drug (IMiD). In another aspect, the present invention provides a kit comprising a first medicament comprising an anti-GPRC5D / anti-CD3 bispecific antibody and a second medicament comprising an immunomodulatory imid drug (IMiD), optionally further comprising a package insert containing instructions for administering the first medicament in combination with the second medicament to treat cancer in an individual.

[0006] 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 moiety that specifically binds GPRC5D, comprising a VH at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a VL 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 moiety that specifically binds CD3, comprising a VH at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24, and a VL 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 and / or second antigen-binding moieties of the anti-GPRC5D / anti-CD3 bispecific antibody are Fab molecules. 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 of the Fab light and heavy chains, in particular the variable domains VL and VH, are replaced by each other.In one embodiment, the first antigen-binding moiety is a Fab molecule in which, in the constant domain, 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 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 yet another embodiment, 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.

[0007] 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. The first, second, and, if present, third antigen-binding moieties are each 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.

[0008] In one embodiment of any one of the above aspects, 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 creating a protuberance in the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of a second subunit, and 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 creating a cavity in the CH3 domain of the second subunit that can be positioned within the protuberance in the CH3 domain of the first subunit. In one embodiment, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or decrease effector function.

[0009] In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody of any one of the aspects 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 the aspects is formtamig.

[0010] In one embodiment, the IMiD of any one of the above aspects is a first generation IMiD or a cereblon E3 ligase modulator (CELMoD). In one embodiment, the IMiD is selected from the group of lenalidomide, pomalidomide, iberdomide, and mezigdomide. In another aspect, the combination of any one of the above aspects further comprises a glucocorticosteroid. In one embodiment, the glucocorticosteroid is dexamethasone. [Brief explanation of the drawings]

[0011] [Figure 1A-B] Figure 1 shows the results of an efficacy / PD study evaluating GPRC5D-TCB as a single agent and in combination with lenalidomide. (A) The multiple myeloma cell line OPM-2 was subcutaneously injected into stem cell-humanized NSG mice to test for tumor growth inhibition. GPRC5D-TCB was injected intravenously once weekly at 0.05 mg / kg, and lenalidomide was administered daily via oral gavage at 20 mg / kg, and tumor growth was compared over 18 days. (B) Tumor burden in individual mice was assessed at the end of the study (day 18). Statistical analysis: ordinary one-way ANOVA, Tukey's test: p = <0.0001 (****); p = 0.0001 to 0.001 (***); p = 0.001 to 0.01 (**); p = 0.01 to 0.05 (*); p = ≥ 0.05 (ns). [Figure 1C]Figure 1 shows the results of an efficacy / PD study evaluating GPRC5D-TCB as a single agent and in combination with lenalidomide. Tumors from five scouts per group were harvested 48 hours after the second GPRC5D-TCB injection, and intratumoral T cell counts were assessed by flow cytometry. Statistical analysis: ordinary one-way ANOVA, Tukey's test: p=<0.0001 (****); p=0.0001 to 0.001 (***); p=0.001 to 0.01 (**); p=0.01 to 0.05 (*); p=≥0.05 (ns). [Figure 2A] Figure 1 shows the results of an efficacy study evaluating GPRC5D-TCB as a single agent and in combination with lenalidomide, with or without additional dexamethasone, against KMS-12BM multiple myeloma tumors subcutaneously implanted in stem cell-humanized NSG mice. GPRC5D-TCB was administered intravenously at 1 mg / kg once weekly and combined with daily lenalidomide at 20 mg / kg administered via oral gavage, with or without additional oral dexamethasone at 2 mg / kg. Statistical analysis: ordinary one-way ANOVA, Tukey's test: p=<0.0001 (****); p=0.0001 to 0.001 (***); p=0.001 to 0.01 (**); p=0.01 to 0.05 (*); p=≥0.05 (ns). [Figure 2B] Figure 1 shows the results of an efficacy study evaluating GPRC5D-TCB as a single agent and in combination with lenalidomide, with or without additional dexamethasone, against KMS-12BM multiple myeloma tumors implanted subcutaneously in stem cell-humanized NSG mice. 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 to 0.001 (***); p=0.001 to 0.01 (**); p=0.01 to 0.05 (*); p=≥0.05 (ns). [Figure 3]Figure 1 shows the results of an efficacy study evaluating GPRC5D-TCB as a single agent and in combination with pomalidomide against NCI-H929 multiple myeloma tumors implanted subcutaneously in stem cell-humanized NSG mice. (A) Growth of NCI-H929 xenograft tumors following weekly intravenous administration of 0.1 mg / kg GPRC5D-TCB and daily injections of 10 mg / kg pomalidomide via oral gavage is shown. Levels of IFN-γ (B), IL-2 (C), and TNF-α (D) in mouse serum were determined 48 hours after the first GPRC5D-TCB injection and 24 hours after pomalidomide injection using multiplexing technology. Statistical analysis, ordinary one-way ANOVA, Tukey's test: p=<0.0001 (****); p=0.0001 to 0.001 (***); p=0.001 to 0.01 (**); p=0.01 to 0.05 (*); p=≥0.05 (ns). [Figure 4] Figure 1 shows the results of an efficacy study evaluating GPRC5D-TCB as a single agent and in combination with iverdin against NCI-H929 multiple myeloma tumors implanted subcutaneously in stem cell-humanized NSG mice. (A) Growth of NCI-H929 xenograft tumors following weekly intravenous administration of GPRC5D-TCB at 0.1 mg / kg and daily injections of iverdin at 10 mg / kg via oral gavage is shown. Levels of IFN-γ (B), IL-2 (C), and TNF-α (D) in mouse serum were determined 48 hours after the first GPRC5D-TCB injection and 24 hours after iverdin injection using multiplexing technology. Statistical analysis, ordinary one-way ANOVA, Tukey's test: p=<0.0001 (****); p=0.0001 to 0.001 (***); p=0.001 to 0.01 (**); p=0.01 to 0.05 (*); p=≥0.05 (ns). [Figure 5] Tumor growth rates of individual mice treated with vehicle (A), GPRC5D-TCB monotherapy (B), or combinations with pomalidomide (C) or iverdomide (D) as described in Figures 3 and 4, respectively, are shown. [Figure 6A-B]Figure 1 shows the results of an efficacy experiment evaluating GPRC5D-TCB as a single agent and in combination with high- or low-dose mezigdomide against NCI-H929 multiple myeloma tumors implanted subcutaneously in stem cell-humanized NSG mice. (A) NCI-H929 tumor growth in individual mice with weekly subcutaneous (sc) injections of vehicle; (B) weekly subcutaneous (sc) injections of GPRC5D-TCB using escalating doses of 0.0005-0.002-0.04 mg / kg followed by a maintenance dose of 0.04 mg / kg. [Figure 6C-E] Figure 1 shows the results of an efficacy experiment evaluating GPRC5D-TCB as a single agent and in combination with high- or low-dose mezigdomide against NCI-H929 multiple myeloma tumors implanted subcutaneously in stem cell-humanized NSG mice. NCI-H929 tumor growth in individual mice following administration of GPRC5D-TCB in combination with 3 mg / kg mezigdomide (C) 5 days per week (5q7d); (D) 3 days per week (3q7d); (E) 1 day per week (1q7d). [Figure 6F-H] Figure 1 shows the results of an efficacy experiment evaluating GPRC5D-TCB as a single agent and in combination with high- or low-dose mezigdomide against NCI-H929 multiple myeloma tumors implanted subcutaneously in stem cell-humanized NSG mice. NCI-H929 tumor growth in individual mice following administration of GPRC5D-TCB in combination with 1 mg / kg mezigdomide (F) 5 days per week (5q7d); (G) 3 days per week (3q7d); (H) 1 day per week (1q7d). [Figure 7A] Figure 7 shows the results of cytokine analysis performed in blood NCI-H929 stem cell-engrafted humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with mezigdomide. Using multiplexing technology, the cytokine IL-2 (Figure 7A) was measured in the serum of mice 48 hours after GPRC5D-TCB dosing on Cycle 1 Day 1 (C1D1, 0.0005 mg / kg), Cycle 1 Day 8 (C1D8, 0.002 mg / kg), and Cycle 1 Day 15 (C1D15, 0.04 mg / kg) and 24 hours after administration of 3 mg / kg or 1 mg / kg mezigdomide. [Figure 7B] Figure 7 shows the results of cytokine analysis performed in stem cell-humanized NSG mice engrafted with blood NCI-H929 treated with GPRC5D-TCB as a single agent and in combination with mezigdomide. Using multiplexing technology, the cytokine IP-10 (Figure 7B) was measured in the serum of mice 48 hours after GPRC5D-TCB dosing on Cycle 1 Day 1 (C1D1, 0.0005 mg / kg), Cycle 1 Day 8 (C1D8, 0.002 mg / kg), and Cycle 1 Day 15 (C1D15, 0.04 mg / kg) and 24 hours after administration of 3 mg / kg or 1 mg / kg mezigdomide. [Figure 7C] Figure 7 shows the results of cytokine analysis performed in blood NCI-H929 stem cell-engrafted humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with mezigdomide. Using multiplexing technology, the cytokine IL-10 (Figure 7C) was measured in the serum of mice 48 hours after GPRC5D-TCB dosing on Cycle 1 Day 1 (C1D1, 0.0005 mg / kg), Cycle 1 Day 8 (C1D8, 0.002 mg / kg), and Cycle 1 Day 15 (C1D15, 0.04 mg / kg) and 24 hours after administration of 3 mg / kg or 1 mg / kg mezigdomide. [Figure 7D] Figure 7 shows the results of cytokine analysis performed in blood NCI-H929 stem cell-engrafted humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with mezigdomide. Using multiplexing technology, the cytokine MIP-1a (Figure 7D) was measured in the serum of mice 48 hours after GPRC5D-TCB dosing on Cycle 1 Day 1 (C1D1, 0.0005 mg / kg), Cycle 1 Day 8 (C1D8, 0.002 mg / kg), and Cycle 1 Day 15 (C1D15, 0.04 mg / kg) and 24 hours after administration of 3 mg / kg or 1 mg / kg mezigdomide. [Figure 8A-D]This figure shows the results of quantitative flow cytometry analysis performed on blood NCI-H929 stem cell-engrafted humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with mezigdomide. Using flow cytometry, CD8a+ T cells (A), regulatory T cells (B), B cells (C), and general CD4+ cells (D) were quantified in the blood of mice 164 hours after cycle 3 (=C4 predose) and cycle 5 (=C6 predose) administration of 0.04 mg / kg GPRC5D-TCB, and 48 hours (5q7d), 96 hours (3q7d), or 144 hours (1q7d) after administration of 3 mg / kg or 1 mg / kg mezigdomide. [Figure 8E] We present the results of quantitative flow cytometry analysis performed on blood NCI-H929 stem cell-engrafted humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with mezigdomide. Using flow cytometry, general NK cells were quantified in the blood of mice 164 hours after cycle 3 (=C4 predose) and cycle 5 (=C6 predose) administration of 0.04 mg / kg GPRC5D-TCB, and 48 hours (5q7d), 96 hours (3q7d), or 144 hours (1q7d) after administration of 3 mg / kg or 1 mg / kg mezigdomide. [Figure 9A-B] This figure shows the results of phenotypic flow cytometry analysis performed on blood NCI-H929 stem cell-engrafted humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with mezigdomide. Using flow cytometry, the phenotype of circulating immune cells was analyzed in the blood of mice 164 hours after cycle 3 (=C4 predose) and cycle 5 (=C6 predose) administration of 0.04 mg / kg GPRC5D-TCB, and 48 hours (5q7d), 96 hours (3q7d), or 144 hours (1q7d) after administration of 3 mg / kg or 1 mg / kg mezigdomide. The percentage of CD8a+ T cells expressing TIGIT (A) and Lag3 (B) was assessed. [Figure 9C-D]This figure shows the results of phenotypic flow cytometry analysis performed on blood NCI-H929 stem cell-engrafted humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with mezigdomide. Using flow cytometry, the phenotype of circulating immune cells was analyzed in the blood of mice 164 hours after Cycle 3 (=C4 predose) and Cycle 5 (=C6 predose) administration of 0.04 mg / kg GPRC5D-TCB, and 48 hours (5q7d), 96 hours (3q7d), or 144 hours (1q7d) after administration of 3 mg / kg or 1 mg / kg mezigdomide. The percentage of general CD4+ T cells expressing TIGIT (C) and Lag3 (D) was assessed. DETAILED DESCRIPTION OF THE INVENTION

[0012] definition Unless otherwise defined below, terms are used herein as commonly used in the art.

