Combination therapy of GPRC5d tcb and imid

Abstract

The present invention relates to a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody and an IMiD. The combination therapy may further comprise a glucocorticoid.

Description

Technical Field

[0001] The present invention relates to a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD). Glucocorticoids may be added to the combination therapy. Background Art

[0002] Multiple myeloma (MM) is one of the most common hematological malignancies, affecting about 75,000 new patients each year in the European Union and the United States, but its medical needs are still unmet. Multiple myeloma is characterized by terminally differentiated plasma cells secreting non-functional monoclonal immunoglobulins. In the short term, immunomodulatory drugs (such as lenalidomide and pomalidomide) and proteasome inhibitors (such as carfilzomib or bortezomib) may still be the mainstay of first-line therapy for multiple myeloma (Moreau, P. and SV Rajkumar, multiple myeloma-translation of trial results into reality. Lancet, 2016. 388 (10040): 111-3 pages). However, these drugs are not specifically directed to diseased tumor cells, such as diseased plasma cells (PC). A lot of efforts have been made in selectively exhausting plasma cells in multiple myeloma. The lack of surface proteins that specifically mark plasma cells has hampered the development of antibody or cell therapies for multiple myeloma. So far, there are few successful cases of biologics, including daratumumab (anti-CD38) and elotuzumab (anti-CD319), and it should be noted that these two molecules are not exclusively expressed by plasma cells. Therefore, RNA sequencing was used to identify novel targets from plasma cells in multiple myeloma, such as G protein-coupled receptor class C group 5 member D (GPRC5D), which is differentially expressed by plasma cells in multiple myeloma and plasma cells from healthy donors. It has been reported that GPRC5D is associated with the prognosis and tumor load of patients with multiple myeloma (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): 953-60; and Cohen, Y., et al., GPRC5D is a promising marker for monitoring the tumour load and to target multiple myeloma cells. Hematology, 2013. 18(6): 348-51).

[0003] GPRC5D is an orphan receptor with no known ligands, and its biological properties in general men and in cancer in particular are largely unknown. The GPRC5D encoding gene is located on chromosome 12p13.3, contains three exons, and spans about 9.6 kb (Brauner-Osborne, H., et al., Cloning and characterization of ahuman orphan family C G-protein coupled receptor GPRC5D. Biochim Biophys Acta, 2001. 1518 (3): p. 237-48). The first large exon encodes seven transmembrane domains. GPRC5D has been shown to be involved in the formation of keratin 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): 565-73).

[0004] WO 2018 / 017786 A2 and WO 2021 / 018859 A1 disclose GPRC5D-specific antibodies or antigen-binding fragments that bind to GPRC5D on target cells and activated T cell antigens (such as CD3) on T cells. When such an antibody binds to its two targets at the same time, a T cell synapse will be formed, resulting in the activation of (cytotoxic) T cells and the subsequent lysis of target cells. Compared with CAR-T cell therapy, T cell bispecific antibodies (TCBs) have become a new treatment option for patients with relapsed refractory myeloma (RRMM) based on their promising objective response rate (ORR), good safety profile and ready availability (vande Donk, NWCJ et al., T-cell redirecting bispecific and trispecific antibodies in multiple myeloma beyond BCMA. Curr Opin Oncol, 2023.35:000–000). Although TCBs targeting BCMA and GPRC5D have been reported to induce deep clinical responses, 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).

[0005] Considering that all standard of care treatments are incurable for multiple myeloma patients, there is a clear need to develop potent and specific new therapies. Accordingly, the present invention provides a combination of an anti-GPRC5D / anti-CD3 bispecific antibody with an IMiD and optionally a glucocorticoid. Summary of the invention

[0006] In a first aspect, the present invention provides a combination of an anti-GPRC5D / anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD) for use as a combination therapy for treating cancer. In a further aspect, the present invention provides a combination of an anti-GPRC5D / anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD) for use in the manufacture of a medicament for treating cancer. In a further aspect, the present invention provides a method for treating cancer in an individual, the method comprising administering a combination of an anti-GPRC5D / anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD) to the individual. On the other hand, the present invention provides a medicine box comprising a first drug containing an anti-GPRC5D / anti-CD3 bispecific antibody and a second drug containing an immunomodulatory imide drug (IMiD), and optionally further comprising a package insert comprising instructions for administering a combination of the first drug and the second drug for treating cancer in an individual.

[0007] In embodiments of any 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 complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 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 complementary determining region (HCDR) 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20, and a light chain variable region (VL) comprising SEQ ID NO: 21. Light chain complementarity determining region (LCDR) 1 of SEQ ID NO:21, LCDR 2 of SEQ ID NO:22, and LCDR3 of SEQ ID NO:23. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody comprises (i) a first antigen binding portion that specifically binds to GPRC5D, the first antigen binding portion comprising a VH and a VL, the VH being at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and the VL being 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 to CD3, the second antigen binding portion comprising a VH and a VL, the VH being at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24, and the VL being at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the first antigen binding moiety and / or the second antigen binding moiety of the anti-GPRC5D / anti-CD3 bispecific antibody is a Fab molecule. In a further embodiment, the second antigen binding moiety is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are replaced with each other.In one embodiment, the first antigen binding moiety is a Fab molecule, wherein in the constant domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to 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) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index numbering). In another embodiment, the first antigen binding moiety and the second antigen binding moiety are fused to each other, optionally fused to each other via a peptide linker. In yet another embodiment, the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule, and 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, or (ii) the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety.

[0008] In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody according to any of the above aspects comprises a third antigen binding portion. In a further embodiment, the third antigen binding portion is the same as the first antigen binding portion. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody comprises an Fc domain consisting of a first subunit and a second subunit. In one aspect, the first antigen binding moiety, the second antigen binding moiety, and the third antigen binding moiety, when present, are each a Fab molecule; and 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 wherein 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 when present. In one embodiment, the Fc domain is an IgG Fc domain. In one embodiment, the Fc domain is an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain.

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

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

[0011] In one embodiment, the IMiD of any of the above aspects is a first generation IMiD or a Cereblon E3 ligase modulator (CELMoD). In one embodiment, the IMiD is selected from lenalidomide, pomalidomide, iberdomide and mezigdomide.

[0012] In another aspect, the combination as described in any one of the above aspects further comprises a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A , Figure 1B and Figure 1C : Results of the efficacy / PD trial evaluating GPRC5D-TCB as a single agent and in combination with lenalidomide. ( Figure 1A ) Multiple myeloma cell line OPM-2 was injected subcutaneously into stem cell humanized NSG mice to study tumor growth inhibition. GPRC5D-TCB was injected intravenously at 0.05 mg / kg once a week and lenalidomide was administered daily using oral gavage at 20 mg / kg, and tumor growth was compared over a period of 18 days. ( Figure 1B ) Tumor burden in individual mice was assessed at the end of the study (day 18). Figure 1C) Tumors of 5 scout mice per group were harvested 48 hours after the second GPRC5D-TCB injection, and the number of intratumoral T cells was assessed by flow cytometry. Statistical analysis, ordinary one-way ANOVA, Tukey 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).

[0014] Figure 2A and Figure 2B : Results of an efficacy experiment 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. Figure 2A ) 1 mg / kg of GPRC5D-TCB was injected intravenously once a week, and 20 mg / kg of lenalidomide (with or without 2 mg / kg of oral dexamethasone) was administered in combination daily using oral gavage. ( Figure 2B ) Tumor burden of individual mice was assessed on day 35 (end of study). Statistical analysis, general one-way ANOVA, Tukey 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).

[0015] Figure 3A , Figure 3B , Figure 3C and Figure 3D : Results of an efficacy experiment 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. Figure 3A ) Growth of NCI-H929 xenograft tumors after weekly intravenous administration of 0.1 mg / kg of GPRC5D-TCB and daily oral gavage of 10 mg / kg of pomalidomide. Using multiplex techniques, IFN-γ ( Figure 3B )、IL-2( Figure 3C ) and TNF-α( Figure 3D ) level. Statistical analysis, ordinary one-way ANOVA, Tukey 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).

[0016] Figure 4A , Figure 4B , Figure 4C and Figure 4D : Results of an efficacy experiment evaluating GPRC5D-TCB as a single agent and in combination with ibrolimumab against NCI-H929 multiple myeloma tumors implanted subcutaneously in stem cell humanized NSG mice. Figure 4A ) Growth of NCI-H929 xenograft tumors after weekly intravenous administration of 0.1 mg / kg of GPRC5D-TCB and daily oral gavage of 10 mg / kg of ibodomide. Using multiplex techniques, IFN-γ ( Figure 4B )、IL-2( Figure 4C ) and TNF-α( Figure 4D ) level. Statistical analysis, ordinary one-way ANOVA, Tukey 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).

[0017] Figure 5A , Figure 5B , Figure 5C and Figure 5D : Use solvent ( Figure 5A ), GPRC5D-TCB monotherapy ( Figure 5B ) or with pomalidomide ( Figure 5C ) or ibrolimumab ( Figure 5D ) in combination with , are depicted in Figures 3 and 4, respectively.

[0018] Fig. 6A , Figure 6B , Figure 6C , Fig.6D , Fig. 6E , Fig. 6F , Figure 6G and 6H Results of an efficacy experiment evaluating GPRC5D-TCB as a single agent and in combination with high or low doses of mezigamide against NCI-H929 multiple myeloma tumors implanted subcutaneously in stem cell humanized NSG mice. NCI-H929 tumor growth in individual mice after weekly subcutaneous (sc) injection of vehicle ( Fig. 6A ); GPRC5D-TCB (using 0.0005 mg / kg–0.002 mg / kg–0.04 mg / kg escalation followed by maintenance at 0.04 mg / kg) Figure 6B); and GPRC5D-TCB and 5 days a week (5q7d; Figure 6C ), 3 days (3q7d; Fig.6D ), 1 day (1q7d; Fig. 6E )3 mg / kg or 5 days a week (5q7d; Fig. 6F ), 3 days (3q7d; Figure 6G ), 1 day (1q7d; Figure 6H )1 mg / kg of mezigamide was administered in combination.