[0013] The term "antigen-binding molecule" as used herein, in its broadest sense, refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and derivatives thereof, such as fragments.

[0014] The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each specific for a different antigenic determinant. In some embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0015] As used herein, the term "valency" refers to the presence of a specific number of antigen-binding sites within an antigen-binding molecule. Thus, the phrase "monovalent binding to an antigen" refers to the presence of one (and not more than one) antigen-binding site specific for the antigen within the antigen-binding molecule.

[0016] "Antigen-binding site" refers to the site of an antigen-binding molecule, i.e., one or more amino acid residues, that interacts with an antigen. For example, the antigen-binding site of an antibody comprises amino acid residues from the complementarity-determining region (CDR). A naturally occurring immunoglobulin molecule generally has two antigen-binding sites, while a Fab molecule generally has a single antigen-binding site.

[0017] 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 the entity to which it is bound (e.g., a second antigen-binding moiety) to a target site, e.g., 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 some embodiments, an antigen-binding moiety can 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.

[0018] 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 three-dimensional structure 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). Unless otherwise indicated, a protein referred to herein as an antigen (e.g., GPRC5D, CD3) refers to 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). In certain embodiments, 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 some embodiments, the bispecific antigen-binding molecule binds to an epitope of CD3 or GPRC5D that is conserved among CD3 or GPRC5D antigens from various species. In certain embodiments, the bispecific antigen-binding molecule binds to human GPRC5D.

[0019] "Specifically binds" means that the binding is antigen-selective and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding moiety to bind 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 (e.g., analysis on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and classical 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 some embodiments, the antigen-binding portion, or antigen-binding molecule comprising the antigen-binding portion, that binds to an antigen 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 to 10 -13 M, e.g., 10 -9 M to 10 -13 Dissociation constant (K D )

[0020] "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, "binding affinity," as used herein, refers to the intrinsic binding affinity, which 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 a molecule X for its partner Y is generally determined by the dissociation and association rate constants (k off and k on ) is the dissociation constant (K D) Thus, equivalent affinities may involve different rate constants, as long as the ratio of the rate constants is 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).

[0021] "Reduced binding," e.g., reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, e.g., as measured by SPR. For clarity, the term also includes a reduction 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.

[0022] As used herein, the term "T cell activation antigen" refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, which can induce T cell activation upon interaction with an antigen-binding molecule. Specifically, the interaction of an antigen-binding molecule with a T cell activation antigen can induce T cell activation by triggering a signal transduction cascade in the T cell receptor complex. In certain embodiments, the T cell activation antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 144), NCBI RefSeq no. NP_000724, SEQ ID NO: 4 for the human sequence; UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, SEQ ID NO: 5 for the cynomolgus monkey [Macaca fascicularis] sequence).

[0023] 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, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein.

[0024] 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 in the tumor stroma. In a specific embodiment, the target cell antigen is GPRC5D, particularly human GPRC5D according to SEQ ID NO: 9.

[0025] The terms "first," "second," or "third" as used herein with respect to Fab molecules and the like are used for ease of distinction when multiple 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 stated.

[0026] 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.

[0027] 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").

[0028] 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 by one another), i.e., the crossover Fab molecule comprises a peptide chain composed of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1 from N-terminal to C-terminal direction), and a peptide chain composed of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL from N-terminal to C-terminal direction). 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.

[0029] In contrast, a "typical" 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.

[0030] 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 chain 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 chain domain or light chain variable region, followed by a constant light chain (CL) domain, also called a light chain constant region. Immunoglobulin heavy chains are assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain connected via an immunoglobulin hinge region.

[0031] The term "antibody" 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.

[0032] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during the production of a monoclonal antibody preparation, which are generally present in minor amounts, the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations, which typically contain 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 may 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.

[0033] An "isolated" antibody is one that has been separated from a component of its natural environment, i.e., one that is not present in its natural 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 natural or recombinant antibodies that have been separated, fragmented, 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 99% purity, as determined, for example, by electrophoretic (SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, etc.) 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).

[0034] 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.

[0035] An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the 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., Plückthun, 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. Patent No. 5,869,046 for a description of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and resulting in 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 some embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent 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.

[0036] The term "antigen-binding domain" refers to a part of an antibody comprising an area 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).

[0037] 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 variable domains of the heavy and light chains (VH and VL, respectively) of a natural antibody generally have a similar structure, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6 th ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. As used herein with respect to variable region sequences, "Kabat numbering" refers to the numbering system set forth by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0038] As used herein, amino acid positions in all constant regions and constant domains of heavy and light chains 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 pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domains CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3), which is further clarified herein by reference to "Kabat EU index numbering."

[0039] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts") ("complementarity determining regions" or "CDRs"; CDRs of heavy chain variable regions / domains are abbreviated, e.g., as HCDR1, HCDR2, and HCDR3; CDRs of light chain variable regions / domains are abbreviated, e.g., as LCDR1, LCDR2, and LCDR3). Generally, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops located 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) an antigen 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). Includes:

[0040] 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.

[0041] "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 generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0042] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human HVRs and human FRs. In some 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 generate properties according to the invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0043] A "human antibody" is one that possesses an amino acid sequence corresponding to an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies, which comprise non-human antigen-binding residues. In some embodiments, a human antibody is derived from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In some 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.

[0044] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its 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.

[0045] The term "Fc domain" or "Fc region" herein is used 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 extend from Cys226, or from Pro230, to the carboxyl 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, antibodies produced by host cells by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain or a truncated variant of the full-length heavy chain (also referred to 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 according to 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 according to the invention, comprises an additional C-terminal glycine residue (G446, numbered according to the EU index of Kabat). Compositions of the invention, such as the 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 with full-length heavy chains and molecules with 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 truncated mutant heavy chains, in which case 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 truncated mutant 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 with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering 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 with an additional C-terminal glycine residue (G446, numbering 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 a heavy chain comprising an Fc domain subunit as specified herein; molecules comprising a heavy chain comprising an Fc domain subunit as specified herein with an additional C-terminal glycine residue (G446, numbering according to the EU index of Kabat); and molecules comprising a heavy chain comprising an Fc domain subunit 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 within 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 that comprises the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable self-association. For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0046] A "modification that promotes association of a first subunit and a second subunit of an Fc domain" refers to a manipulation of the peptide backbone or a 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 homodimers. 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, where such modifications 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 identical. In some embodiments, a modification that promotes association includes an amino acid mutation, specifically an amino acid substitution, within the Fc domain. In certain embodiments, the association-promoting modifications comprise distinct amino acid mutations, typically amino acid substitutions, in each of the two subunits of the Fc domain.

[0047] The term "effector function" refers to biological activities attributable to the Fc region of an antibody, which vary 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.

[0048] As used herein, the terms "modify, modified, modification" and the like are intended to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Modifications include modifications of the amino acid sequence, modifications of the glycosylation pattern, or modifications of the side groups of individual amino acids, as well as combinations of these techniques.

[0049] As used herein, the term "amino acid mutation" 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 a final construct, provided that the final construct has the desired properties, e.g., reduced binding to Fc receptors or increased association with another peptide. Deletions and insertions in the amino acid sequence include deletions and insertions of amino and / or carboxy terminal amino acids. A particular amino acid mutation is an amino acid substitution. 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, e.g., for the purpose of altering the binding characteristics of the Fc region. 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, and gene synthesis. It is anticipated that methods other than genetic modification, such as chemical modification, that alter the side chain group of an amino acid may also be useful. In this specification, various notations may be used to indicate the same amino acid mutation. For example, a proline to glycine substitution at position 329 of the Fc domain is represented by 329G, G329, G 329 , P329G or Pro329Gly.

[0050] "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 by a variety of methods 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 across the full 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 is described in 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, these sequences can be compared using the public server accessible at http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch(global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2) to perform a global, rather than local, alignment. The percent amino acid identity is shown in the output alignment header.

[0051] An "activating Fc receptor" is an Fc receptor that, following binding of the Fc domain of an antibody, initiates a signaling event that stimulates the receptor-bearing cell to carry out an effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0052] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which an antibody or its derivative, including an Fc region, specifically binds, typically via a protein portion 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 by the ADCC mechanism as defined above, at a given antibody concentration in the medium surrounding the target cells, and / or an increase in the antibody concentration in the medium surrounding the target cells required to lyse a given number of target cells in a given time period by the ADCC mechanism. Reduced ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell, but unmodified, using the same standard production, purification, formulation, and storage methods (known to those skilled in the art). For example, reduced ADCC mediated by an antibody containing an ADCC-reducing amino acid substitution in its Fc domain is relative 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).

[0053] "Formtamig" refers to the specific GPRC5D-TCB listed in the Recommended International Nonproprietary Name: List 89 (WHO Drug Information, Vol. 37, No. 1, 2023).

[0054] An "effective amount" of an agent refers to the amount necessary to cause a physiological change in the cells or tissue to which it is administered.

[0055] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents adverse effects of a disease.

[0056] 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.

[0057] The term "pharmaceutical composition" refers to a preparation in a form that is suitable for the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0058] 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, additives, stabilizers, or preservatives.

[0059] As used herein, "treatment" (and grammatical variations thereof) refers to clinical intervention in an attempt to alter the natural course of disease in the individual being treated and can be performed prophylactically 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, reducing the direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating disease symptoms, and achieving remission or improving prognosis. In some embodiments, the antibody-antibody bispecific antigen-binding molecules of the invention are used to delay the onset of disease or slow the progression of disease.

[0060] 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.

[0061] Bispecific antigen-binding molecules that bind to GPRC5D and CD3 The anti-GPRC5D / anti-CD3 bispecific antigen-binding molecules used in the combination therapies described herein, also referred to herein as "GPRC5D TCB," comprise at least two antigen-binding moieties capable of specifically binding to two distinct 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.

[0062] According to certain embodiments of the invention, the antigen-binding moieties comprised in the bispecific antigen-binding molecule are Fab molecules (i.e., antigen-binding domains composed of a heavy chain and a light chain, each comprising a variable 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 certain embodiments, the Fab molecules are humanized. In yet another embodiment, the Fab molecules comprise human heavy and light chain constant domains.

[0063] Preferably, at least one of the antigen-binding moieties is a crossover Fab molecule. Such modification reduces mispairing between heavy and light chains from different Fab molecules, thereby improving 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 moieties 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 certain by-products due to so-called Bence-Jones type interactions between mispaired heavy and light chains (see Schaefer et al., PNAS, 108 (2011) 1118711191). To further reduce mispairing of heavy and light chains from different Fab molecules and thus 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 one or more Fab molecules that bind to the first antigen (GPRC5D) or the Fab molecules that bind to the second antigen (CD3). This is described below. Charge modifications are made in one or more common Fab molecules contained in the bispecific antigen-binding molecule or in one or more VH / VL crossover Fab molecules (but not in both) contained in the bispecific antigen-binding molecule. In certain embodiments, charge modifications are made in one or more common Fab molecules (which in certain embodiments bind to the first antigen, i.e., GPRC5D) contained in the bispecific antigen-binding molecule.

[0064] 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 to GPRC5D and a T cell activation antigen, the bispecific antigen-binding molecule can crosslink T cells and target cells. Such simultaneous binding results in T cell activation in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of lysis, proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers of target cells, particularly GPRC5D-expressing tumor cells.

[0065] In one embodiment, the bispecific antigen binding molecule is capable of redirecting the cytotoxic activity of T cells to target cells, which in certain embodiments is independent of MHC-mediated peptide antigen presentation by the target cells and / or the specificity of the T cells.

[0066] 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.

[0067] 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 some embodiments, a bispecific antigen-binding molecule comprises two antigen-binding portions, particularly Fab molecules, that bind to GPRC5D. In certain such embodiments, each of these antigen-binding portions binds to the same antigenic determinant. In further specific embodiments, all of these antigen-binding portions are identical, i.e., they comprise the same amino acid sequence, including the same amino acid substitutions in the CH1 and CL domains (if present), as described herein. In one embodiment, a bispecific antigen-binding molecule comprises no more than two antigen-binding portions, particularly Fab molecules, that bind to GPRC5D.

[0068] In certain embodiments, one or more antigen-binding moieties that bind to GPRC5D are generic Fab molecules, in such embodiments, one or more antigen-binding moieties that bind to a second antigen are crossover Fab molecules as described herein, 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 by each other.

[0069] In alternative embodiments, one or more antigen-binding moieties that bind to GPRC5D are crossover Fab molecules as described herein, 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 by each other. In such embodiments, one or more antigen-binding moieties that bind to a second antigen are generic Fab molecules.

[0070] The GPRC5D-binding moiety can direct the bispecific antigen-binding molecule to a target site, for example, to a particular type of tumor cell that expresses GPRC5D.

[0071] Unless clearly scientifically unreasonable or impossible, 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.

[0072] In one aspect, the dual specificity 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.

[0073] In some embodiments, the first antigen-binding portion 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 portion comprises the CDRs of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0074] 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.

[0075] In one embodiment, the first 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: 10 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: 11.

[0076] 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.

[0077] 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.

[0078] In certain embodiments, 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 certain embodiments, the first antigen-binding portion comprises the VH sequence of SEQ ID NO: 48 and the VL sequence of SEQ ID NO: 11.

[0079] 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, 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 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, particularly 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 amino acid mutations described herein under "charge modifications" 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 first antigen-binding portion comprises a heavy chain constant region comprising an amino acid sequence that is 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 (specifically the CH1 domain) may comprise the amino acid mutations described herein under "charge modifications."

[0080] Second antigen-binding moiety The bispecific antigen-binding molecule comprises at least one antigen-binding moiety, in particular a Fab molecule, that binds to a second antigen (CD3).

[0081] In certain 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 with each other. In such embodiments, one or more antigen-binding portions that bind to the first antigen (i.e., GPRC5D) are preferably a common Fab molecule. In embodiments in which multiple antigen-binding portions, particularly Fab molecules, that bind to GPRC5D are present 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 common Fab molecule.

[0082] In alternative embodiments, the antigen-binding moiety that binds to the second antigen is a generic Fab molecule. In such embodiments, one or more of the antigen-binding moieties that bind to the first antigen (i.e., GPRC5D) are crossover Fab molecules as described herein, 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 are swapped / replaced by each other. In embodiments in which there are multiple antigen-binding moieties, particularly Fab molecules, that bind to the second antigen contained in the bispecific antigen-binding molecule, the antigen-binding moiety that binds to GPRC5D is preferably a crossover Fab molecule, and the antigen-binding moiety that binds the second antigen is a generic Fab molecule.

[0083] The second antigen, i.e., CD3, is a T cell activation antigen (also referred to herein as a "T cell activation antigen-binding portion, or a T cell activation 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 a T cell activation antigen. In one embodiment, the bispecific antigen-binding molecule provides monovalent binding to the T cell activation antigen.

[0084] The second antigen is CD3, particularly human CD3 (SEQ ID NO: 4) or cynomolgus CD3 (SEQ ID NO: 5), most particularly human CD3. In one embodiment, the second antigen-binding moiety is cross-reactive with (i.e., specifically binds to) human and cynomolgus CD3. In some embodiments, the second antigen is the epsilon subunit of CD3 (CD3 epsilon).

[0085] In one embodiment, the second antigen-binding moiety comprises an HCDR1 of SEQ ID NO: 18, an HCDR2 of SEQ ID NO: 19, and an HCDR3 of SEQ ID NO: 20, and 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 CDRs of 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 is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of VL sequence 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 first 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.

[0086] 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 an amino acid mutation described herein under "charge modification" and / or, in the case of a crossover Fab molecule, may comprise deletion or substitution 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 that is 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 (specifically the CH1 domain) may comprise the amino acid mutations described herein under "charge modifications."

[0087] 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 by 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 third, if present) antigen-binding moiety is a generic Fab molecule.

[0088] 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).

[0089] charge modification Bispecific antigen-binding molecules may contain amino acid substitutions in the Fab molecules contained therein that are effective in reducing mismatching of mismatched heavy and light chains (Bens-Jones by-products) that can occur in the generation of Fab-based bi / multispecific antigen-binding molecules, particularly those having a VH / VL domain exchange in one of their binding arms (or two or more in the case of molecules containing three or more antigen-binding Fab molecules) (see also WO 2015 / 150447, particularly the Examples, which are incorporated herein by reference in their entirety). The ratio of desired bispecific antigen-binding molecules compared to undesired by-products, particularly Bens-Jones by-products, present in bispecific antigen-binding molecules having a VH / VL domain exchange in one of their 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").

[0090] Thus, in some embodiments, both the first and second antigen-binding moieties of the bispecific antigen-binding molecule are 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 by each other: i) in the constant domain CL of said 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 said 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).

[0091] A bispecific antigen-binding molecule cannot 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 by each other (i.e., remain unexchanged).

[0092] In a more particular embodiment, i) in the constant domain CL of said 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 said 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).

[0093] In one such embodiment, 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).

[0094] 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 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 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0095] In certain embodiments, 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).

[0096] In a more particular 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) and 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).

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

[0098] In a particular 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.

[0099] Alternatively, the amino acid substitutions according to the above embodiments may be made in the constant domains CL and CH1 of the second antigen-binding moiety instead of the constant domains CL and CH1 of the first antigen-binding moiety. In certain such embodiments, the constant domain CL of the second antigen-binding moiety is of the kappa isotype.

[0100] Thus, in one embodiment, the constant domain CL of the second antigen-binding moiety has an independently substituted amino acid at position 124 by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and the constant domain CH1 of the second antigen-binding moiety has an independently substituted amino acid at position 147 or 213 by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0101] 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 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 is substituted independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0102] 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).

[0103] 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) (Kabat numbering) and the amino acid at position 123 is substituted by lysine (K) (Kabat numbering), and in the constant domain CHI of said second antigen-binding moiety the amino acid at position 147 is substituted by glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat EU index numbering).

[0104] 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) (Kabat numbering) and the amino acid at position 123 is substituted by arginine (R) (Kabat numbering); and in the constant domain CHI of said second antigen-binding moiety the amino acid at position 147 is substituted by glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat EU index numbering).

[0105] In certain embodiments, 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; and (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 light chain and Fab heavy chain are replaced by each other. Includes; 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) (in certain embodiments, independently by lysine (K) or arginine (R)), and the amino acid at position 123 is independently substituted by lysine (K), arginine (R), or histidine (H) (Kabat numbering) (in certain embodiments, independently by lysine (K) or arginine (R)); 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).

[0106] Bispecific antigen-binding molecule formats In some 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 some embodiments, multispecific antibodies have three or more binding specificities. In some 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.

[0107] 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-into-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 be produced by modifying 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 generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using common 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)); al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and by using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0108] Engineered antibodies with three or more antigen-binding sites are also included herein, including, for example, "octopus antibodies" or DVD-Igs (see, e.g., WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies with 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).

[0109] Multispecific antibodies can also be provided in an 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) 11871191 and Klein at al., MAbs 8 (2016) 101020). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to induce correct Fab pairing. See, for example, WO 2016 / 172485.

[0110] 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).

[0111] Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, so-called "BiTE" (bispecific T cell inducer) molecules in which two scFv molecules are fused by a flexible linker (e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567; Nagorsen and Baeuerle, Exp Cell Res 317, 12551260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299305 (1996)) and their derivatives, such as tandem diabodies ("TandAb"; Kipriyanov et al., J Mol Biol 293, 4156 (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, 436449 (2010)), as well as so-called triomabs, which are all-hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458467 (2010)). Specific T cell bispecific antibody formats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0112] The components of the bispecific antigen-binding molecule can be fused to each other in a variety of configurations.

[0113] In certain 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.

[0114] In one embodiment, the first and second antigen-binding moieties 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 moieties are each Fab molecules. In one such embodiment, the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain. In another such embodiment, the first antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen-binding moiety at the C-terminus of the Fab heavy chain. In embodiments in which (i) the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain, or (ii) the first antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen-binding moiety at the C-terminus of the Fab heavy chain, the Fab light chain of the first antigen-binding moiety and the Fab light chain of the second antigen-binding moiety may additionally be fused to each other, optionally via a peptide linker.

[0115] 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 particularly useful when internalization of the target cell antigen is expected following 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 may enhance internalization of the target cell antigen, thereby reducing its availability.

[0116] However, in other cases, it may be advantageous to have a bispecific antigen-binding molecule that contains two or more antigen-binding portions (e.g., Fab molecules) specific for target cell antigens, in order to target a target site or to enable cross-linking of target cell antigens.

[0117] Thus, in certain embodiments, the bispecific antigen-binding molecule comprises a third antigen-binding moiety.

[0118] 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.

[0119] In one embodiment, the third antigen-binding moiety is identical to the first antigen-binding moiety.

[0120] Unless scientifically clearly unreasonable or impossible, 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.

[0121] 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.

[0122] 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 CDRs of any of the above embodiments and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0123] 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.

[0124] In one embodiment, the third 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: 10 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: 11.

[0125] 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.

[0126] 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.

[0127] In certain embodiments, 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 certain embodiments, the third antigen-binding portion comprises the VH sequence of SEQ ID NO: 48 and the VL sequence of SEQ ID NO: 11.

[0128] 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 an amino acid mutation described herein under "charge modification" and / or, in the case of a crossover Fab molecule, may comprise deletion or substitution 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 that is 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 (specifically the CH1 domain) may comprise an amino acid mutation described herein under "charge modification."

[0129] In certain embodiments, the third and first antigen-binding moieties are each Fab molecules, and the third antigen-binding moiety is identical to the first antigen-binding moiety. Thus, in such embodiments, the first and third antigen-binding moieties comprise the same heavy and light chain amino acid sequences and have the same domain organization (i.e., common or crossover). Furthermore, in such embodiments, the third antigen-binding moiety, if present, comprises the same amino acid substitutions as the first antigen-binding moiety. For example, amino acid substitutions described herein as "charge modifications" may occur in the constant domains CL and CHI of each of the first and third antigen-binding moieties. Alternatively, the amino acid substitutions may occur in the constant domains CL and CHI of the second antigen-binding moiety (which in certain embodiments is also a Fab molecule), but not in the constant domains CL and CHI of the first and third antigen-binding moieties.

[0130] Like the first antigen-binding portion, the third antigen-binding portion is particularly a generic 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 generic Fab molecule) are also contemplated. Thus, in certain embodiments, the first and third antigen-binding portions are each generic 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 have been swapped / replaced by each other. In other embodiments, the first and third antigen-binding portions are each crossover Fab molecules, and the second antigen-binding portion is a generic Fab molecule.

[0131] When a third antigen-binding moiety is present, in certain embodiments, the first and third antigen-binding moieties bind to GPRC5D and the second antigen-binding moiety binds to CD3, particularly CD3 epsilon.

[0132] In certain embodiments, the bispecific antigen-binding molecule comprises an Fc domain composed of a first and a second subunit, the first and second subunits of the Fc domain being capable of stable association.

[0133] Bispecific antigen-binding molecules can have different configurations, i.e., the first, second (and optionally third) antigen-binding moieties can be fused to each other and to the Fc domain in different ways. These components can be fused to each other directly or, preferably, via one or more suitable peptide linkers. When a Fab molecule is fused to the N-terminus of a Fab molecule of a subunit of the Fc domain, this is typically via the immunoglobulin hinge region.

[0134] In some embodiments, the first and second antigen-binding moieties are each 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 first or second subunit of the Fc domain. In such embodiments, 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 or to the N-terminus of the other subunit of the Fc domain. 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 have been swapped / replaced by each other. In other such embodiments, the first Fab molecule is a crossover Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0135] In one embodiment, the first and second antigen-binding moieties 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 first and second subunits, and optionally one or more peptide linkers, wherein 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 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 further be fused to each other.

[0136] In another embodiment, the first and second antigen-binding moieties are each Fab molecules, and each of the first and second antigen-binding moieties is fused to the N-terminus of one of the Fc domain subunits at the C-terminus of the Fab heavy chain. 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 each of the first and second Fab molecules is fused to the N-terminus of one of the Fc domain subunits at the C-terminus of the Fab heavy chain. The first and second Fab molecules may be fused to the Fc domain directly or indirectly via a peptide linker. In a specific embodiment, the first and second Fab molecules are fused to the Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, particularly when the Fc domain is an IgG1 Fc domain.