[0019] Fig. 7A , Figure 7B , Figure 7C and Fig.7D : Results of cytokine analysis performed in blood NCI-H929 implanted stem cell humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with mezigamide. Using multiplex technology, the cytokine IL-2 (IL-23) was measured in mouse serum 48 hours after administration of GPRC5D-TCB on Day 1 of Cycle 1 (C1D1, 0.0005 mg / kg), Day 8 of Cycle 1 (C1D8, 0.002 mg / kg), and Day 15 of Cycle 1 (C1D15, 0.04 mg / kg) and 24 hours after administration of 3 mg / kg or 1 mg / kg of mezigamide. Fig. 7A )、IP-10( Figure 7B )、IL-10( Figure 7C ) and MIP-1a( Fig.7D ).

[0020] Fig. 8A , Figure 8B , Figure 8C , Fig.8D and Fig. 8E : Presents the results of quantitative flow cytometric analysis performed in blood NCI-H929 implanted stem cell humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with mezigamide. Using flow cytometry, CD8a was quantified in the blood of mice 164 hours after administration of 0.04 mg / kg of GPRC5D-TCB in cycle 3 (= before C4 administration) and cycle 5 (= before C6 administration) and 48 hours (5q7d), 96 hours (3q7d) or 144 hours (1q7d) after administration of 3 mg / kg or 1 mg / kg of mezigamide. + T cells ( Fig. 8A ), regulatory T cells ( Figure 8B ), B cells ( Figure 8C ), conventional CD4 + cell( Fig.8D ) and NK cells ( Fig. 8E ).

[0021] Fig.9A , Fig. 9B , Fig. 9C and Fig.9D :The results of phenotypic flow cytometric analysis performed in blood NCI-H929 implanted stem cell humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with mezigamide are presented. Using flow cytometry, the phenotype of circulating immune cells was analyzed in the blood of mice 164 hours after administration of 0.04 mg / kg of GPRC5D-TCB in cycle 3 (= before C4 administration) and cycle 5 (= before C6 administration) and 48 hours (5q7d), 96 hours (3q7d) or 144 hours (1q7d) after administration of 3 mg / kg or 1 mg / kg of mezigamide. Expression of TIGIT ( Fig.9A ) and Lag3( Fig. 9B ) + T cells and TIGIT expression Fig. 9C ) and Lag3( Fig.9D ) of conventional CD4 + The percentage of T cells. DETAILED DESCRIPTION

[0022] definition

[0023] Unless otherwise defined below, the terms used herein are generally as used in the art.

[0024] As used herein, the term "antigen binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and their derivatives, such as fragments thereof.

[0025] The term "bispecific" refers to an antigen binding molecule that is able to specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for different antigenic determinants. In certain embodiments, a bispecific antigen binding molecule is able to simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two unique cells.

[0026] The term "valency" as used herein means that there is a specified number of antigen binding sites in an antigen binding molecule. Thus, the term "monovalently binds to an antigen" means that there is one (and no more than one) antigen binding site specific for an antigen in an antigen binding molecule.

[0027] "Antigen binding site" refers to the site of an antigen binding molecule that provides interaction with an antigen, i.e., one or more amino acid residues. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining region (CDR). Natural immunoglobulin molecules typically have two antigen binding sites, and Fab molecules typically have a single antigen binding site.

[0028] As used herein, the term "antigen binding portion" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen binding portion is capable of directing the entity to which it is attached (e.g., a second antigen binding portion) to a target site, such as to a specific type of tumor cell carrying an antigenic determinant. In another embodiment, the antigen binding portion is capable of activating signal transduction by its target antigen (e.g., a T cell receptor complex antigen). The antigen binding portion includes antibodies and fragments thereof as further defined herein. A specific antigen binding portion includes an antigen binding domain of an antibody, which includes an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding portion may include an antibody constant region as further defined herein and known in the art. Available heavy chain constant regions include any of the following five isotypes: α, δ, ε, γ, or μ. Available light chain constant regions include any of the following two isotypes: κ and λ.

[0029] 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 stretch of continuous amino acids or a conformational configuration consisting of different regions of non-continuous amino acids), to which an antigen-binding portion binds, thereby forming an antigen-binding portion-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, in free matter in serum and / or in the extracellular matrix (ECM). Unless otherwise indicated, proteins (e.g., GPRC5D, CD3) as antigens described herein refer to any natural protein form 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 a specific embodiment, the antigen is a human protein. When referring to a specific protein herein, the term encompasses "full-length", unprocessed proteins, and any form of protein produced by intracellular processing. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants. Exemplary human proteins that can be used as antigens are CD3, in particular the epsilon subunit of CD3 (see UniProt No. P07766 (Release 185), NCBI RefSeq No. NP_000724.1, SEQ ID NO: 4 for human sequences; or UniProt No. Q95LI5 (Release 69), NCBI GenBank No. BAB71849.1, SEQ ID NO: 5 for cynomolgus monkey [Macaca fascicularis] sequences) or GPRC5D (see UniProt No. Q9NZD1 (Release 115), NCBI RefSeq No. NP_061124.1, SEQ ID NO: 9 for human sequences). In certain embodiments, the bispecific antigen binding molecule binds to an epitope of CD3 or GPRC5D that is conserved among CD3 or GPRC5D antigens from different species. In specific embodiments, the bispecific antigen binding molecule binds to human GPRC5D.

[0030] By "specific binding" is meant that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding portion to bind to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance (SPR) technology (e.g., analysis on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree of binding of an antigen binding portion to an unrelated protein is less than about 10% of the degree of binding of the antigen binding portion to the antigen, as measured, for example, by SPR. In certain embodiments, the dissociation constant (K) of an antigen binding portion or an antigen binding molecule comprising the antigen binding portion that binds to an antigen is measured. D ) is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or lower, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M).

[0031] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to 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 can generally be expressed in terms of the dissociation constant (K D ) indicates that the dissociation constant is the sum of the dissociation rate constant and the association rate constant (k off and k on ). Thus, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0032] "Decreased binding" (e.g., reduced binding to an Fc receptor) refers to a decrease in affinity for the corresponding interaction, as measured, for example, by SPR. For clarity, the term also includes a decrease in affinity to zero (or below the detection limit of the analytical method), i.e., complete elimination of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the corresponding interaction.

[0033] As used herein, "activating T cell antigen" refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, which can induce T cell activation when interacting with an antigen binding molecule. Specifically, the interaction of an antigen binding molecule with an activating T cell antigen can induce T cell activation by triggering the signal transduction cascade of the T cell receptor complex. In a specific embodiment, the activating T cell antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt number P07766 (version 144), NCBI RefSeq number NP_000724.1, SEQ ID NO: 4 for human sequences; or UniProt number Q95LI5 (version 49), NCBI GenBank number BAB71849.1, SEQ ID NO: 5 for cynomolgus monkey [Macaca fascicularis] sequence).

[0034] 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.

[0035] As used herein, "target cell antigen" refers to an antigenic determinant present on the surface of a target cell, such as a cell in a tumor (such as a cancer cell or a cell of a tumor stroma). In a specific embodiment, the target cell antigen is GPRC5D, in particular human GPRC5D according to SEQ ID NO:9.

[0036] As used herein, the terms "first", "second" or "third" with respect to Fab molecules, etc., are used for convenience in distinguishing when there is more than one of each type of part. Unless explicitly stated, the use of these terms is not intended to confer a particular order or orientation to the bispecific antigen binding molecules.

[0037] By "fused", it is meant that the components (eg, Fab molecule and Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0038] "Fab molecule" refers to a protein composed of the VH and CH1 domains of the heavy chain ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain") of an immunoglobulin.

[0039] The so-called "cross" Fab molecule (also referred to as "Crossfab") refers to the following Fab molecules: wherein the variable domains or constant domains of the Fab heavy chain and light chain are exchanged (i.e., replaced with each other), i.e., the cross Fab molecule comprises a peptide chain (VL-CH1, in the N-terminal to C-terminal direction) consisting of the light chain variable domain VL and the heavy chain constant domain 1CH1, and a peptide chain (VH-CL, in the N-terminal to C-terminal direction) consisting of the heavy chain variable domain VH and the light chain constant domain CL. For clarity, in the cross Fab molecule in which the variable domains of the Fab light chain and the variable domains of the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the (cross) Fab molecule. Conversely, in the cross Fab molecule in which the constant domains of the Fab light chain and the constant domains of the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (cross) Fab molecule.

[0040] In contrast, the so-called "conventional" Fab molecule refers to a Fab molecule in its natural form, i.e., comprising a heavy chain consisting of a heavy chain variable domain and a constant domain (VH-CH1, in the N-terminal to C-terminal direction), and a light chain consisting of a light chain variable domain and a constant domain (VL-CL, in the N-terminal to C-terminal direction).

[0041] The term "immunoglobulin molecule" refers to a protein with the structure of a naturally occurring antibody. For example, the IgG class immunoglobulin is a heterotetrameric glycoprotein of about 150,000 daltons, which consists of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH) (also referred to as a variable heavy chain domain or a heavy chain variable region), followed by three constant domains (CH1, CH2, and CH3) (also referred to as a heavy chain constant region). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL) (also referred to as a variable light chain domain or a light chain variable region), followed by a constant light chain (CL) domain (also referred to as a light chain constant region). The heavy chains of immunoglobulins can be assigned to one of five types: called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types based on the amino acid sequence of their constant domains: called kappa (κ) and lambda (λ). Immunoglobulins are essentially composed of two Fab molecules and an Fc domain connected by the immunoglobulin hinge region.

[0042] The term "antibody" herein is used in the broadest sense and encompasses various 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.

[0043] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for possible variant antibodies, each antibody included in the population is identical and / or binds to the same epitope, and the possible variant antibodies, for example, contain naturally occurring mutations or are produced during the production process of monoclonal antibody preparations, and such variants are usually present in trace amounts. Contrary to the polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the modifier "monoclonal" represents that the feature of the antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to the present invention can be prepared 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 human immunoglobulin loci, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0044] "Isolated" antibodies are antibodies that have been separated from the components of their natural environment, i.e. antibodies that are not in their natural environment. No specific purification level is required. For example, separated antibodies can be taken out from the natural or natural environment of the antibody. Recombinant antibodies expressed in host cells are considered to be separated for the purposes of the present invention, and natural or recombinant antibodies that have been separated, graded, or partially or substantially purified by any suitable technique are also considered to be separated for the purposes of the present invention. Therefore, the antibodies and bispecific antigen binding molecules of the present invention are separated. In some embodiments, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC) methods, antibodies are purified to a purity greater than 95% or 99%. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0045] The terms "full length antibody," "intact antibody," and "complete antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure.