[0137] In some embodiments, the first and second antigen-binding moieties 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 Fc domain subunit. In such embodiments, 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 second antigen-binding moiety, or to the N-terminus of the other Fc domain subunit (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 have been swapped / replaced by each other. In other such embodiments, the first Fab molecule is a crossover Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0138] In one embodiment, the first and second antigen-binding moieties are each Fab molecules, and the first 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 second 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 first antigen-binding moiety. In a specific embodiment, the bispecific antigen-binding molecule consists essentially of first and second Fab molecules, an Fc domain consisting 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 its 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 its 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 further be fused to each other.

[0139] 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 a conventional Fab molecule, 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 by each other. In other such embodiments, the first and third Fab molecules are each a crossover Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0140] 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 Fc domain subunits, 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 one particular 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 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 second and third Fab molecules may be fused to the Fc domain directly or through peptide linkers. In a particular embodiment, the second and third Fab molecules are each fused to an Fc domain through an immunoglobulin hinge region. In a particular embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, particularly when 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 further be fused to each other.

[0141] 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 through peptide linkers. In a specific embodiment, the first and third Fab molecules are each fused to an Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, particularly when 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 further be fused to each other.

[0142] In the construction of a bispecific antigen-binding molecule in which a Fab molecule is fused at the C-terminus of the Fab heavy chain through the immunoglobulin hinge region to the N-terminus of each of the Fc domain subunits, the two Fab molecules, hinge region, and 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.

[0143] 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 configuration 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 mismatched Fab heavy and Fab light chains and reduces the number of plasmids required to express some bispecific antigen-binding molecules.

[0144] The antigen-binding portions may be fused to the Fc domain or to each other, either directly or through peptide linkers, which comprise 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, 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 mwherein G=glycine, S=serine, and (x=3, n=3, 4, 5, or 6, and m=0, 1, 2, or 3) or (x=4, n=2, 3, 4, or 5, and m=0, 1, 2, or 3), in one embodiment, x=4 and n=2 or 3, and in a further embodiment, x=4 and n=2. In one embodiment, the peptide linker is (G4S)2. A particularly suitable peptide linker 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 connecting the Fab heavy chains of the first and second Fab molecules 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 (a portion 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.

[0145] 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 a 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), and the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with a subunit of an Fc domain (VL (2) -CH1 (2) -CH2-CH3(-CH4)) and a polypeptide in which the Fab heavy chain of the first Fab portion shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CH1 (1) 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. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1)In some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0146] In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab heavy chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with a Fab light 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 constant region is replaced by a light chain constant region), and the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with a subunit of an Fc domain (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 a subunit of the Fc domain (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 the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) In some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0147] 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 (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), 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 the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with a subunit of an Fc domain (VL (1) -CH1 (1) -VH (2) -CH1(2) In other embodiments, the bispecific antigen-binding molecule comprises a polypeptide 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, 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 in which the heavy chain variable region is replaced by a light chain variable region), and the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with a subunit of an Fc domain (VH (1) -CH1 (2) -VL (1) -CH1 (1) -CH2-CH3(-CH4)).

[0148] 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) of the first Fab molecule (1) -CL (1) In others of these embodiments, the bispecific antigen-binding molecule optionally 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, and 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, and 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) ) further includes.

[0149] The bispecific antigen-binding molecules according to these embodiments may further comprise (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 a Fab light chain polypeptide (VL (3) -CL (3) In some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0150] 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 (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), 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, and the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CL (1) -VH (2) -CH1 (2) In other embodiments, the bispecific antigen-binding molecule comprises a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a second Fab molecule, the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with a 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), and the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with a subunit of an Fc domain (VH (1) -CH1 (1) -VH (2) -CL(2) -CH2-CH3(-CH4)).

[0151] 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) of the first Fab molecule (1) -CL (1) In others of these embodiments, the bispecific antigen-binding molecule optionally 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, and 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, and 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) -CL (2) ) further includes.

[0152] The bispecific antigen-binding molecules according to these embodiments may further comprise (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 a Fab light chain polypeptide (VL (3) -CL (3)In some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0153] In some embodiments, the bispecific antigen-binding molecule does not comprise an Fc domain. In certain such embodiments, the first Fab molecule and, if present, the third Fab molecule are each a generic Fab molecule, 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 by each other. In other such embodiments, the first Fab molecule and, if present, the third Fab molecule are each a crossover Fab molecule, and the second Fab molecule is a generic Fab molecule.

[0154] In one such embodiment, the bispecific antigen-binding molecule consists essentially of a first and a second antigen-binding moiety, optionally comprising 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.

[0155] In another such embodiment, the bispecific antigen-binding molecule consists essentially of a first and a second antigen-binding moiety, optionally comprising 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.

[0156] 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 being fused at its C-terminus to the N-terminus of the Fab heavy chain of the first Fab molecule. In some such embodiments, the bispecific antigen-binding molecule consists essentially of first, second, and third Fab molecules, optionally comprising one or more peptide linkers, and 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.

[0157] 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 some such embodiments, the bispecific antigen-binding molecule consists essentially of first, second, and third Fab molecules, optionally comprising 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.

[0158] In some 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 light chain variable region of a second Fab molecule, and 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.

[0159] 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 in which the heavy chain variable region is replaced by a light chain variable region), and 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. (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.

[0160] 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 in which the heavy chain constant region is replaced by a light chain constant region), and 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 the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) and further.

[0161] In some embodiments, a bispecific antigen-binding molecule according to the invention 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 in which the heavy chain variable region is replaced by a light chain variable region), and 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. (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.

[0162] In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VH) in which 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, the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, and 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). (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.

[0163] In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide (VH) in which 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, 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, and 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). (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 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.

[0164] In some 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 in which the heavy chain variable region is replaced by a light chain variable region), the Fab heavy 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 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.

[0165] In some 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 in which the heavy chain constant region is replaced by a light chain constant region), 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 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 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.

[0166] In some 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 light chain variable region of the first Fab molecule, 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 in which the heavy chain variable region is replaced by a light chain variable region), the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a third Fab molecule, and 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 (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region). (2) -CH1 (1) -VL (2) -CH1 (2) -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 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 (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.

[0167] In some 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, 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 in which the heavy chain constant region is replaced by a light chain constant region), 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, and 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 a crossover Fab heavy chain in which 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 the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the 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.

[0168] In some 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 the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), 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 the first Fab molecule, and 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 in which the heavy chain variable region is replaced by a light chain variable region), and 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 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 (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 some 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 the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), the Fab light 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, 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 in which the heavy chain constant region is replaced by a light chain constant region), and 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 the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the 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 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 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 by each other, 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, and a light chain complementarity determining region (HCDR) 21 of SEQ ID NO: 22. a) a light chain variable region (VL) comprising an LCDR1 of SEQ ID NO: 22, an LCDR2 of SEQ ID NO: 23, and a light chain variable region (VL) comprising an LCDR3 of SEQ ID NO: 24; 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 the second 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), 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).

[0171] In another embodiment, the invention provides 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 comprising a Fab light chain and a Fab heavy chain variable domain VL and VH or a constant domain CL. and (c) a second antigen-binding portion, wherein 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, 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 (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.

[0172] In all of the various configurations of bispecific antigen-binding molecules, the amino acid substitutions described herein, if present, may be present in the CH1 and CL domains of the first and (if present) third antigen-binding moiety / Fab molecule, or in the CH1 and CL domains of the second antigen-binding moiety / Fab molecule. Preferably, such substitutions are present 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, none of such amino acid substitutions is 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, none of such amino acid substitutions is made in the first (and if present, third) antigen-binding moiety / Fab molecule. 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 by each other.

[0173] In certain embodiments, particularly where the amino acid substitutions described herein are made in the first (and, if present, the third) antigen-binding moiety / Fab molecule, 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. In other embodiments of bispecific antigen-binding molecules according to the invention, particularly 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.

[0174] In one embodiment, the present invention provides a first antigen-binding moiety comprising: 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 replaced by 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 a light chain variable region (VL). a second antigen-binding portion comprising a heavy chain variable region (VH) comprising 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; c) a bispecific antigen-binding molecule comprising an Fc domain composed of a first and a second subunit; in the constant domain CL of the first antigen-binding portion 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) (numbering according to Kabat) (most particularly by arginine (R)); in the constant domain CHI of the first antigen-binding portion of a), 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);(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) is 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), 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) is 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);

[0175] In a specific embodiment, the first antigen-binding moiety is selected from the group consisting of: 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 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 replaced by each other, 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, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 16, and an LCDR3 of SEQ ID NO: 17. a) a second antigen-binding portion comprising a light chain variable region (VL) comprising a complementarity determining region (LCDR) 1, a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 2 of SEQ ID NO: 22, a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 3 of SEQ ID NO: 23; c) a third antigen-binding portion that binds to a first antigen and is identical to the first antigen; 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) (numbering according to Kabat) (most particularly by arginine (R)), 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) (Kabat numbering). (EU index numbering), and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat EU index numbering);(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);

[0176] In another embodiment, the present invention provides 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 replaced by 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 a light chain variable region (VL). a second antigen-binding portion comprising a heavy chain variable region (VH) comprising 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; c) a bispecific antigen-binding molecule comprising an Fc domain composed of a first and a second subunit; in the constant domain CL of the first antigen-binding portion 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) (numbering according to Kabat) (most particularly by arginine (R)), and in the constant domain CH1 of the first antigen-binding portion of a), the amino acid at position 147 is substituted by glutamic acid (E) (Kabat numbering). 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)

[0177] In any of the above embodiments, the components of the bispecific antigen-binding molecule (e.g., Fab molecules, Fc domains) can be fused directly or via a variety of linkers described herein or known in the art, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids. 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.

[0178] In a particular embodiment, the present invention provides a) a first and third antigen-binding moiety that binds to a first antigen; wherein the first antigen is GPRC5D, and the first and second antigen-binding moieties are each a (generic) Fab molecule 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 (generic) Fab molecule comprising a heavy chain variable region comprising 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, in which the variable domains VL and VH of the Fab light and heavy chains are replaced by each other. a) a second antigen-binding moiety which is a Fab molecule comprising a first and a second subunit; c) a bispecific antigen-binding molecule comprising an Fc domain composed of a first and a second subunit; wherein in the constant domains CL of the first and third antigen-binding moieties 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) (numbering according to Kabat) (most particularly by arginine (R)), and in the constant domains CH1 of the first and third antigen-binding moieties 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); further 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).

[0179] In one embodiment, in the first subunit of the Fc domain of the bispecific antigen-binding molecule, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index).

[0180] In yet 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 with a cysteine ​​residue (S354C) or a replacement of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C), particularly a 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).

[0181] 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).

[0182] In yet a further embodiment, the Fc domain is a human IgG1 Fc domain.

[0183] 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 formtamig.

[0184] Fc domain In certain embodiments, the bispecific antigen-binding molecule comprises an Fc domain composed of a first and a second subunit.

[0185] The Fc domain of a bispecific antigen-binding molecule consists of a pair of polypeptide chains comprising the heavy chain domains 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, the bispecific antigen-binding molecule of the present invention comprises no more than one Fc domain.

[0186] 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 (numbering according to the Kabat EU index), in particular the amino acid substitution S228P. 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 set forth in SEQ ID NO: 6.

[0187] Fc domain modifications that promote heterodimerization Bispecific antigen-binding molecules contain multiple different antigen-binding moieties, which 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. Therefore, to improve the yield and purity of bispecific antigen-binding molecules during recombinant production, it would be advantageous to introduce modifications to the Fc domain of the bispecific antigen-binding molecule that promote the association of the desired polypeptides.