[0046] "Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269 to 315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab fragments and F(ab')2 fragments that contain salvage receptor binding epitope residues and have extended in vivo half-lives, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B1). Antibody fragments can be prepared by various 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.

[0047] The term "antigen binding domain" refers to a portion of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. The antigen binding domain can be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). In particular, the antigen binding domain includes an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0048] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. As used herein, "Kabat numbering" in relation to variable region sequences refers to the numbering system proposed by Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0049] As used herein, the amino acid positions of all constant regions and constant domains of heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "numbered according to Kabat" or "Kabat numbering." Specifically, the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) pages 647 to 660) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661 to 723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3), which is further clarified herein by being referred to in this case as "numbered according to the Kabat EU index."

[0050] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR"; CDRs of heavy chain variable regions / domains are abbreviated as, for example, HCDR1, HCDR2, and HCDR3; CDRs of light chain variable regions / domains are abbreviated as, for example, LCDR1, LCDR2, and LCDR3) and / or form structurally defined loops ("hypervariable loops") and / or contain antigen-contact residues ("antigen contacts"). Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0051] (a) hypervariable loops occurring at the following amino acid residues: 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2), and 96 to 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0052] (b) CDRs occurring 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));

[0053] (c) antigenic contact points occurring 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

[0054] (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).

[0055] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0056] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain is usually composed of the following four FR domains: FR1, FR2, FR3 and FR4. Therefore, the HVR sequence and FR sequence usually appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0057] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will substantially comprise at least one, usually two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to HVRs of non-human antibodies, and all or substantially all FRs correspond to FRs of human antibodies. 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 in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived), for example 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 antibodies in which the constant region has been additionally modified or altered relative to the original antibody to produce properties according to the present invention, particularly properties with respect to C1q binding and / or Fc receptor (FcR) binding.

[0058] "Human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from an amino acid sequence of an antibody of non-human origin utilizing a full library of human antibodies or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. In certain embodiments, human antibodies are derived from non-human transgenic mammals, such as mice, rats, or rabbits. In certain embodiments, human antibodies are derived from hybridoma cell lines. Antibodies or antibody fragments isolated from human antibody libraries are also considered to be human antibodies or human antibody fragments herein.

[0059] The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0060] The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes a native sequence Fc region and a variant Fc region. Although the boundaries of the IgG heavy chain Fc region may be slightly different, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the antibody produced by the host cell can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Therefore, the antibody produced by the host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain can include a full-length heavy chain, or the antibody can include a cleavage variant (also referred to herein as "a cleaved variant heavy chain") of the full-length heavy chain. This may be a situation where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index numbering). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) in Fc region may or may not exist.If not otherwise specified, the amino acid sequence of the heavy chain comprising the Fc domain (or the subunit of the Fc domain as defined herein) is represented herein as having no C-terminal glycine-lysine dipeptide.In one embodiment of the invention, the heavy chain comprising the subunit of the Fc domain as specified herein is included in the antibody or bispecific antigen binding molecule according to the present invention, and the heavy chain comprises other C-terminal glycine-lysine dipeptide (G446 and K447, according to Kabat EU index numbering).In one embodiment of the invention, the heavy chain comprising the subunit of the Fc domain as specified herein is included in the antibody or bispecific antigen binding molecule according to the present invention, and the heavy chain comprises other C-terminal glycine residues (G446, according to Kabat EU index numbering).The composition of the present invention, such as pharmaceutical composition as described herein, comprises the antibody or bispecific antigen binding molecule colony of the present invention. The antibody or bispecific antigen binding molecule population may include molecules with full-length heavy chains and molecules with cut variant heavy chains. The antibody or bispecific antigen binding molecule population may be composed of a mixture of molecules with full-length heavy chains and molecules with cut variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the antibodies or bispecific antigen binding molecules have cut variant heavy chains. In one embodiment of the invention, the composition comprising the antibody or bispecific antigen binding molecule population of the present invention includes such an antibody or bispecific antigen binding molecule, and the antibody or bispecific antigen binding molecule includes such a heavy chain, and the heavy chain includes a subunit of the Fc domain as specified herein and an additional C-terminal glycine-lysine dipeptide (G446 and K447, according to Kabat EU index numbering).In one embodiment of the invention, the composition comprising the antibody or bispecific antigen binding molecule population of the present invention comprises such an immunoconjugate, the antibody or bispecific antigen binding molecule comprising such a heavy chain, the heavy chain comprising a subunit of the Fc domain as specified herein and an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index). In one embodiment of the invention, this composition comprises an antibody or bispecific antigen binding molecule population, the immunoconjugate population being composed of the following molecules: a molecule comprising such a heavy chain, the heavy chain comprising a subunit of the Fc domain as specified herein; a molecule comprising such a heavy chain, the heavy chain comprising a subunit of the Fc domain as specified herein and an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index); and a molecule comprising such a heavy chain, the heavy chain comprising a subunit of the Fc domain as specified herein and an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain, which polypeptide is capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0061] "Modifications that promote the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the association of a polypeptide comprising the Fc domain subunit with the same polypeptide to form a homodimer. As used herein, modifications that promote association particularly include separate modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, wherein the modifications complement each other to promote the association of the two Fc domain subunits. For example, modifications that promote association may change the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (heterologous) dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be different in the sense that the additional components (e.g., antigen binding moieties) fused to each subunit are not the same. In some embodiments, modifications that promote association include amino acid mutations, particularly amino acid substitutions, in the Fc domain. In a specific embodiment, the modifications promoting association comprise individual amino acid mutations, in particular amino acid substitutions, of each of the two subunits of the Fc domain.

[0062] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with 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, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0063] As used herein, the terms "engineering, engineered, engineered" are considered to include any manipulation of the peptide backbone, or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications to the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, as well as combinations of these methods.

[0064] As used herein, the term "amino acid mutation" means that amino acid substitution, deletion, insertion and modification are covered. Any combination of substitution, deletion, insertion and modification can be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as reduced binding to Fc receptors, or increased association with another peptide. Amino acid sequence deletions and insertions include amino terminal and / or carboxyl terminal deletions and insertions of amino acids. Specific amino acid mutations are amino acid substitutions. For the purpose of changing the binding characteristics of, for example, Fc regions, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structures and / or chemical properties, are particularly preferred. Amino acid substitutions include replacement with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be produced using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is also useful to envision methods for changing amino acid side chain groups by methods other than genetic engineering (such as chemical modification). Various names can be used herein to indicate the same amino acid mutation. For example, substitution of proline at position 329 of the Fc domain with glycine can be represented as 329G, G329, G 329 , P329G or Pro329Gly.

[0065] "Percentage (%) of amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignments for determining percentages of amino acid sequence identity can be achieved in various ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA package. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for purposes herein, the BLOSUM50 comparison matrix is ​​used to generate values ​​for % amino acid sequence identity using the ggsearch program of the FASTA package version 36.3.8c or higher. The FASTA package was developed by W. R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; W. R. Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available from http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml. Alternatively, sequences may be compared using a 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 ensure that a global rather than a local alignment is performed. The amino acid identity percentage is given in the output alignment header.

[0066] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc domain of an antibody, initiates a signaling event that stimulates cells bearing the receptor to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0067] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells that specifically bind to antibodies or derivatives thereof comprising Fc regions, and the specific binding is usually through the protein portion of the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as a reduction in the number of target cells lysed by the ADCC mechanism defined above at a given antibody concentration in the culture medium surrounding the target cells within a given time, and / or an increase in the antibody concentration necessary to achieve lysis of a given number of target cells by the ADCC mechanism in the culture medium surrounding the target cells within a given time. ADCC reduction is relative to ADCC mediated by the same antibody produced by the same type of host cells but not yet engineered using the same standard production, purification, formulation and storage methods (such methods are known to those skilled in the art). For example, the reduction of ADCC mediated by an antibody comprising an amino acid substitution that reduces ADCC in the Fc domain is relative to ADCC mediated by the same antibody without the amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT Publication No. WO 2006 / 082515 or PCT Publication No. WO 2012 / 130831).

[0068] “Voritumimab” refers to the specific GPRC5D-TCB listed in the Recommended International Nonproprietary Medicines Names: List 89 (World Health Organization Drug Information, Volume 37, Issue 1, 2023).

[0069] An "effective amount" of an agent is that amount required to produce a physiological change in the cell or tissue to which it is administered.

[0070] 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 the desired therapeutic or preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0071] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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.

[0072] The term "pharmaceutical composition" refers to a preparation which is in such form that the biological activity of the active ingredient contained therein is effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered.

[0073] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0074] As used herein, "treatment" (and grammatical variants thereof such as treat or treat) refers to an attempt to alter the natural course of the disease of the individual being treated, and may be performed for prevention or clinical interventions that may be performed during clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, weakening any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies or bispecific antigen binding molecules of the invention are used to delay the development of a disease or slow down the progression of a disease.

[0075] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0076] Bispecific antigen binding molecules that bind to GPRC5D and CD3

[0077] The anti-GPRC5D / anti-CD3 bispecific antigen binding molecules for the combination therapy described herein are also referred to herein as "GPRC5D TCBs", comprising at least two antigen binding moieties that are capable of specifically binding to two different antigenic determinants (a first antigen and a second antigen). Suitable bispecific antigen binding molecules for use in the present invention in combination with GPRC5D and CD3 are described in, for example, WO 2021 / 018859A1, WO 2019 / 154890A1, and WO 2018017786 A2.

[0078] According to a specific embodiment of the present invention, the antigen binding moiety contained in the bispecific antigen binding molecule is a Fab molecule (i.e., an antigen binding domain consisting of a heavy chain and a light chain, each antigen binding domain comprising a variable domain and a constant domain). In one embodiment, the first and / or second antigen binding moiety is a Fab molecule. In one embodiment, the Fab molecule is human. In a specific embodiment, the Fab molecule is humanized. In another embodiment, the Fab molecule comprises human heavy chain and light chain constant domains.