[0188] That is, in certain embodiments, the Fc domain of the bispecific antigen-binding molecule comprises a modification that promotes association of the first and second subunits of the Fc domain. The site of greatest protein-protein interaction between the two subunits of the Fc domain of human IgG is the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0189] There are several approaches for modifications in the CH3 domain of an Fc domain to enhance heterodimerization, which are described in detail in, for example, WO 96 / 27011, WO 98 / 050431, EP1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, and WO 2013096291. Typically, in all of these approaches, the CH3 domain of the first Fc domain subunit and the CH3 domain of the second Fc domain subunit are complementarily modified so that each CH3 domain (or the heavy chain containing it) can no longer homodimerize with itself but heterodimerize with another complementarily modified CH3 domain (i.e., the first and second CH3 domains heterodimerize and homodimerization between the two first CH3 domains or the two second CH3 domains is prevented). These different approaches to improving heavy chain heterodimerization are considered as different alternatives in combination with heavy-light chain modifications in bispecific antigen-binding molecules to reduce heavy / light chain mispairing and Bence-Jones by-products (e.g., swapping / replacing VH and VL in one binding arm and introducing oppositely charged amino acid substitutions at the CH1 / CL interface).

[0190] In a specific embodiment, the modification that promotes association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, comprising 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.

[0191] Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this method involves introducing a protuberance ("knob") into the interface of a first polypeptide and a cavity ("hole") into the interface of a second polypeptide, such that the protuberance is positioned within the corresponding cavity ("hole"), promoting heterodimer formation and preventing homodimer formation. The protuberance is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protuberance is created in the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine).

[0192] Thus, in one 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 creating a protuberance within the CH3 domain of the first subunit that can be positioned within the cavity within 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 creating a cavity within the CH3 domain of the second subunit that can be positioned within the protuberance within the CH3 domain of the first subunit.

[0193] 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).

[0194] 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).

[0195] The protuberances and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0196] In one particular embodiment, in (the CH3 domain of) the first subunit ("knob" subunit) of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in (the CH3 domain of) the second subunit ("hole" subunit) of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, the second subunit of the Fc domain also 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).

[0197] 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)).

[0198] 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).

[0199] In certain embodiments, an antigen-binding moiety that binds a second antigen (e.g., a T cell activation 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, such as a T cell activation antigen, to a knob-containing subunit of an Fc domain will (further) minimize the generation of antigen-binding molecules comprising two antigen-binding moieties that bind to the T cell activation antigen (steric clash of the two knob-containing polypeptides).

[0200] Other techniques of CH3 modification to enhance heterodimerization are contemplated as alternatives according to 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, WO 2013 / 096291.

[0201] In one embodiment, the heterodimerization technique described in EP1870459 is used instead. This technique 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 comprises the amino acid mutations R409D;K370E in one of the two CH3 domains (of the Fc domain) and D399K;E357K (numbering according to the Kabat EU index) in the other CH3 domain of the Fc domain.

[0202] 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 and the amino acid mutations 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 (numbering according to Kabat EU index).

[0203] 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 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).

[0204] 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).

[0205] 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 and Y407A, and the second CH3 domain comprises the amino acid mutations T366A and K409F. In a further embodiment, the second CH3 domain comprises, at positions T411, D399, S400, F405, N390, or K392, e.g., a) T411N, T411R, T411Q, T411K, T411D, T411E, or T411W; b) D399R, D399W, D399Y, or D399K; c) S400E, S400F, or S400G; 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 mutations 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).

[0206] In one embodiment, the heterodimerization approach described in WO 2011 / 143545 is alternatively used, e.g., using amino acid modifications at positions selected from the group consisting of 368 and 409 (numbering according to the Kabat EU index).

[0207] In one embodiment, the heterodimerization approach described in WO 2011 / 090762 is alternatively used, which also employs the knob-into-hole technique described above. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (numbering according to the Kabat EU index). 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 alternatively used.

[0208] In an alternative embodiment, the modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates an electrostatic steering effect, e.g., as described in WO 2009 / 089004. Typically, 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 and heterodimerization is electrostatically favorable. In one such embodiment, the first CH3 domain comprises an amino acid substitution of 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 of 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 of 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 additionally 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).

[0209] In still further embodiments, the heterodimerization approach described in WO 2007 / 147901 is alternatively used. 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).

[0210] In yet another embodiment, the heterodimerization approach described in WO 2007 / 110205 can alternatively be used.

[0211] 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).

[0212] Fc domain modifications that reduce Fc receptor binding and / or decrease effector function The Fc domain confers desirable pharmacokinetic properties to bispecific antigen-binding molecules, including a long serum half-life that contributes to favorable accumulation in target tissues and a favorable tissue-to-blood distribution ratio. However, it can also cause undesirable targeting of bispecific antigen-binding molecules to cells that express Fc receptors rather than to preferred antigen-bearing cells. Furthermore, coactivation of Fc receptor signaling pathways, combined with activated T cell properties (e.g., in bispecific antigen-binding molecule embodiments in which the second antigen-binding moiety binds to a T cell-activating antigen) and the long half-life of the bispecific antigen-binding molecule, can lead to cytokine release, resulting in excessive cytokine receptor activation and severe side effects upon systemic administration. Activation of immune cells other than T cells (Fc receptor-bearing) can potentially further reduce the efficacy of bispecific antigen-binding molecules (e.g., bispecific antigen-binding molecules in which the second antigen-binding moiety binds to a T cell-activating antigen) due to the potential for T cell destruction by, for example, NK cells.

[0213] Thus, in certain embodiments, the Fc domain of the bispecific antigen-binding molecule exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to the Fc domain of a native IgG1. 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 the 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 the 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 and / or induce effector function of Fc receptors. 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 one embodiment, 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 a binding affinity to FcRn that is greater than about 70%, particularly greater than about 80%, and more particularly greater than about 90% of that of a native IgG1 Fc domain (or a bispecific antigen-binding molecule comprising a native IgG1 Fc domain).

[0214] In some embodiments, the Fc domain is modified to have reduced binding affinity to an Fc receptor and / or reduced effector function compared to an unmodified Fc domain. In certain embodiments, the Fc domain of the bispecific antigen-binding molecule comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor and / or the effector function. 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 to an Fc receptor. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where multiple amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor are present, the combination of these amino acid mutations reduces the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or at least 50-fold. In one embodiment, a bispecific antigen-binding molecule comprising a modified Fc domain exhibits less than 20%, particularly less than 10%, or even less than 5% of the binding affinity to an Fc receptor compared to a bispecific antigen-binding molecule comprising an unmodified 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, most particularly human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to a complementary component, 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 said Fc domain) exhibits greater than about 70% of the binding affinity to FcRn of the unmodified form of the Fc domain (or a bispecific antigen-binding molecule comprising said unmodified form of the Fc domain). Fc domains or bispecific antigen-binding molecules of the invention comprising said Fc domains may exhibit greater than about 80%, and in some cases greater than about 90%, of such affinity. In some embodiments, the Fc domain of the bispecific antigen-binding molecule is modified to have reduced effector function compared to the unmodified Fc domain. Reduced effector function can include, but is not limited to, one or more of: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent T 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 that induces apoptosis, reduced cross-linking 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 an unmodified Fc domain (or a bispecific antigen-binding molecule comprising an unmodified Fc domain).

[0215] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or 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 the 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 the Kabat EU index). In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to the 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 the Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and a further 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 further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more particular embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA," "PGLALA," or "LALAPG").Specifically, in certain embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., in each of the first and second subunits of the Fc domain, 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) (Kabat EU index numbering).

[0216] 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.

[0217] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function compared to IgG1 antibodies. Thus, 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 its binding affinity to Fc receptors and / or its effector function, 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.

[0218] In certain embodiments, the Fc domain that exhibits reduced binding affinity to an Fc receptor 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).

[0219] In some embodiments, N-glycosylation of the Fc domain is eliminated, hi one such embodiment, the Fc domain comprises an amino acid substitution at position N297, specifically replacing asparagine with alanine (N297A) or aspartic acid (N297D) (numbering according to the Kabat EU index).

[0220] In addition to the Fc domains described herein and in WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or reduced effector function also include those with substitutions at 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 so-called "DANA" Fc variants with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0221] 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 can include site-directed mutagenesis of the encoding DNA sequence, PCR, and gene synthesis. The exact nucleotide changes can be verified, for example, by sequencing.

[0222] Binding to Fc receptors 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 such Fc receptors can be obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain, or a bispecific antigen-binding molecule comprising an Fc domain, to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor, for example, human NK cells expressing the FcγIIIa receptor.

[0223] The effector function of an Fc domain or a bispecific antigen-binding molecule comprising 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; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays may be used (e.g., ACTI for flow cytometry). TM non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0224] In some embodiments, binding of the Fc domain to complement components, specifically C1q, is reduced. Thus, in some embodiments in which the Fc domain is modified to reduce effector function, the reduced effector function includes reduced CDC. C1q binding assays can be performed to determine whether an Fc domain, or a bispecific antigen-binding molecule comprising an Fc domain, can bind to C1q and thereby have CDC activity. See, e.g., 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)).

[0225] FcRn binding and in vivo clearance / half-life determinations 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).

[0226] 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, for use in, for example, any of the therapeutic methods described below. 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, for example, as described below.

[0227] 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) formulating the antibody or bispecific antigen-binding molecule with at least one pharmaceutically acceptable carrier, wherein the antibody or bispecific antigen-binding molecule preparation is formulated for in vivo administration.

[0228] 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 term "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that are nontoxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic, or other undesirable responses when administered to animals, e.g., humans, as needed. Preparation of pharmaceutical compositions comprising an antibody or bispecific antigen-binding molecule, and optionally additional active ingredients, will be known to those skilled 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, for administration to animals (e.g., humans), preparations must meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards or the corresponding authorities of other countries. 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), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, 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). Any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions, provided that it is not incompatible with the active ingredient.

[0229] The immunoconjugates of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, intranasal, or, if localized treatment is desired, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., injections, such as intravenous or subcutaneous injections, depending in part on whether administration is brief or chronic.

[0230] Parenteral compositions include those designed for administration by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, the antibodies or bispecific antigen-binding molecules of the present invention can be formulated in aqueous solutions, preferably physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solutions may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the antibodies or bispecific antigen-binding molecules may be in powder form suitable for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the antibodies or bispecific antigen-binding molecules of the present invention in the required amount into an appropriate solvent with various other ingredients, as listed below, as needed. 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 liquid medium that has already been sterile-filtered. The liquid medium should be appropriately buffered, if necessary, and the liquid diluent must first be 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. Endotoxin contamination must be kept to a safe level, e.g., less than 0.5 ng / mg protein.Suitable pharmaceutically acceptable carriers include, but are not limited to, buffers such as 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 methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; tannins, etc. 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 dextrin; 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 non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound 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.

[0231] The active ingredient can be encapsulated in microcapsules, such as hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules, prepared by, for example, coacervation techniques or interfacial polymerization methods, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. 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 products include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films or microcapsules. In certain embodiments, prolonged absorption of injectable compositions can be achieved by using agents delaying absorption in the compositions, such as aluminum monostearate, gelatin, or combinations thereof.

[0232] In addition to the aforementioned compositions, antibodies or bispecific antigen-binding molecules can also be formulated as sustained-release preparations. Such long-acting preparations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by 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 a sparingly soluble salt.

[0233] Pharmaceutical compositions containing the antibodies or bispecific antigen-binding molecules of the present invention can be prepared by common mixing, dissolving, emulsifying, encapsulating, entrapment, or lyophilization methods. Pharmaceutical compositions can be formulated in a common manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate processing of proteins into pharmaceutically usable preparations. The appropriate formulation depends on the selected route of administration.

[0234] Antibodies or bispecific antigen-binding molecules can be formulated into compositions in the form of free acids or bases, either neutral or in salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acids or bases. 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.

[0235] Immunomodulatory imide drugs (IMiDs) The term "immunomodulatory imide drugs (IMiDs)" refers to a class of immunomodulators (drugs that modulate immune responses) containing an imide group. IMiDs refer to both first-generation IMiDs and cereblon E3 ligase modulators (CELMoDs; also called next-generation IMiDs), both of which contain a conserved glutarimide ring. First-generation and next-generation IMiDs bind to cereblon (CRBN), a receptor for the cullin-ring 4 ubiquitin ligase (CRL4) complex, and regulate ubiquitin ligase activity. The specificity of the ligase is redirected to non-physiological protein targets, also called "neosubstrates," for subsequent ubiquitination and / or degradation. The conserved glutarimide ring binds to CRBN, and variable side groups interact with CRBN and the neosubstrate. The term "first-generation IMiDs" refers to thalidomide and its derivatives, lenalidomide and pomalidomide. The term "CELMoD" or "next generation IMiD" refers to thalidomide derivatives with extended side groups that can improve interactions with CRBN and / or neosubstrates, including, but not limited to, iberdomide (also known as CC-220), avadomide (also known as CC-122), mezigdomide (also known as CC-92480), CC885, CC647, CC-90009, and CC3060.