[0079] Preferably, at least one antigen binding moiety in the antigen binding moiety is a cross Fab molecule. This modification reduces the mispairing of the heavy chain and light chain from different Fab molecules, thereby improving the yield and purity of the bispecific antigen binding molecules of the present invention in recombinant production. In the specific cross Fab molecules that can be used for the bispecific antigen binding molecules of the present invention, the variable domains (VL and VH, respectively) of the Fab light chain and the Fab heavy chain are exchanged. However, even if the domain exchange is performed, due to the so-called Bence Jones type interaction between the heavy chain and the light chain of the mispairing, the preparation of the bispecific antigen binding molecules may include some by-products (see Schaefer et al., PNAS, 108 (2011) 11187-11191). In order to further reduce the mispairing of the heavy chain and light chain from different Fab molecules and thus improve the purity and yield of the desired bispecific antigen binding molecules, it is possible to introduce oppositely charged charged amino acids at specific amino acid positions in the CH1 and CL domains of any one of the Fab molecules combined with the first antigen (GPRC5D) or the Fab molecules combined with the second antigen (CD3), as further described herein. The charge modification is performed in a conventional Fab molecule contained in the bispecific antigen binding molecule or in a VH / VL crossover Fab molecule contained in the bispecific antigen binding molecule (but not in both). In a specific embodiment, the charge modification is performed in a conventional Fab molecule contained in the bispecific antigen binding molecule (which in a specific embodiment binds to the first antigen, i.e., GPRC5D).

[0080] Bispecific antigen binding molecules can be combined with the first antigen (i.e. GPRC5D) and the second antigen (i.e. CD3) simultaneously. Bispecific antigen binding molecules can crosslink T cells and target cells by combining GPRC5D and activated T cell antigens simultaneously. This kind of simultaneous combination causes the cracking of target cells (particularly tumor cells expressing GPRC5D), the activation of T cells and the cellular response of T lymphocytes (particularly cytotoxic T lymphocytes), which is selected from the following groups: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity and the expression of activation markers.

[0081] In one embodiment, the bispecific antigen binding molecule is capable of redirecting the cytotoxic activity of T cells to target cells. In a specific embodiment, the redirection is independent of MHC-mediated peptide antigen presentation of the target cell and / or the specificity of the T cell.

[0082] In particular, the T cells according to any embodiment of the present invention are cytotoxic T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells, especially CD8 + T cells.

[0083] First antigen binding moiety

[0084] The bispecific antigen binding molecule comprises at least one antigen binding moiety that binds to GPRC5D (first antigen), particularly a Fab molecule. In certain embodiments, the bispecific antigen binding molecule comprises two antigen binding moieties that bind to GPRC5D, particularly a Fab molecule. In a specific embodiment of this type, each of these antigen binding moieties binds to the same antigenic determinant. In an even more specific embodiment, all of these antigen binding moieties are identical, i.e., they comprise the same amino acid sequence, including the same amino acid substitutions (if any) in the CH1 and CL domains as described herein. In one embodiment, the bispecific antigen binding molecule comprises no more than two antigen binding moieties that bind to GPRC5D, particularly a Fab molecule.

[0085] In certain embodiments, the antigen binding moiety that binds to GPRC5D is a conventional Fab molecule. In such embodiments, the antigen binding moiety that binds to a second antigen is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged / replaced with each other.

[0086] In alternative embodiments, the antigen binding moiety that binds to GPRC5D 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 are exchanged / replaced with each other. In such embodiments, the antigen binding moiety that binds to the second antigen is a conventional Fab molecule.

[0087] The GPRC5D binding moiety is capable of directing the bispecific antigen binding molecule to a target site, for example, to a specific type of tumor cell that expresses GPRC5D.

[0088] The first antigen binding portion of the bispecific antigen binding molecule may incorporate any of the features described herein for antibodies that bind GPRC5D, alone or in combination, unless clearly scientifically unreasonable or impossible.

[0089] In one aspect, 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 comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and the light chain variable region comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; and (b) a second antigen binding portion that binds to CD3.

[0090] In some embodiments, the first antigen binding portion is (derived from) a humanized antibody. In one embodiment, VH is a humanized VH and / or VL is a humanized VL. In one embodiment, the first antigen binding portion comprises the CDRs in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0091] 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.

[0092] In one embodiment, the first antigen binding portion comprises a VH sequence and a VL sequence, wherein the VH sequence 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 sequence is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:11.

[0093] In one embodiment, the first antigen binding portion comprises VH and VL, wherein VH comprises the amino acid sequence of SEQ ID NO:10 and VL comprises the amino acid sequence of SEQ ID NO:11.

[0094] 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.

[0095] In a specific embodiment, the first antigen binding moiety comprises: a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 53. In a specific embodiment, the first antigen binding moiety comprises a VH sequence of SEQ ID NO: 48 and a VL sequence of SEQ ID NO: 11.

[0096] In one embodiment, the first antigen binding portion comprises a human constant region. In one embodiment, the first antigen binding portion 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 NO: 1 and SEQ ID NO: 2 (human κ and λ CL domains, respectively) and SEQ ID NO: 3 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In some embodiments, the first antigen binding portion comprises a light chain constant region, the amino acid sequence of which is 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 SEQ ID NO: 1. Specifically, the light chain constant region may comprise amino acid mutations under "charge modification" as described herein and / or if in a cross-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. Specifically, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations under "charge modification" as described herein.

[0097] Second antigen binding moiety

[0098] The bispecific antigen binding molecule comprises at least one antigen binding moiety, particularly a Fab molecule, that binds to a second antigen (CD3).

[0099] In a specific embodiment, the antigen binding moiety 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 are exchanged / replaced with each other. In such embodiments, the antigen binding moiety that binds to the first antigen (i.e., GPRC5D) is preferably a conventional Fab molecule. In embodiments where there is more than one antigen binding moiety that binds to GPRC5D in the bispecific antigen binding molecule, particularly a Fab molecule, the antigen binding moiety that binds to the second antigen is preferably a crossover Fab molecule, and the antigen binding moiety that binds to GPRC5D is a conventional Fab molecule.

[0100] In alternative embodiments, the antigen binding moiety that binds to the second antigen is a conventional Fab molecule. In a specific embodiment, the antigen binding moiety that binds to the first antigen (i.e., GPRC5D) is a cross-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 are exchanged / replaced with each other. In embodiments where there is more than one antigen binding moiety that binds to the second antigen in the bispecific antigen binding molecule, particularly a Fab molecule, the antigen binding moiety that binds to GPRC5D is preferably a cross-Fab molecule, and the antigen binding moiety that binds to the second antigen is a conventional Fab molecule.

[0101] The second antigen (i.e., CD3) is an activating T cell antigen (also referred to herein as an "activating T cell antigen binding portion, or an activating T cell antigen binding Fab molecule"). In a specific embodiment, the bispecific antigen binding molecule comprises no more than one antigen binding portion that is capable of specifically binding to an activating T cell antigen. In one embodiment, the bispecific antigen binding molecule provides monovalent binding to an activating T cell antigen.

[0102] 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 cross-reacts with (i.e. specifically binds to) human and cynomolgus CD3. In some embodiments, the second antigen is the epsilon subunit of CD3 (CD3 epsilon).

[0103] In one embodiment, the second antigen binding moiety comprises HCDR 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, HCDR 3 of SEQ ID NO: 20, LCDR 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23. In one embodiment, the second antigen binding moiety comprises a VH comprising HCDR 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, HCDR 3 of SEQ ID NO: 20; and a VL comprising LCDR 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23. In some embodiments, the second antigen binding moiety is (derived from) a humanized antibody. In one embodiment, VH is a humanized VH and / or VL is a humanized VL. In one embodiment, the second antigen binding moiety comprises the CDRs in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In one embodiment, the second antigen binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24. In one embodiment, the second antigen binding portion comprises a VL sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the second antigen binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24, and a VL sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the VH of the second antigen binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24, and the VL of the second antigen binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the second antigen binding portion comprises: a VH comprising an amino acid sequence such as SEQ ID NO: 24 and a VL comprising an amino acid sequence such as SEQ ID NO: 25. In one embodiment, the second antigen binding portion comprises a VH amino acid sequence such as SEQ ID NO: 24 and a VL amino acid sequence such as SEQ ID NO: 25.

[0104] 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 NO: 1 and SEQ ID NO: 2 (human κ and λ 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, the amino acid sequence of which is 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 SEQ ID NO: 1. Specifically, the light chain constant region may comprise amino acid mutations under "charge modification" as described herein and / or if in a cross-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. Specifically, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations under "charge modification" as described herein.

[0105] 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, in particular the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are replaced with each other (i.e., according to such an embodiment, the second antigen binding moiety is a crossover Fab molecule in which the variable domains or the constant domains of the Fab light chain and the Fab heavy chain are exchanged). In one such embodiment, the first (and third, if any) antigen binding moiety is a conventional Fab molecule.

[0106] In one embodiment, there is no more than one antigen binding moiety in the bispecific antigen binding molecule that binds a second antigen (ie, CD3) (ie, the bispecific antigen binding molecule provides monovalent binding to the second antigen).

[0107] Charge modification

[0108] The bispecific antigen-binding molecule may comprise amino acid substitutions in the Fab molecules contained therein that are particularly effective in reducing mispairing of the light chain with an unmatched heavy chain (Bence-Jones type byproducts), which mispairing may occur in the production of Fab-based bi / multispecific antigen-binding molecules having a VH / VL exchange in one (or multiple, if the molecule comprises more than two antigen-binding Fab molecules) of its binding arms (see also PCT Publication No. WO 2015 / 150447, in particular the examples therein, the entire contents of which are incorporated herein by reference). The ratio of the desired bispecific antigen-binding molecule to undesirable byproducts, in particular Bence Jones type byproducts occurring in bispecific antigen-binding molecules having a VH / VL domain exchange in one of the binding arms of the bispecific antigen-binding molecule, can be increased by introducing charged amino acids with opposite charges (sometimes referred to herein as "charge modification") at specific amino acid positions in the CH1 and CL domains.

[0109] Therefore, in some embodiments, wherein the first antigen-binding moiety and the second antigen-binding moiety of the bispecific antigen-binding molecule are both Fab molecules, and the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other in one of the antigen-binding moieties (particularly the second antigen-binding moiety),

[0110] i) in the constant domain CL of the first antigen binding moiety, the amino acid at position 124 is substituted by a positively charged amino acid (according to Kabat numbering), and wherein in the constant domain CH1 of the first antigen binding moiety, the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (according to the Kabat EU index numbering); or

[0111] ii) in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is substituted by a positively charged amino acid (according to Kabat numbering), and wherein in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (according to the Kabat EU index numbering).