[0236] The term "lenalidomide" refers to a compound having the following chemical structure: TIFF2025532806000001.tif42170

[0237] The empirical formula of lenalidomide is C 13 H 13 N3O3, CAS Registry Number 191732-72-6, with a gram molecular weight of 259.3. Lenalidomide is a thalidomide analog sold under the trade name REVLIMID®.

[0238] The term "pomalidomide" refers to a compound having the following chemical structure: TIFF2025532806000002.tif42170

[0239] The empirical formula of pomalidomide is C 13 H 11 N3O4, CAS Registry Number 19171-19-8, with a gram molecular weight of 273.24. Pomalidomide is a thalidomide analogue sold under the trade name Imnovid in Europe and Pomalyst in the United States.

[0240] The term "iverdomide" refers to a compound having the following chemical structure: TIFF2025532806000003.tif68170

[0241] The empirical formula of iverdomide is C 25 H 27 N3O5, CAS Registry Number 1323403-33-3, with a gram molecular weight of 449.5.

[0242] The term "mezigdomide" refers to a compound having the following chemical structure: TIFF2025532806000004.tif80170

[0243] The empirical formula of mezigdomide is C 32 H 30FN5O4, CAS Registry Number 2259648-80-9, and gram molecular weight 567.6.

[0244] The present invention provides a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody and an immunomodulatory imid drug (IMiD). The IMiD used in this combination therapy described herein can be a first-generation IMiD or a CELMoD. In one embodiment, the IMiD is a first-generation IMiD or a CELMoD. In a further embodiment, the IMiD is a first-generation IMiD and is selected from the group of thalidomide, lenalidomide, and pomalidomide. In one embodiment, the IMiD is a CELMoD and is selected from the group of iberdomide, avadomide, mezigdomide (CC-92480), CC885, CC647, CC-90009, and CC3060. In one embodiment, the IMiD is selected from the group of lenalidomide, pomalidomide, iberdomide, and mezigdomide.

[0245] Glucocorticosteroids The present invention further provides a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody with an immunomodulatory imid drug (IMiD) and a glucocorticosteroid.

[0246] 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 substitutes.

[0247] The term "dexamethasone" refers to a compound having the following chemical structure: TIFF2025532806000005.tif52170

[0248] The empirical formula for dexamethasone is C 22H 29 FO5, CAS Registry Number 50-02-2, gram molecular weight 392.46.

[0249] The present invention further provides a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody with an immunomodulatory imid drug (IMiD) and a glucocorticoid, hi one embodiment, the glucocorticosteroid is dexamethasone.

[0250] Therapeutic methods and compositions The present invention includes combination therapies that combine an anti-GPRC5D / anti-CD3 bispecific antibody with an IMiD. Optionally, the combination therapies described herein may further include a glucocorticosteroid.

[0251] The present invention includes a method for treating a patient in need of therapy, comprising administering to the patient a therapeutically effective amount of a combination therapy combining an anti-GPRC5D / anti-CD3 bispecific antibody and an IMiD.The present invention includes a method for treating a patient in need of therapy, comprising administering to the patient a therapeutically effective amount of a combination therapy combining an anti-GPRC5D / anti-CD3 bispecific antibody with an IMiD and a glucocorticosteroid.

[0252] One preferred embodiment of the present invention is a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody with an IMiD 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 an IMiD and a glucocorticosteroid for use in the treatment of cancer or tumors.

[0253] One embodiment of the present invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody described herein with an IMiD described herein for use in treating cancer or tumors. One embodiment of the present invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody described herein with an IMiD described herein and a glucocorticosteroid described herein for use in treating cancer or tumors.

[0254] Another embodiment of the invention is a combination of an IMiD described herein with an anti-GPRC5D / anti-CD3 bispecific antibody described herein for use in the treatment of cancer or tumors. Another embodiment of the invention is a combination of an IMiD described herein with an anti-GPRC5D / anti-CD3 bispecific antibody described herein and a glucocorticosteroid described herein for use in the treatment of cancer or tumors.

[0255] A further embodiment is a combination of a glucocorticosteroid as described herein with an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and an IMiD as described herein for use in treating cancer or tumors.

[0256] 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 proliferative disorders that can be treated using the combination therapy of the present invention include, but are not limited to, neoplasms 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, breast, and genitourinary system. Precancerous conditions or lesions and cancer metastasis are also included. In some 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.

[0257] One embodiment of the present invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody described herein with an IMiD described herein for use in the treatment of any of the above-mentioned cancers or tumors. One embodiment of the present invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody described herein with an IMiD and a glucocorticoid described herein for use in the treatment of any of the above-mentioned cancers or tumors.

[0258] One embodiment of the present invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody described herein with an IMiD described herein for use in the treatment of multiple myeloma.One embodiment of the present invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody described herein with an IMiD and a glucocorticoid described herein for use in the treatment of multiple myeloma.

[0259] The invention includes a method for treating a patient in need of therapy, comprising administering to the patient a therapeutically effective amount of a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody described herein and an IMiD described herein. The invention further includes a method for treating a patient in need of therapy, comprising administering to the patient a therapeutically effective amount of a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody described herein, an IMiD described herein, and a glucocorticosteroid described herein.

[0260] The invention includes methods of treating cancer in an individual comprising administering to said individual a combination of an anti-GPRC5D / anti-CD3 bispecific antibody described herein and an IMiD described herein. The invention further includes methods of treating cancer in an individual comprising administering to said individual a combination of an anti-GPRC5D / anti-CD3 bispecific antibody described herein, an IMiD described herein, and a glucocorticosteroid described herein.

[0261] The invention includes methods for preventing or treating metastasis in a patient in need of therapy, comprising administering to the patient a therapeutically effective amount of a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody described herein and an IMiD described herein. The invention further includes methods for preventing and treating metastatic patients in need of therapy, comprising administering to the patient a therapeutically effective amount of a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody described herein, an IMiD described herein, and a glucocorticosteroid described herein.

[0262] The present invention includes the use of an anti-GPRC5D / anti-CD3 bispecific antibody according to the invention and an IMiD for the combination therapy described. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination therapy 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. In a further embodiment, the IMiD used in the above-mentioned combination therapy and medical applications is selected from the group of lenalidomide, pomalidomide, iberdomide and mezigdomide. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination therapy 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 IMiD is selected from the group of lenalidomide, pomalidomide, iberdomide and mezigdomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination treatments and medical applications comprises the polypeptide sequences of SEQ ID NOs: 26, 27, 28, and 29, and the IMiD is lenalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination treatments and medical applications comprises the polypeptide sequences of SEQ ID NOs: 26, 27, 28, and 29, and the IMiD is pomalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination treatments and medical applications comprises the polypeptide sequences of SEQ ID NOs: 26, 27, 28, and 29, and the IMiD is iveldomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination treatments and medical applications comprises the polypeptide sequences of SEQ ID NOs: 26, 27, 28, and 29, and the IMiD is mezigdomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination treatments and medical uses is formtamig and the IMiD is lenalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination treatments and medical uses is formtamig and the IMiD is pomalidomide.In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination treatments and medical uses is formtamig and the IMiD is iberdomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination treatments and medical uses is formtamig and the IMiD is mezigdomide.

[0263] The present invention includes the use of an anti-GPRC5D / anti-CD3 bispecific antibody according to the invention with an IMiD and a glucocorticosteroid for the combination therapy described.

[0264] In a preferred embodiment, the anti-GPRC5D / anti-CD3 bispecific antibodies used in the above-mentioned combination treatments and medical applications comprise the polypeptide sequences of SEQ ID NOs: 26, 27, 28, and 29. In a further embodiment, the IMiD used in the above-mentioned combination treatments and medical applications is selected from the group of lenalidomide, pomalidomide, iverdomide, and mezigdomide. In a further embodiment, the glucocorticoid used in the above-mentioned combination treatments and medical applications is dexamethasone. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibodies used in the above-mentioned combination treatments and medical applications comprise the polypeptide sequences of SEQ ID NOs: 26, 27, 28, and 29, and the IMiD used in the above-mentioned combination treatments and medical applications is selected from the group of lenalidomide, pomalidomide, iverdomide, and mezigdomide. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NOs: 26, 27, 28 and 29, wherein the IMiD is selected from the group of lenalidomide, pomalidomide, iverdomide and mezigdomide, and the glucocorticosteroid is dexamethasone. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NOs: 26, 27, 28 and 29, wherein the IMiD is lenalidomide, and the glucocorticosteroid is dexamethasone. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the above-mentioned combination treatments and medical uses is formtamig, the IMiD is lenalidomide, and the glucocorticosteroid is dexamethasone.

[0265] The present invention includes a combination of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein with an IMiD as described herein for use in the manufacture of a medicament for the treatment of cancer.The present invention includes a combination of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein with an IMiD as described herein and a glucocorticoid as described herein for use in the manufacture of a medicament for the treatment of cancer.

[0266] In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising an anti-GPRC5D / anti-CD3 bispecific antibody described herein and an IMiD described herein, formulated together with a pharmaceutically acceptable carrier. In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising an anti-GPRC5D / anti-CD3 bispecific antibody described herein, an IMiD described herein, and a glucocorticosteroid described herein, formulated together with a pharmaceutically acceptable carrier.

[0267] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption / resorption delaying agents, etc. Preferably, the carrier is suitable for injection or infusion.

[0268] The compositions of the present invention can be administered by a variety of methods known in the art. As one of skill in the art will recognize, the route and / or mode of administration will vary depending on the desired results.

[0269] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of 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, the carrier can be, for example, isotonic buffered saline.

[0270] Regardless of the selected route of administration, 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 general methods known to those skilled in the art.

[0271] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient (effective amount). Dosage may include ascending dosage cycles of the active ingredient. The term "dosage" refers to the amount, i.e., dose, and frequency of administration of the active ingredient. The term "escalating dosage cycle" refers to a treatment period in which the dosage of the active ingredient is gradually increased over the treatment period. This may be achieved by increasing the dose and / or increasing the frequency of administration of the active ingredient. Thus, in one embodiment, the dosage of an anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD described herein includes at least one ascending dosage cycle. In one embodiment, the dosage of an anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD described herein includes at least one ascending dosage cycle of the anti-GPRC5D / anti-CD3 bispecific antibody. In one embodiment, the dosage of the anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD described herein comprises at least one ascending dosage cycle of the anti-GPRC5D / anti-CD3 bispecific antibody. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody used in the combination treatments and medical uses described herein is administered in an effective amount, and the dosage comprises at least one ascending dosage cycle. In one embodiment, the formtamig used in the combination treatments and medical uses described herein is administered in an effective amount, and the dosage comprises at least one ascending dosage cycle. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the invention or its ester, salt, or amide employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, other drugs, compounds, and / or substances used in combination with the particular composition employed, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0272] The present invention includes a combination of an IMiD described herein and an anti-GPRC5D / anti-CD3 bispecific antibody described herein for use in the manufacture of a medicament for the treatment of cancer. The present invention includes a combination of an IMiD described herein, an anti-GPRC5D / anti-CD3 bispecific antibody described herein, and a glucocorticoid described herein for use in the manufacture of a medicament for the treatment of cancer. The present invention includes a combination of a glucocorticoid described herein, an anti-GPRC5D / anti-CD3 bispecific antibody described herein, and an IMiD described herein. In one embodiment, an anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of 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 the treatment of cancer according to the present invention is formtamig. In one embodiment, an IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is selected from the group consisting of lenalidomide, pomalidomide, iverdomide, and mezigdomide. In one embodiment, the glucocorticoid for use in the manufacture of a medicament for the treatment of 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 the treatment of 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 IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is selected from the group of lenalidomide, pomalidomide, iverdomide and mezigdomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of 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 IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is lenalidomide.In one embodiment, an anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of 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 an IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is pomalidomide. In one embodiment, an anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of 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 an IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is iveldomide. In one embodiment, an anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of 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 an IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is mezigdomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the present invention is formtamig and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is lenalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the present invention is formtamig and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is pomalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the present invention is formtamig and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is iberdomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the present invention is formtamig and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is mezigdomide.In one embodiment, an anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of 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, an IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is selected from the group of lenalidomide, pomalidomide, iverdomide and mezigdomide, and a glucocorticoid for use in the manufacture of a medicament for the treatment of cancer according to the present invention is dexamethasone. In one embodiment, an anti-GPRC5D / anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of 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, an IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is lenalidomide, and a glucocorticoid for use in the manufacture of a medicament for the treatment of 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 the treatment of cancer according to the present invention is formtamig, the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the present invention is lenalidomide, and the glucocorticoid for use in the manufacture of a medicament for the treatment of cancer according to the present invention is dexamethasone.