[0112] The bispecific antigen binding molecule does not comprise the two modifications mentioned in i) and ii). The constant domains CL and CH1 of the antigen binding part with VH / VL exchange are not replaced with each other (ie remain unexchanged).

[0113] In a more specific embodiment,

[0114] i) in the constant domain CL of the first antigen binding portion, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first antigen binding portion, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index numbering); or

[0115] ii) in the constant domain CL of the second antigen-binding portion, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen-binding portion, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0116] In one such embodiment, in the constant domain CL of the first antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first antigen binding portion, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0117] In another embodiment, in the constant domain CL of the first antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first antigen binding portion, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0118] In a specific embodiment, in the constant domain CL of the first antigen binding portion, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first antigen binding portion, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0119] In a more specific embodiment, in the constant domain CL of the first antigen binding portion, the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted by lysine (K) (according to Kabat numbering), and in the constant domain CH1 of the first antigen binding portion, the amino acid at position 147 is substituted by glutamic acid (E) (according to the Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (according to the Kabat EU index numbering).

[0120] In an even more specific embodiment, in the constant domain CL of the first antigen binding portion, the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted by arginine (R) (according to Kabat numbering), and in the constant domain CH1 of the first antigen binding portion, the amino acid at position 147 is substituted by glutamic acid (E) (according to the Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (according to the Kabat EU index numbering).

[0121] In a specific embodiment, if 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.

[0122] Alternatively, the amino acid substitutions according to the above embodiments may be made in the constant domain CL and constant domain CH1 of the second antigen binding moiety instead of in the constant domain CL and constant domain CH1 of the first antigen binding moiety. In specific such embodiments, the constant domain CL of the second antigen binding moiety is of the kappa isotype.

[0123] Thus, in one embodiment, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0124] In another embodiment, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0125] In another embodiment, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0126] In one embodiment, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted by lysine (K) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 is substituted by glutamic acid (E) (according to the Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (according to the Kabat EU index numbering).

[0127] In another embodiment, in the constant domain CL of the second antigen binding portion, the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted by arginine (R) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding portion, the amino acid at position 147 is substituted by glutamic acid (E) (according to the Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (according to the Kabat EU index numbering).

[0128] In a particular embodiment, the bispecific antigen binding molecule comprises

[0129] (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) and a light chain variable region (VL), the heavy chain variable region comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, the light chain variable region comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17, and

[0130] (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, the Fab molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and HCDR3 of SEQ ID NO: 20, the light chain variable region comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other;

[0131] wherein in the constant domain CL of the first antigen binding portion, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) (in a specific embodiment, it is independently substituted by lysine (K) or arginine (R)) and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) (in a specific embodiment, it is independently substituted by lysine (K) or arginine (R)); and in the constant domain CH1 of the first antigen binding portion, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0132] Bispecific antigen-binding molecule formats

[0133] In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity to at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In certain embodiments, multispecific antibodies have three or more binding specificities. In certain embodiments, bispecific antibodies can bind to two (or more) different epitopes of GPRC5D. Multispecific (e.g., bispecific) antibodies can also be used to localize cytotoxic agents to cells expressing GPRC5D. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0134] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, 305:537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be prepared by engineering electrostatic manipulation effects for preparing antibody Fc-heterodimer molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using common light chain technology to avoid light chain mispairing problems (see, e.g., WO 98 / 50431); using "diabody" technology for preparing bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described in Tutt et al., J. Immunol. 147:60 (1991).

[0135] Also included herein are engineered antibodies with three or more antigen binding sites, including, for example, "octopus antibodies" or DVD-Ig (see, for example, 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, WO2010 / 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, for example, US2008 / 0069820 and WO 2015 / 095539).

[0136] Multispecific antibodies can also be provided in an asymmetric form, where there is a domain swap in one or more binding arms with the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253), or complete Fab arms (see, e.g., WO2009 / 080251, WO 2016 / 016299, see also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interface to direct the correct Fab pairing. See, e.g., WO 2016 / 172485.

[0137] Various other 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).

[0138] Examples of bispecific antibody formats that can be used for this purpose include, but are not limited to, so-called "BiTE" (bispecific T-cell engager) molecules in which two scFv molecules are fused via a flexible linker (see, e.g., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 119567; Nagorsen and Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAb"; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); "DART" (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which are fully hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Specific T cell bispecific antibody formats encompassed herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0139] The components of the bispecific antigen binding molecule can be fused to each other in a variety of configurations.

[0140] In certain embodiments, the antigen binding moiety contained in the bispecific antigen binding molecule is a Fab molecule. In such embodiments, the first, second, third antigen binding moiety, etc., may be referred to herein as the first, second, third Fab molecule, etc., respectively.

[0141] In one embodiment, the first antigen binding moiety and the second antigen binding moiety of the bispecific antigen binding molecule are fused to each other, optionally via a peptide linker. In a specific embodiment, each of the first and second antigen binding moieties is a Fab molecule. 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 the embodiment where (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 can be optionally fused to each other via a peptide linker.

[0142] Bispecific antigen binding molecules (such as Fab molecules) with a single antigen binding portion that is capable of specifically binding to a target cell antigen (such as GPRC5D) are useful, particularly where internalization of the target cell antigen is expected following binding of the high affinity antigen binding portion. In this case, the presence of more than one antigen binding portion specific for the target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availability.

[0143] However, in other cases it will be advantageous to have a bispecific antigen binding molecule (such as a Fab molecule) comprising two or more antigen binding portions specific for a target cell antigen, for example to optimize targeting to a target site or to allow cross-linking of target cell antigens.

[0144] Thus, in certain embodiments, the bispecific antigen binding molecule comprises a third antigen binding moiety.

[0145] In one embodiment, the third antigen binding moiety binds the first antigen, i.e. GPRC5D. In one embodiment, the third antigen binding moiety is a Fab molecule.

[0146] In one embodiment, the third antigen binding moiety is identical to the first antigen binding moiety.

[0147] The third antigen binding moiety of the bispecific antigen binding molecule may incorporate any of the features described herein for the first antigen binding moiety and / or antibody that binds GPRC5D, alone or in combination, unless clearly scientifically unreasonable or impossible.

[0148] In one embodiment, the third antigen binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and the light chain variable region comprises a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17.

[0149] In some embodiments, the third antigen binding portion is (derived from) a humanized antibody. In one embodiment, VH is a humanized VH and / or VL is a humanized VL. In one embodiment, the third antigen binding portion comprises the CDRs in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0150] 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.

[0151] In one embodiment, the third antigen binding portion comprises a VH sequence and a VL sequence, wherein the VH sequence 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 sequence is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:11.

[0152] In one embodiment, the third antigen binding portion comprises VH and VL, wherein VH comprises the amino acid sequence of SEQ ID NO:10 and VL comprises the amino acid sequence of SEQ ID NO:11.

[0153] 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.

[0154] In a specific embodiment, the third antigen binding moiety comprises: a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 53. In a specific embodiment, the third antigen binding moiety comprises a VH sequence of SEQ ID NO: 48 and a VL sequence of SEQ ID NO: 11.

[0155] 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 NO: 1 and SEQ ID NO: 2 (human κ and λ 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, the amino acid sequence of which is 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 SEQ ID NO: 1. Specifically, the light chain constant region may comprise amino acid mutations under "charge modification" as described herein and / or if in a cross-Fab molecule, may comprise deletions or substitutions of one or more (particularly two) N-terminal amino acids. In some embodiments, the third antigen binding portion comprises a heavy chain constant region, the 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. Specifically, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations under "charge modification" as described herein.

[0156] In a specific embodiment, the third and first antigen binding moieties are each a Fab molecule and the third antigen binding moiety is identical to the first antigen binding moiety. Therefore, in these embodiments, the first and third antigen binding moieties comprise the same heavy chain and light chain amino acid sequences and have the same domain arrangement (i.e., conventional or crossover). In addition, in these embodiments, the third antigen binding moiety comprises the same amino acid substitutions (if any) as the first antigen binding moiety. For example, the amino acid substitutions described herein as "charge modification" will be carried out in the constant domains CL and constant domains CH1 of the first antigen binding moiety and the third antigen binding moiety respectively. Alternatively, the amino acid substitutions may be carried out in the constant domains CL and constant domains CH1 of the second antigen binding moiety (which is also a Fab molecule in a specific embodiment), but not in the constant domains CL and constant domains CH1 of the first antigen binding moiety and the third antigen binding moiety.

[0157] As with the first antigen binding moiety, the third antigen binding moiety is particularly a conventional Fab molecule. However, embodiments in which the first and third antigen binding moieties are cross Fab molecules (and the second antigen binding moiety is a conventional Fab molecule) are also contemplated. Therefore, in a particular embodiment, the first and third antigen binding moieties are each a conventional Fab molecule, and the second antigen binding moiety is a cross Fab molecule as described herein, i.e., such a Fab molecule, in which the variable domains VH and VL or constant domains CL and CH1 of Fab heavy and light chains are exchanged / replaced with each other. In other embodiments, the first and third antigen binding moieties are each a cross Fab molecule and the second antigen binding moiety is a conventional Fab molecule.

[0158] If a third antigen binding moiety is present, in a specific embodiment the first and third antigen binding moieties bind to GPRC5D and the second antigen binding moiety binds to CD3 (particularly CD3ε).

[0159] In certain embodiments, the bispecific antigen binding molecule comprises an Fc domain composed of a first subunit and a second subunit. The first subunit and the second subunit of the Fc domain are capable of stably associating.

[0160] Bispecific antigen binding molecules can have different configurations, i.e. the first and second (and optionally the third) antigen binding moieties can be fused to each other and to the Fc domain in different ways. The components can be directly fused to each other, or preferably fused via one or more suitable peptide linkers. When the Fab molecule is fused to the N-terminus of the subunit of the Fc domain, the fusion is typically via an immunoglobulin hinge region.

[0161] In some embodiments, the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule, and the second antigen binding moiety is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. In such embodiments, the first antigen binding moiety can be 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 or fused to the N-terminus of another subunit of the Fc domain. In such specific embodiments, the first antigen binding moiety is a conventional Fab molecule, and the second antigen binding moiety is a cross Fab molecule as described herein, i.e., such a Fab molecule, in which the variable domains VH and VL of the Fab heavy chain and light chain or the constant domains CL and CH1 are exchanged / replaced with each other. In other such embodiments, the first Fab molecule is a cross Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0162] In one embodiment, the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule, the second antigen binding moiety is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain, and 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 a specific embodiment, the bispecific antigen binding molecule is substantially composed of the first and second Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the second Fab molecule is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can be fused to each other in addition.

[0163] In another embodiment, the first antigen binding moiety and the second antigen binding moiety are each Fab molecules, and the first antigen binding moiety and the second antigen binding moiety are each fused to the N-terminus of a subunit of the Fc domain at the C-terminus of the Fab heavy chain. In a specific embodiment, the bispecific antigen binding molecule is substantially composed of the first Fab molecule and the second Fab molecule, the Fc domain consisting of the first subunit and the second subunit, and optionally one or more peptide linkers, wherein the first Fab molecule and the second Fab molecule are each fused at the C-terminus of the Fab heavy chain with the N-terminus of a subunit in the subunit of the Fc domain. The first Fab molecule and the second Fab molecule can be fused with the Fc domain directly or through a peptide linker. In a specific embodiment, the first Fab molecule and the second Fab molecule are each fused with the Fc domain by 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 IgG1Fc domain.

[0164] In some embodiments, the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule, and the first antigen binding moiety is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. In such embodiments, the second antigen binding moiety can be 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 or (as described above) to the N-terminus of another subunit of the Fc domain. In such specific embodiments, the first antigen binding moiety is a conventional Fab molecule, and the second antigen binding moiety is a cross Fab molecule as described herein, i.e., such a Fab molecule, in which the variable domains VH and VL of the Fab heavy chain and light chain or the constant domains CL and CH1 are exchanged / replaced with each other. In other such embodiments, the first Fab molecule is a cross Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0165] In one embodiment, the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule, the first antigen binding moiety is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain, and 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 a specific embodiment, the bispecific antigen binding molecule is substantially composed of the first and second Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the first Fab molecule is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can be fused to each other in addition.

[0166] In some embodiments, the third antigen binding moiety, in particular the third Fab molecule, is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. In such specific embodiments, each of the first and third Fab molecules is 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 chain and light chain are exchanged / replaced with each other. In other such embodiments, each of the first and third Fab molecules is a crossover Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0167] In such specific embodiments, the second and third antigen binding moieties are each fused to the N-terminus of a subunit of the Fc domain at the C-terminus of the Fab heavy chain, and the first antigen binding moiety is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain. In a specific embodiment, the bispecific antigen binding molecule is substantially composed of the first, second and third Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the second Fab molecule is fused to the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and wherein the third Fab molecule is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain. The second and third Fab molecules can be fused to the Fc domain directly or by a peptide linker. In a specific embodiment, the second and third Fab molecules are each fused to the Fc domain by an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, 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 additionally be fused to each other.

[0168] In another such embodiment, the first and third antigen binding moieties are each fused to the N-terminus of a subunit of the Fc domain at the C-terminus of the Fab heavy chain, and 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 a specific embodiment, the bispecific antigen binding molecule is substantially composed of the first, second and third Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the first Fab molecule is fused to the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and wherein the third Fab molecule is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain. The first and third Fab molecules can be fused to the Fc domain directly or by a peptide linker. In a specific embodiment, the first Fab molecule and the third Fab molecule are each fused to the Fc domain by an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, 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 additionally be fused to each other.

[0169] In the configuration of the bispecific antigen binding molecule, wherein the Fab molecule is fused to each of the N-terminal ends of the subunits of the Fc domain at the C-terminal end of the Fab heavy chain through an immunoglobulin hinge region, the two Fab molecules, the hinge region and the Fc domain essentially form an immunoglobulin molecule. In a specific embodiment, the immunoglobulin molecule is an IgG class immunoglobulin. In an even more specific embodiment, the immunoglobulin is an IgG1 subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG4 subclass immunoglobulin. In another 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.

[0170] 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. According to the configuration of the first Fab molecule and the second Fab molecule, the Fab light chain of the first Fab molecule can be fused to the N-terminal of the Fab light chain of the second Fab molecule at its C-terminus, or the Fab light chain of the second Fab molecule can be fused to the N-terminal of the Fab light chain of the first Fab molecule at its C-terminus. The fusion of the Fab light chain of the first and second Fab molecules further reduces the mismatch of the unmatched Fab heavy chain and light chain, and also reduces the number of plasmids required for expressing some bispecific antigen binding molecules.

[0171] The antigen binding portion can be fused to the Fc domain (or to each other) directly or through a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n 、(SG4) n 、(G4S) n or G4(SG4) n Peptide linker. "n" is generally an integer from 1 to 10, and is generally from 2 to 4. In one embodiment, the peptide linker is at least 5 amino acids long, in one embodiment 5 to 100 amino acids long, and in another embodiment 10 to 50 amino acids long. In one embodiment, the peptide linker is (GxS) n or (GxS) n G m, wherein 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, in another 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 Fab molecule and the second Fab molecule to each other is (G4S)2. An exemplary peptide linker suitable for connecting the Fab heavy chains of the first Fab fragment and the second Fab fragment comprises the sequence (D)-(G4S)2 (SEQ ID NO:7 and SEQ ID NO: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, in the case of fusion of the Fab molecule to the N-terminus of the Fc domain subunit, the fusion may be via an immunoglobulin hinge region or a portion thereof with or without an additional peptide linker.

[0172] In certain embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxyl-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), which in turn shares a carboxyl-terminal peptide bond (VL (2) -CH1 (2) -CH2-CH3(-CH4)), and a polypeptide in which the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond (VH (1) -CH1 (1) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ). In certain embodiments, the polypeptides are covalently linked, for example, via a disulfide bond.

[0173] In certain embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxyl-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), which in turn shares a carboxyl-terminal peptide bond with an Fc domain subunit (VH (2) -CL (2) -CH2-CH3(-CH4)), and a polypeptide in which the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond (VH (1) -CH1 (1) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VL (2) -CH1 (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ). In certain embodiments, the polypeptides are covalently linked, for example, via a disulfide bond.

[0174] 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 carboxyl-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), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fc domain subunit (VL (2) -CH1 (2) -VH (1) -CH1 (1) -CH2-CH3(-CH4)). In other embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxyl-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, which in turn shares a carboxyl-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 the light chain variable region), which in turn shares a carboxyl-terminal peptide bond with an Fc domain subunit (VH (1) -CH1 (1) -VL (2) -CH1 (2)-CH2-CH3(-CH4)).

[0175] In some such embodiments, the bispecific antigen binding molecule further comprises a crossover Fab light chain polypeptide of a second Fab molecule in which the Fab heavy chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) In other such embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, which in turn shares a carboxyl-terminal peptide bond (VH (2) -CL (2) -VL (1) -CL (1) ), or wherein the Fab light chain polypeptide of a first Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxyl-terminal peptide bond (VL (1) -CL (1) -VH (2) -CL (2) ), as the case may be.

[0176] 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 carboxyl-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)), and the Fab light chain polypeptide (VL (3) -CL (3) ). In certain embodiments, the polypeptides are covalently linked, for example, via a disulfide bond.

[0177] 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 carboxyl-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), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fc domain subunit (VH (2) -CL (2) -VH (1) -CH1 (1) -CH2-CH3(-CH4)). In other embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxyl-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 the light chain constant region), which in turn shares a carboxyl-terminal peptide bond with an Fc domain subunit (VH (1) -CH1 (1) -VH (2) -CL (2) -CH2-CH3(-CH4)).

[0178] In some such embodiments, the bispecific antigen binding molecule further comprises a crossover Fab light chain polypeptide of a second Fab molecule in which the Fab light chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VL (2) -CH1 (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) In other such embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, which in turn shares a carboxyl-terminal peptide bond (VL (2) -CH1 (2) -VL (1) -CL (1)), or wherein the Fab light chain polypeptide of a first Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxyl-terminal peptide bond (VL (1) -CL (1) -VL (2) -CH1 (2) ), as the case may be.

[0179] 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 carboxyl-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)), and the Fab light chain polypeptide (VL (3) -CL (3) ). In certain embodiments, the polypeptides are covalently linked, for example, via a disulfide bond.

[0180] In certain embodiments, the bispecific antigen binding molecule does not include an Fc domain. In such specific embodiments, each of the first and third Fab molecules (if present) is a conventional Fab molecule, and the second Fab molecule is a cross Fab molecule as described herein, i.e., such a Fab molecule, in which the variable domains VH and VL of the Fab heavy chain and light chain or the constant domains CL and CH1 are exchanged / replaced with each other. In other such embodiments, each of the first and third Fab molecules (if present) is a cross Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0181] In one such embodiment, the bispecific antigen binding molecule consists essentially of a first and a second antigen binding moiety and optionally one or more peptide linkers, wherein the first and second antigen binding moieties are both Fab molecules and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety.

[0182] In another such embodiment, the bispecific antigen binding molecule consists essentially of a first and a second antigen binding moiety and optionally one or more peptide linkers, wherein the first and the second antigen binding moiety 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.

[0183] In some embodiments, the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the bispecific antigen binding molecule further comprises a third antigen binding moiety, in particular a third Fab molecule, wherein the third Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain. In certain such embodiments, the bispecific antigen binding molecule consists essentially of the first, second and third Fab molecules and optionally one or more peptide linkers, wherein the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain.

[0184] In some embodiments, the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the bispecific antigen binding molecule further comprises a third antigen binding moiety, in particular a third Fab molecule, wherein the third Fab molecule is fused to the C-terminus of the Fab heavy chain of the first Fab molecule at the N-terminus of the Fab heavy chain. In certain such embodiments, the bispecific antigen binding molecule consists essentially of the first, second and third Fab molecules and optionally one or more peptide linkers, wherein the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule is fused to the C-terminus of the Fab heavy chain of the first Fab molecule at the N-terminus of the Fab heavy chain.

[0185] In certain embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxyl-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, which in turn shares a carboxyl-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) (VH (1) -CH1 (1) -VL (2) -CH1 (2) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ).

[0186] In certain embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxyl-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), which in turn shares a carboxyl-terminal peptide bond (VL ) 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 further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ).

[0187] In certain embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxyl-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), which in turn shares a carboxyl-terminal peptide bond (VH) 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 further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VL (2) -CH1 (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ).