[0273] The present invention further provides the use of an anti-GPRC5D / anti-CD3 bispecific antibody according to the invention as described herein and an IMiD according to the invention as 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 as described herein and an IMiD according to the invention as described herein and a glucocorticosteroid according to the invention as described herein for the manufacture of a medicament, preferably together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.

[0274] 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) an IMiD described herein, optionally further comprising (c) a package insert comprising printed instructions directing the use of the combination therapy 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) an IMiD described herein, and (c) a glucocorticosteroid described herein, optionally further comprising (d) a package insert comprising printed instructions directing the use of the combination therapy as a method for treating the disease.

[0275] Furthermore, the kit may 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 an IMiD described herein; and, optionally, (c) a third container containing a composition, the composition comprising an additional cytotoxic or other therapeutic agent. The kit of this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or additionally, 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.

[0276] Furthermore, the kit may 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 an IMiD 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 may comprise an additional cytotoxic or other therapeutic agent. The kit of this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or additionally, 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. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0277] In one aspect, the invention provides a kit for treating a disease comprising (a) a container comprising an anti-GPRC5D / anti-CD3 bispecific antibody described herein, and (b) a package insert comprising instructions directing the use of the anti-GPRC5D / anti-CD3 bispecific antibody in combination therapy with an IMiD described herein as a method for treating the disease. In one aspect, the invention provides a kit for treating a disease comprising (a) a container comprising an anti-GPRC5D / anti-CD3 bispecific antibody described herein, and (b) a package insert comprising instructions directing the use of the anti-GPRC5D / anti-CD3 bispecific antibody in combination therapy with an IMiD described herein and a glucocorticosteroid as a method for treating the disease.

[0278] In another aspect, the invention provides a kit for treating a disease comprising (a) a container comprising an IMiD as described herein, and (b) a package insert comprising instructions directing the use of the IMiD in combination therapy with an anti-GPRC5D / anti-CD3 bispecific antibody as described herein as a method for treating the disease. In another aspect, the invention provides a kit for treating a disease comprising (a) a container comprising an IMiD as described herein, and (b) a package insert comprising instructions directing the use of the IMiD in combination therapy with an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and a glucocorticoid as a method for treating the disease.

[0279] In another aspect, the present invention provides a kit for treating 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 an IMiD as described herein as a method for treating the disease.

[0280] 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, said medicament for use in combination therapy with an IMiD as described herein, and optionally comprising a package insert comprising printed instructions directing the use of the combination treatment 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, said medicament for use in combination therapy with an IMiD as described herein and a glucocorticosteroid, and optionally comprising a package insert comprising printed instructions directing the use of the combination treatment as a method for treating the disease.

[0281] The term "method of treating," or equivalent terms, when applied to cancer, for example, refers to a procedure 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 the cancer cells or other lesions are actually eliminated, that the number of cells or disease is actually reduced, or that the symptoms of cancer or other disease are actually alleviated. In many cases, a method of treating cancer will have a low probability of success but will still be judged to induce an overall beneficial course of action, taking into account the patient's medical history and expected survival time.

[0282] The terms "administered in combination with" or "co-administration," or "combination therapy" or "combination treatment," refer to the administration of an anti-GPRC5D / anti-CD3 bispecific antibody described herein with an IMiD described herein and, optionally, a glucocorticosteroid, e.g., as separate formulations / dosage regimens (or as a single formulation / dosage regimen). Co-administration can be simultaneous or sequential in any order, although it is preferred that there is 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)) via continuous infusion or given orally. When all therapeutic agents are co-administered sequentially, the doses are split into two separate administrations administered on the same day, or one of the agents is administered on day 1 and the second on days 2 through 7, preferably days 2 through 4. Thus, in one embodiment, the term "sequentially" means within 7 days of the dose of the first component, preferably within 4 days of the dose of the first component; the term "concurrently" means at the same time point. The term "co-administration" with respect to a maintenance dose of an anti-GPRC5D / anti-CD3 bispecific antibody and / or an IMiD and / or optionally a glucocorticosteroid means that the maintenance doses can be co-administered simultaneously, e.g., weekly, provided that treatment cycles are appropriate for all drugs.

[0283] It will be appreciated that antibodies will be administered to a patient in a "therapeutically effective amount" (or simply "effective amount"), which is the amount of each compound or combination that will elicit the biological or medical effect in a tissue, system, animal or human that is desired by a researcher, veterinarian, physician or other clinician.

[0284] The amount and timing of co-administration will depend on the type (race, sex, age, weight, etc.) and condition of the patient being treated, as well as the severity of the disease or condition being treated. The anti-GPRC5D / anti-CD3 bispecific antibody and / or IMiD and / or glucocorticosteroid are suitably co-administered to the patient, for example, on the same day or on subsequent days or every other week, once or over a series of treatments, as needed.

[0285] Those skilled in the art will readily recognize that in many cases, combination therapy may not provide a cure but may provide partial benefit. In some embodiments, any physiological change that has any benefit is also considered therapeutically beneficial. Thus, in some embodiments, the amount of a therapeutic combination that produces a physiological change is considered an "effective amount" or a "therapeutically effective amount."

[0286] Aspects of the present invention: Aspect 1. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an immunomodulatory imid drug (IMiD) for use as a combination therapy in the treatment of cancer.

[0287] Aspect 2. Use of an anti-GPRC5D / anti-CD3 bispecific antibody in combination with an immunomodulatory imid drug (IMiD) in the manufacture of a medicament for the treatment of cancer.

[0288] Embodiment 3. A method of treating cancer in an individual, comprising administering to the individual an anti-GPRC5D / anti-CD3 bispecific antibody in combination with an immunomodulatory imid drug (IMiD).

[0289] Embodiment 4. A kit comprising a first medicament comprising an anti-GPRC5D / anti-CD3 bispecific antibody and a second medicament comprising an immunomodulatory imid drug (IMiD), optionally further comprising a package insert comprising instructions for administering the first medicament in combination with the second medicament to treat cancer in an individual.

[0290] Embodiment 5. 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. 5. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit according to any one of aspects 1 to 4, comprising:

[0291] Embodiment 6. The anti-GPRC5D / anti-CD3 bispecific antibody comprises: (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) 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. 6. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit according to any one of aspects 1 to 5, comprising:

[0292] Aspect 7. An anti-GPRC5D / anti-CD3 bispecific antibody for use in combination with an IMiD, use, method or kit according to aspect 5 or 6, wherein the first and / or second antigen-binding moiety of the anti-GPRC5D / anti-CD3 bispecific antibody is a Fab molecule.

[0293] Embodiment 8. An anti-GPRC5D / anti-CD3 bispecific antibody for use in combination with an IMiD according to any one of embodiments 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 and Fab heavy chains, in particular the variable domains VL and VH, are replaced by each other.

[0294] Embodiment 9. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit according to any one of embodiments 5 to 8, wherein the first antigen-binding moiety is a Fab molecule in which in the constant domains 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 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).

[0295] Embodiment 10. An anti-GPRC5D / anti-CD3 bispecific antibody for use in combination with an IMiD according to any one of embodiments 5 to 9, use, method or kit, wherein the first antigen-binding portion and the second antigen-binding portion are fused to each other, optionally via a peptide linker.

[0296] Embodiment 11. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit according to any one of embodiments 5 to 10, wherein 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.

[0297] Embodiment 12. An anti-GPRC5D / anti-CD3 bispecific antibody for use in combination with an IMiD, use, method or kit according to embodiment 1 to 11, wherein the anti-GPRC5D / anti-CD3 bispecific antibody comprises a third antigen-binding moiety.

[0298] Embodiment 13. An anti-GPRC5D / anti-CD3 bispecific antibody for use in combination with an IMiD, use, method or kit according to embodiment 12, wherein the third antigen-binding portion is identical to the first antigen-binding portion.

[0299] Aspect 14. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit 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.

[0300] Embodiment 15. The first, second, and, if present, third antigen-binding moieties 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; a third antigen-binding portion, if present, fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain; 15. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit according to aspects 5 to 14.

[0301] Embodiment 16. An anti-GPRC5D / anti-CD3 bispecific antibody for use, in combination with an IMiD, use, method or kit according to embodiment 14 or 15, wherein the Fc domain is an IgG Fc domain.

[0302] Embodiment 17. An anti-GPRC5D / anti-CD3 bispecific antibody for use, in combination with an IMiD, use, method or kit according to any one of embodiments 14 to 16, wherein the Fc domain is an IgG1 Fc domain.

[0303] Embodiment 18. An anti-GPRC5D / anti-CD3 bispecific antibody for use, in combination with an IMiD, use, method or kit according to any one of embodiments 14 to 17, wherein the Fc domain is a human Fc domain.

[0304] Embodiment 19. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit according to any one of embodiments 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 creating a protuberance in the CH3 domain of the first subunit that can be positioned in 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 creating a cavity in the CH3 domain of the second subunit that can be positioned in the protuberance in the CH3 domain of the first subunit.

[0305] Aspect 20. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit 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 reduce effector function.

[0306] Aspect 21. An anti-GPRC5D / anti-CD3 bispecific antibody for use in combination with an IMiD, use, method or kit according to any one of aspects 1 to 19, wherein 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.

[0307] Aspect 22. An anti-GPRC5D / anti-CD3 bispecific antibody for use, in combination with an IMiD, use, method or kit according to any one of aspects 1 to 21, wherein the IMiD is a first generation IMiD or a cereblon E3 ligase modulator (CELMoD).

[0308] Aspect 23. An anti-GPRC5D / anti-CD3 bispecific antibody for use in combination with an IMiD, use, method or kit according to any one of aspects 1 to 22, wherein the IMiD is selected from the group consisting of lenalidomide, pomalidomide and iverdomide.

[0309] Embodiment 24. An anti-GPRC5D / anti-CD3 bispecific antibody for use, in combination with an IMiD, use, method or kit according to any one of embodiments 1 to 23, wherein the combination further comprises a glucocorticosteroid.

[0310] Aspect 25. An anti-GPRC5D / anti-CD3 bispecific antibody for use in combination with an IMiD, use, method or kit according to aspect 24, wherein the glucocorticosteroid is dexamethasone.