[0188] In certain embodiments, the bispecific antigen binding molecules according to the present invention comprise a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxyl-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), which in turn shares a carboxyl-terminal peptide bond (VL ) 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 further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ).

[0189] In certain embodiments, the bispecific antigen binding molecules according to the present invention comprise a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxyl-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) (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VL (2) -CH1 (2) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) In some embodiments, the bispecific antigen binding molecule further comprises a Fab light chain polypeptide (VL (3) -CL (3) ).

[0190] In certain embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxyl-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 the light chain constant region) (VH (3) -CH1 (3) -VH(1) -CH1 (1) -VH (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 second Fab molecule shares a carboxyl-terminal peptide bond (VL (2) -CH1 (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) In some embodiments, the bispecific antigen binding molecule further comprises a Fab light chain polypeptide (VL (3) -CL (3) ).

[0191] In certain embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxyl-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), which Fab heavy chain constant region in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the third Fab molecule (VL (2) -CH1 (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) In some embodiments, the bispecific antigen binding molecule further comprises a Fab light chain polypeptide (VL (3) -CL (3) ).

[0192] In certain embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxyl-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), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the third Fab molecule (VH (2) -CL (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VL (2) -CH1 (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) In some embodiments, the bispecific antigen binding molecule further comprises a Fab light chain polypeptide (VL (3) -CL (3) ).

[0193] In certain embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab heavy chain of the second Fab molecule shares a carboxyl-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, which in turn shares a carboxyl-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 the light chain variable region), which in turn shares a carboxyl-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 the light chain variable region) (VH (2) -CH1 (2) -VL (1) -CH1 (1) -VL (3) -CH1 (3) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab heavy chain variable region of the first Fab molecule shares a carboxyl-terminal peptide bond (VH (1) -CL (1)) and shares a carboxyl-terminal peptide bond (VL (2) -CL (2) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxyl-terminal peptide bond (VH (3) -CL (3) ).

[0194] In certain embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab heavy chain of the second Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxyl-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, wherein the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxyl-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, wherein the heavy chain constant region is replaced by the light chain constant region) (VH (2) -CH1 (2) -VH (1) -CL (1) -VH (3) -CL (3) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule shares a carboxyl-terminal peptide bond (VL (1) -CH1 (1) ) and shares a carboxyl-terminal peptide bond (VL (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 carboxyl-terminal peptide bond (VL (3) -CH1 (3) ).

[0195] In certain embodiments, the bispecific antigen binding molecule comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule shares a carboxyl-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), which in turn shares a carboxyl-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, which in turn shares a carboxyl-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), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (3) -CH1 (3) -VL (1) -CH1 (1) -VH (2) -CH1 (2) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab heavy chain variable region of the first Fab molecule shares a carboxyl-terminal peptide bond (VH (1) -CL (1) ) and shares a carboxyl-terminal peptide bond (VL (2) -CL (2) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxyl-terminal peptide bond (VH (3) -CL (3) ).

[0196] In certain embodiments, the bispecific antigen binding molecule according to the present invention comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxyl-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), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the first 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), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the second Fab molecule (VH (3) -CL (3) -VH (1)-CL (1) -VH (2) -CH1 (2) In some embodiments, the bispecific antigen binding molecule further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule shares a carboxyl-terminal peptide bond (VL (1) -CH1 (1) ) and shares a carboxyl-terminal peptide bond (VL (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 carboxyl-terminal peptide bond (VL (3) -CH1 (3) ).

[0197] In a specific embodiment, the present invention provides a bispecific antigen binding molecule 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, the Fab molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and the light chain variable region comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; 3; 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, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other or the constant domains CL and CH1 are replaced with each other, and wherein the Fab molecule comprises a heavy chain variable region (VH) and a light chain variable region (Vl), the heavy chain variable region comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20, and the light chain variable region comprises a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR2 of SEQ ID NO: 22, and LCDR3 of SEQ ID NO: 23. 3; c) a third antigen binding moiety that binds to the first antigen and is identical to the first antigen binding moiety; and d) an Fc domain composed of a first subunit and a second subunit; wherein (i) the first antigen binding moiety under 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 moiety under b), and the second antigen binding moiety under b) and the third antigen binding moiety under 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 under d), or (ii) the second antigen binding moiety under 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 moiety under a), and the first antigen binding moiety under a) and the third antigen binding moiety under 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 under d).

[0198] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and the light chain variable region comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; 3; 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, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other or the constant domains CL and CH1 are replaced with each other, and wherein the Fab molecule comprises a heavy chain variable region (VH) and a light chain variable region (Vl), the heavy chain variable region comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20, and the light chain variable region comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23; c) an Fc domain composed of a first subunit and a second subunit; wherein (i) the first antigen-binding portion under a) and the second antigen-binding portion under b) 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 under c).

[0199] In all different configurations of bispecific antigen binding molecules, the amino acid substitutions described herein (if any) can be in the CH1 and CL domains of the first and (if any) third antigen binding moiety / Fab molecules, or in the CH1 and CL domains of the second antigen binding moiety / Fab molecules. Preferably, they are in the CH1 and CL domains of the first and (if any) third antigen binding moiety / Fab molecules. According to the concept of the present invention, if amino acid substitutions as described herein are performed in the first (and if any third) antigen binding moiety / Fab molecule, such amino acid substitutions are not performed in the second antigen binding moiety / Fab molecule. On the contrary, if amino acid substitutions as described herein are performed in the second antigen binding moiety / Fab molecule, such amino acid substitutions are not performed in the first (and if any third) antigen binding moiety / Fab molecule. Amino acid substitutions are particularly performed in bispecific antigen binding molecules comprising such Fab molecules, in which the variable domains VL and VH1 of the Fab light chain and the Fab heavy chain replace each other.

[0200] In specific embodiments, particularly wherein the amino acid substitutions as described herein are carried out in the first (and if present the third) antigen binding moiety / Fab molecule, and the constant domain CL of the first (and if present the third) Fab molecule of the bispecific antigen binding molecule is of a κ isotype. In other embodiments of the bispecific antigen binding molecule according to the present invention, particularly wherein the amino acid substitutions as described herein are carried out in the second antigen binding moiety / Fab molecule, and the constant domain CL of the second antigen binding moiety / Fab molecule is of a κ 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 a κ isotype.

[0201] In one embodiment, the bispecific antigen binding molecule comprises: a) a first antigen binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen binding moiety is a Fab molecule, the Fab molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and the light chain variable region comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; 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, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and wherein the Fab molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising SEQ ID NO:18 heavy chain complementary determining region (HCDR) 1, HCDR 2 of SEQ ID NO:19 and HCDR 3 of SEQ ID NO:20, the light chain variable region comprises a light chain complementary determining region (LCDR) 1 of SEQ ID NO:21, LCDR 2 of SEQ ID NO:22 and LCDR 3 of SEQ ID NO:23; c) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding portion under a), the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (according to Kabat numbering) (most particularly substituted by arginine (R)), and wherein in the constant domain CH1 of the first antigen-binding portion under a), the amino acid at position 147 is substituted by glutamic acid (E) (according to Kabat EU index numbering), and the amino acid at position 213 is substituted by glutamic acid (E) (according to Kabat EU index numbering). EU index numbering); and wherein (i) the first antigen binding moiety under 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 moiety under b), and the second antigen binding moiety under 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 under c), or (ii) the second antigen binding moiety under 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 moiety under a), and the first antigen binding moiety under 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 under c).

[0202] In a specific embodiment, the bispecific antigen binding molecule comprises: a) a first antigen binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen binding moiety is a Fab molecule, the Fab molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and the light chain variable region comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; 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, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and wherein the Fab molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising SEQ ID NO: The heavy chain variable region comprises a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20, and the light chain variable region comprises a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23. 3; c) a third antigen binding moiety that binds to the first antigen and is identical to the first antigen binding moiety; and d) an Fc domain composed of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen binding moiety under a) and of the third antigen binding moiety under c), the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (according to Kabat numbering) (most particularly by arginine (R)), and wherein in the constant domain CH1 of the first antigen binding moiety under a) and of the third antigen binding moiety under c), the amino acid at position 147 is substituted by glutamic acid (E) (according to the Kabat EU index numbering), and the amino acid at position 213 is substituted by glutamic acid (E) (according to the Kabat EU index numbering);and wherein (i) the first antigen binding moiety under 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 moiety under b), and the second antigen binding moiety under b) and the third antigen binding moiety under 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 under d), or (ii) the second antigen binding moiety under 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 moiety under a), and the first antigen binding moiety under a) and the third antigen binding moiety under 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 under d).;

[0203] In another embodiment, the bispecific antigen binding molecule comprises: a) a first antigen binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen binding moiety is a Fab molecule, the Fab molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and the light chain variable region comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; 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, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and wherein the Fab molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising SEQ ID NO: NO:18 heavy chain complementary determining region (HCDR) 1, HCDR 2 of SEQ ID NO:19 and HCDR 3 of SEQ ID NO:20, the light chain variable region comprises a light chain complementary determining region (LCDR) 1 of SEQ ID NO:21, LCDR 2 of SEQ ID NO:22 and LCDR 3 of SEQ ID NO:23; c) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding portion under a), the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (according to Kabat numbering) (most particularly substituted by arginine (R)), and wherein in the constant domain CH1 of the first antigen-binding portion under a), the amino acid at position 147 is substituted by glutamic acid (E) (according to Kabat EU index numbering), and the amino acid at position 213 is substituted by glutamic acid (E) (according to Kabat EU index numbering). EU index numbering); and wherein the first antigen binding moiety under a) and the second antigen binding moiety under b) 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 under c).

[0204] According to any of the above embodiments, the components of the bispecific antigen binding molecule (e.g., Fab molecules, Fc domains) can be fused directly or through various 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 Peptide linkers, wherein "n" is generally an integer from 1 to 10, usually from 2 to 4.