[0311] Amino acid sequence TIFF2025532806000006.tif222170TIFF2025532806000007.tif252170TIFF2025532806000008.tif248170 [Example]

[0312] The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0313] 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 housed in the Roche Innovation Center Munich animal facility transfected with human CD34 +Mice were delivered 14–20 weeks after hematopoietic stem cell transplantation. After arrival, animals were maintained for one week for acclimatization and observation. Mice were maintained under specific pathogen-free conditions with a 12-hour light / 12-hour dark cycle daily in accordance with relevant guidelines (GV-Solas; Felasa; TierschG). Continuous health monitoring was performed regularly. The experimental study protocol was reviewed and approved by the local authorities (ROB-55.22532.Vet_03-16-10 or ROB-55.2-2532.Vet_03-20-170). To evaluate therapeutic effects on established multiple myeloma tumors, humanized NSG mice were subcutaneously implanted with human tumor cell lines. Tumor cell lines were obtained from various sources, expanded, and deposited in Roche Munich's in-house cell bank (Table 1). All tumor cells were cultured at 37°C in a water-saturated atmosphere of 5% CO2 and co-injected into the right flank of anesthetized humanized NSG mice with 50 μl of Matrigel at different cell numbers and >90% viability (Table 1). Subcutaneous tumors were grown to 200–300 mm2. 3 When the mean tumor volume reached 180 mm, the humanized mice were randomized into different treatment groups based on tumor volume and body weight. For the evaluation of the combination of GPRC5D-TCB and mezigdomide, animals were randomized to receive treatment with subcutaneous tumors of 180 mm. 3When the mean tumor volume reached 1000 mg / kg, animals were randomized into eight different treatment groups (Table 3). At randomization, animals were treated with GPRC5D-TCB (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29; disclosed in WO 2021 / 018859; RO7425781, formtamig or "formim"), a standard of care (SoC) agent for the treatment of multiple myeloma, either as monotherapy or in combination with an IMiD. Additionally, the addition of dexamethasone (Dex) to the combination of GPRC5D-TCB and lenalidomide was investigated. The treatment schedule, dose, and route of administration for each treatment are summarized in Tables 2 and 3. All treatments were prepared fresh before injection. Animals were controlled daily for clinical symptoms and adverse effects. Animal termination criteria were visible illness (dirty fur, hunched back, respiratory failure, impaired movement), >20% weight loss, or tumor size. Tumor growth was monitored twice weekly using caliper measurements. To quantify tumor-infiltrating lymphocytes, in some experiments investigating GPRC5D-TCB in combination with lenalidomide (Len), pomalidomide, and iverdomide, tumors from scout animals were harvested, and single-cell suspensions were subjected to flow cytometry using a FACSFortessa device and FlowJo software. To quantify and characterize peripheral immune cells in animals treated with the combination of GPRC5D-TCB and mezigdomide (Mezi), whole blood was collected and processed for flow cytometry with red blood cell lysates. Flow cytometry was performed using a Cytek Aurora spectrophotometer 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 the results of ImmunoPD, tumor volumes or cytokine levels between different treatment groups were subjected to one-way ANOVA analysis (Tukey's test) corrected for multiple comparisons.

[0314] Table 1: Tumor cell lines TIFF2025532806000009.tif98170

[0315] Table 2: Summary of treatment schedule, doses and routes of administration TIFF2025532806000010.tif248170TIFF2025532806000011.tif40170

[0316] Table 3: Experimental groups for evaluation of the combination of GPRC5D-TCB and mezigdomide TIFF2025532806000012.tif214170

[0317] result Combination of GPRC5D-TCB and immunomodulatory drug (IMiD) First-generation IMiDs, such as lenalidomide and pomalidomide, are approved first-line treatments for patients with multiple myeloma. 1When combined with low-dose GPRC5D-TCB therapy against OPM-2 xenografts in humanized mice, lenalidomide exhibited strong synergistic antitumor activity, confirmed by tumor growth control, and statistically significant reductions in tumor burden compared with monotherapy (Figure 1A and B). Furthermore, the combination with lenalidomide significantly increased the number of intratumoral T cells compared with GPRC5D-TCB monotherapy, demonstrating the synergistic mode of action of both drugs (Figure 1C). IMiDs are frequently combined with dexamethasone for the treatment of multiple myeloma. Using the low-response multiple myeloma tumor model, KMS-12BM, GPRC5D-TCB was combined with lenalidomide and with lenalidomide plus dexamethasone. Compared with the control group, the combination of lenalidomide and GPRC5D-TCB induced statistically significant tumor growth inhibition in a difficult-to-treat xenograft model of multiple myeloma (Figures 2A and 2B). Notably, even more robust efficacy was observed when dexamethasone was added to the GPRC5D-TCB and lenalidomide combination, supporting the clinical development of GPRC5D-TCB using this SoC backbone (Figures 2A and 2B). Despite the highly potent monotherapy response, a high percentage of NCI-H929 xenograft tumors relapsed under GPRC5D-TCB monotherapy (Figures 3A and 5B). When combined with pomalidomide, the frequency of relapsed mice was dramatically reduced (Figures 3A and 5C). The improved efficacy correlated with elevated cytokine levels detected in mouse serum 48 hours after the first TCB and 24 hours after the first pomalidomide injection, indicating enhanced immune activation in the combination (Figures 3B, 3C, and 3D). Interestingly, the combination with pomalidomide did not induce complete tumor eradication, as evidenced by the appearance of tumor growth in individual animals upon cessation of treatment (Figures 3A and 5C).

[0318] Iberdomide belongs to a novel class of IMiDs called "cereblon E3 ligase modulators" (CELMoDs), which are currently being evaluated in early clinical development. Iberdomide prevented NCI-H929 tumor recurrence in response to GPRC5D-TCB monotherapy (Figures 4A and 5D) and induced a stronger boosted T cell response compared to pomalidomide, as confirmed by higher cytokine levels in the combination (Figures 4B, 4C, and 4D). Combination with iberdomide, but not pomalidomide, induced a complete tumor response in humanized mice, as highlighted by the lack of tumor regrowth upon treatment cessation (Figure 5D).

[0319] To evaluate the combination of GPRC5D-TCB and mezigdomide in a clinically relevant in vivo setting, humanized mice bearing NCI-H929 tumors were treated once weekly (1 q7d or q7d) with GPRC5D-TCB for a fixed duration using escalating doses (cycle 1, C1) of 0.0005 mg / kg (escalating dose 1 on day 1; C1D1), 0.002 mg / kg (escalating dose 2 on day 8; C1D8), and 0.04 mg / kg (escalating dose 3 on day 15; C1D15), followed by five cycles (C2–C6) of maintenance doses at 0.04 mg / kg, with a treatment-free follow-up of >2 weeks (Figure 6). Mezigdomide was administered 24 hours after each GPRC5D-TCB injection at 3 mg / kg or 1 mg / kg once weekly (q7d), three times weekly (3q7d), or five times weekly (5q7d). Although GPRC5D-TCB induced transient tumor shrinkage after escalating doses 1 (C1D1) and 3 (C1D15), mice showed progressive disease at the end of cycle 1 (C1), with a progression-free survival (PFS) rate of only 20% at the end of the study (Figure 6B). In contrast, the combination with mezigdomide administered at 3q7d and 5q7d induced rapid onset of tumor shrinkage during C1, which correlated with significantly improved PFS rates of 80% (3q7d; Figure 6D) and 100% (5q7d; Figure 6C) at the 3 mg / kg dose and 60% (3q7d; Figure 6G) and 90% (5q7d; Figure 6F) at the 1 mg / kg dose, respectively. Mezigdomide administered once weekly (1q7d) did not improve the efficacy of GPRC5D-TCB during escalation doses 1 (C1D1) and 2 (C1D8), but achieved a deepening of response after the target dose administration on day 15 of cycle 1, particularly at 3 mg / kg (C1D15; Figure 6H). Correlating with the shallower depth of early response, the PFS rate of the 1q7d combination with mezigdomide was not improved compared with GPRC5D-TCB monotherapy (Figure 6E).To investigate the effect of the combination with mezigdomide on immune activation, cytokine release was measured in the serum of all mice 48 hours after each escalating injection of GPRC5D-TCB at C1D1, C1D8, and C1D15 and 24 hours after mezigdomide administration (Figure 7).When mezigdomide was administered 5q7d or 3q7d, serum levels of IL-10 and IP-10 were comparable to those of GPRC5D-TCB monotherapy (Figures 7B and 7C), and IL-2 levels were slightly elevated at all three time points (Figure 7A). Interestingly, a different trend was observed for MIP-1, with serum levels decreasing in correlation with the number of mezigdomide administrations (Figure 7D). In contrast to the 5q7d and 3q7d schedules, reducing the dosing frequency of mezigdomide (1q7d) induced a robust increase in IL-2, IP-10, and MIP-1a after administration of the target dose at C1D15 (Figures 7A, 7B, and 7D). To perform quantitative and phenotypic analysis of circulating immune cells, we collected blood from all animals at the end of cycle 3 (C4 re-dose) and cycle 5 (C6 re-dose) and performed spectral flow cytometry. Compared to monotherapy, we found that mezigdomide significantly increased peripheral CD8a expression at doses of 3 mg / kg 3 q7d and 1 mg / kg 5 q7d. + (CD8α-positive cells) and general CD4 + The reduction in T cell counts was observed with 1 mg / kg 1q7d dosing (CD8a + and CD4 + ) and 1 mg / kg (CD4 + We observed an increase in the number of regulatory T cells (Tregs) following 3q7d dosing with 3 mg / kg mezigdomide (Figures 8A and 8D). The number of regulatory T cells (Tregs) was slightly decreased following 3q7d dosing with 3 mg / kg mezigdomide and significantly increased following 3q7d dosing with 1 mg / kg mezigdomide (Figure 8B). The number of peripheral B cells was clearly decreased following 3q7d and 5q7d dosing, regardless of the mezigdomide dose level (Figure 8C). In contrast, reducing the dosing frequency of mezigdomide induced a significant increase in the number of not only B cells but also NK cells following C4 re-dosing, especially following C6 re-dosing (Figures 8C and 8E). We next evaluated whether the combination of GPRC5D-TCB and mezigdomide induced a change in the depletion status of circulating T lymphocytes. Compared to GPRC5D-TCB monotherapy, we found that when mice were treated with mezigdomide at 5q7d and 3q7d, but not at 1q7d, LAG3- and TIGIT-positive CD4 + and CD8a +Higher frequencies of T cells were observed (Figures 9A, 9B, 9C, and 9D), indicating that repeated treatment with mezigdomide induces T cell depletion.

[0320] Taken together, our data suggest that the combination of GPRC5D-TCB with mezigdomide significantly improves PFS rates in patients with multiple myeloma. The addition of mezigdomide to GPRC5D-TCB induced profound responses at early time points and enabled patients to overcome tumor recurrence at later time points. The cytokine data indicate that the combination of escalating doses of GPRC5D-TCB with mezigdomide does not pose a major risk factor for the development or exacerbation of cytokine release syndrome (CRS).

[0321] References: 1.Raza S, Safyan RA, Lentzsch S.Immunomodulatory Drugs(IMiDs) in Multiple Myeloma.Curr Cancer Drug Targets.2017;17(9):846-857.doi:10.2174 / 1568009617666170214104426.PMID:28201976.

[0322] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but such descriptions and illustrations 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 entireties.

Claims

1. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an immunomodulatory imid drug (IMiD) for use as a combination therapy in the treatment of cancer.

2. 10. Use of an anti-GPRC5D / anti-CD3 bispecific antibody in combination with an immunomodulatory imidrug (IMiD) in the manufacture of a medicament for the treatment of cancer.

3. A method of treating cancer in an individual, comprising administering to the individual an anti-GPRC5D / anti-CD3 bispecific antibody in combination with an immunomodulatory imid drug (IMiD).

4. 1. A kit comprising a first medicament comprising an anti-GPRC5D / anti-CD3 bispecific antibody and a second medicament comprising an immunomodulatory imid drug (IMiD), optionally further comprising a package insert comprising instructions for administering the first medicament in combination with the second medicament 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.

5. The anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit according to any one of claims 1 to 4, comprising:

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 (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.

6. The anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit according to any one of claims 1 to 5, comprising:

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

8. 8. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit 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. The anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit 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 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 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).

10. 10. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit 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 via a peptide linker.

11. 11. The anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit according to any one of claims 5 to 10, wherein 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.

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

14. 14. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit 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, second, and, if present, third antigen-binding moieties are each Fab molecules; (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; a third antigen-binding moiety, if present, fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain; 15. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit according to any one of claims 5 to 14.

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

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

18. 18. An anti-GPRC5D / anti-CD3 bispecific antibody for use, in combination with an IMiD, use, method or kit 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 in combination with an IMiD for use, use, method or kit 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 replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance in the CH3 domain of the first subunit that can be positioned in 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 creating a cavity in the CH3 domain of the second subunit that can be positioned in the protuberance in the CH3 domain of the first subunit.

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

21. 21. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit according to any one of claims 1 to 20, wherein 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. An anti-GPRC5D / anti-CD3 bispecific antibody in combination with an IMiD for use, use, method or kit 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 in combination with an IMiD for use, use, method or kit according to any one of claims 1 to 22, wherein the IMiD is a first generation IMiD or a cereblon E3 ligase modulator (CELMoD).

24. 24. An anti-GPRC5D / anti-CD3 bispecific antibody for use, use, method or kit in combination with an IMiD according to any one of claims 1 to 23, wherein the IMiD is selected from the group consisting of lenalidomide, pomalidomide, iberdomide and mezigdomide.

25. 25. An anti-GPRC5D / anti-CD3 bispecific antibody for use, use, method or kit in combination with an IMiD according to any one of claims 1 to 24, wherein the combination further comprises a glucocorticosteroid.

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

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