[0205] In a specific aspect, the bispecific antigen binding molecule comprises: a) a first and a third antigen binding moiety that binds to a first antigen; wherein the first antigen is GPRC5D, and wherein the first and the second antigen binding moiety are each a (conventional) Fab molecule, the Fab molecule comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, the light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; b) a second antigen binding moiety that binds to a second antigen; wherein the second antigen is CD3 and wherein the second antigen binding moiety is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, the Fab molecule comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24, the light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. NO:25 amino acid sequence; c) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first and third antigen-binding moieties under a), the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (according to Kabat numbering) (most particularly substituted by arginine (R)); and wherein in the constant domain CH1 of the first and third antigen-binding moieties under a), the amino acid at position 147 is substituted by glutamic acid (E) (according to Kabat EU index numbering), and the amino acid at position 213 is substituted by glutamic acid (E) (according to Kabat EU index numbering). EU index numbering); and wherein further the first antigen binding moiety under 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 moiety under b), and the second antigen binding moiety under b) and the third antigen binding moiety under 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 under c).

[0206] 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), and 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).

[0207] In a further embodiment, in the first subunit of the Fc domain of the bispecific antigen binding molecule, additionally the serine residue at position 354 is replaced by a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (particularly the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second subunit of the Fc domain, additionally the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index).

[0208] In a further embodiment, in each of the first subunit and the second subunit 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).

[0209] In a further embodiment, the Fc domain is a human IgG1 domain.

[0210] In another specific embodiment, the bispecific antigen binding molecule includes: a polypeptide containing an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26, a polypeptide containing an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27, a polypeptide containing an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, and a polypeptide containing an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29. In another specific embodiment, the bispecific antigen binding molecule includes: a polypeptide containing an amino acid sequence such as SEQ ID NO: 26, a polypeptide containing an amino acid sequence such as SEQ ID NO: 27, a polypeptide containing an amino acid sequence such as SEQ ID NO: 28, and a polypeptide containing an amino acid sequence such as SEQ ID NO: 29. In one embodiment, the bispecific antigen binding molecule is voritatumomab.

[0211] Fc domain

[0212] In certain embodiments, the bispecific antigen binding molecule comprises an Fc domain composed of a first subunit and a second subunit.

[0213] The Fc domain of a bispecific antigen binding molecule is composed of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises a CH2 and CH3 IgG heavy chain constant domain. The two subunits of the Fc domain can stably associate with each other. In one embodiment, the bispecific antigen binding molecule of the present invention comprises no more than one Fc domain.

[0214] 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, which comprises an amino acid substitution (Kabat EU index number) at position S228, particularly an amino acid substitution S228P. The amino acid substitution reduces the in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In another 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. The exemplary sequence of the human IgG1 Fc region is given in SEQ ID NO:6.

[0215] Fc domain modifications that promote heterodimerization

[0216] Bispecific antigen binding molecules include different antigen binding moieties fused to one or the other of the two subunits of the Fc domain, so the two subunits of the Fc domain are usually contained in two different polypeptide chains. The recombinant co-expression of these polypeptides and subsequent dimerization have resulted in several possible combinations of two polypeptides. In order to improve the productivity and purity of bispecific antigen binding molecules in recombinant production, it is therefore advantageous to introduce modifications that promote the association of the desired polypeptide in the Fc domain of the bispecific antigen binding molecules.

[0217] Thus, in a specific embodiment, the Fc domain of the bispecific antigen binding molecule comprises a modification that promotes the association of the first subunit and the second subunit of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is located in the CH3 domain of the Fc domain.

[0218] There are several methods for modifying the CH3 domain of the Fc domain to implement heterodimerization, which are described in detail in, for example, WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO2013157954, WO 2013096291. Typically, in all such methods, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are engineered in a complementary manner so that each CH3 domain (or a heavy chain comprising it) can no longer homodimerize with itself, but is forced to heterodimerize with other complementary engineered CH3 domains (so that the first and second CH3 domains heterodimerize and no homodimer is formed between the two first or two second CH3 domains). These different methods for achieving improved heavy chain heterodimerization are considered to be different alternatives combined with heavy-light chain modifications in bispecific antigen-binding molecules (e.g., VH and VL exchange / replacement in one binding arm and introduction of substitutions of charged amino acids with opposite charges in the CH1 / CL interface) that reduce heavy chain / light chain mispairing and Bence Jones-type side products.

[0219] In a specific embodiment, the modification that promotes the association of the first subunit and the second subunit of the Fc domain is a so-called "protrusion into hole" modification, which comprises a "protrusion" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

[0220] The knob-and-hole technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide so that the protrusion can be positioned in the cavity to promote the formation of heterodimers and hinder the formation of homodimers. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity having the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0221] Therefore, in a specific embodiment, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen binding molecule, amino acid residues are substituted by amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion can be positioned in the cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, amino acid residues are substituted by amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit can be positioned in the cavity.

[0222] Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0223] Preferably, the 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).

[0224] Protrusions and cavities can be produced by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis.

[0225] In a specific embodiment, in the first subunit of the Fc domain (the "protrusion" subunit) (the CH3 domain), the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain (the "hole" subunit) (the CH3 domain), the tyrosine residue at position 407 is replaced by a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, additionally, 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).

[0226] In yet another embodiment, in the first subunit of the Fc domain, additionally, the serine residue at position 354 is replaced by a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second subunit of the Fc domain, additionally, the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index). The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0227] In a specific embodiment, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0228] In a specific embodiment, an antigen binding moiety that binds a second antigen (e.g., an activating T cell antigen) is fused (optionally via a first antigen binding moiety that binds GPRC5D and / or a peptide linker) to a first subunit of an Fc domain (comprising a "knob" modification). Without wishing to be bound by theory, fusion of an antigen binding moiety that binds a second antigen (e.g., an activating T cell antigen) to a knob-containing subunit of an Fc domain will (further) minimize the generation of antigen binding molecules comprising two antigen binding moieties that bind an activating T cell antigen (steric clash of two knob-containing polypeptides).

[0229] Other CH3 modification techniques for implementing heterodimerization are envisioned 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, WO2013 / 157954, WO 2013 / 096291.

[0230] In one embodiment, the heterodimerization method described in EP 1870459 is used alternatively. The method is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. A preferred embodiment of the bispecific antigen binding molecule of the present invention is the amino acid mutation R409D; K370E in one of the two CH3 domains (of the Fc domain), and the amino acid mutation D399K; E357K in the other CH3 domain of the Fc domain (numbering according to the Kabat EU index).

[0231] 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, and additionally the amino acid mutation R409D in the CH3 domain of the second subunit of the Fc domain; K370E in the CH3 domain of the first subunit of the Fc domain, and the amino acid mutation D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numbering according to the Kabat EU index).

[0232] 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 the 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 additionally the amino acid mutation R409D; K370E in the CH3 domain of the first subunit of the Fc domain, and the amino acid mutation D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numbered according to the Kabat EU index).

[0233] In one embodiment, the heterodimerization method 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 another embodiment, the first CH3 domain comprises an additional amino acid mutation L351K. In another embodiment, the second CH3 domain further comprises an amino acid mutation selected from the group consisting of Y349E, Y349D and L368E (preferably L368E) (numbering according to the Kabat EU index).

[0234] In one embodiment, the heterodimerization method described in WO 2012 / 058768 is used instead. In one embodiment, the first CH3 domain comprises amino acid mutations L351Y, Y407A, and the second CH3 domain comprises amino acid mutations T366A, K409F. In another embodiment, the second CH3 domain comprises further amino acid mutations at positions T411, D399, S400, F405, N390 or K392, for example selected from the group consisting of: a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numbering according to Kabat EU index). In another embodiment, the first CH3 domain comprises amino acid mutations L351Y, Y407A, and the second CH3 domain comprises amino acid mutations T366V, K409F. In another embodiment, the first CH3 domain comprises amino acid mutations Y407A, and the second CH3 domain comprises amino acid mutations T366A, K409F. In another embodiment, the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (numbered according to the Kabat EU index).

[0235] In one embodiment, the heterodimerization method described in WO 2011 / 143545 is used instead, for example, an amino acid modification is made at a position selected from the group consisting of 368 and 409 (numbered according to the Kabat EU index).

[0236] In one embodiment, the heterodimerization method described in WO 2011 / 090762 is used instead, which also uses the above-mentioned protrusion-into-hole technology. 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).

[0237] In one embodiment, the bispecific antigen binding molecule or its Fc domain is of IgG2 subclass and the heterodimerization method described in WO 2010 / 129304 is used instead.

[0238] In an alternative embodiment, the modification that promotes the association of the first subunit and the second subunit of the Fc domain includes a modification that mediates an electrostatic steering effect, for example as described in PCT Publication WO 2009 / 089004. Typically, the 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 becomes electrostatically unfavorable, but heterodimerization is electrostatically favorable. In one such embodiment, the first CH3 domain comprises an amino acid substitution with a negatively charged amino acid pair K392 or N392 (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain comprises an amino acid substitution with a positively charged amino acid pair D399, E356, D356 or E357 (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, more preferably D399K and E356K). In another 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 another embodiment, the first CH3 domain further or alternatively comprises an amino acid substitution of 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).

[0239] In yet another embodiment, the heterodimerization method described in WO 2007 / 147901 is used instead.In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K and K322D, and the second CH3 domain comprises the amino acid mutations D239K, E240K and K292D (numbered according to the Kabat EU index).

[0240] In yet another embodiment, the heterodimerization method described in WO 2007 / 110205 may be used instead.

[0241] In one embodiment, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to the Kabat EU index).

[0242] Fc domain modifications that reduce Fc receptor binding and / or effector function

[0243] The Fc domain gives the bispecific antigen binding molecule favorable pharmacokinetic properties, including a long serum half-life and a favorable tissue-blood distribution ratio that contribute to good accumulation in the target tissue. However, at the same time it may result in the bispecific antigen binding molecule being undesirably targeted to cells expressing Fc receptors, rather than cells that preferably carry antigens. In addition, the co-activation of the Fc receptor signaling pathway can lead to cytokine release, which is combined with T cell activation characteristics (e.g., in the embodiment of the bispecific antigen binding molecule, wherein the second antigen binding moiety is combined with activated T cell antigens) and the long half-life of the bispecific antigen binding molecule, resulting in excessive activation of cytokine receptors and serious side effects during systemic administration. Due to the potential destruction of T cells (e.g., by NK cells), the activation of immune cells (with Fc receptors) other than T cells may even reduce the efficacy of the bispecific antigen binding molecule (particularly the bispecific antigen binding molecule in which the second antigen binding moiety is combined with activated T cell antigens).

[0244] Thus, in a particular embodiment, the Fc domain of the bispecific antigen binding molecule exhibits reduced binding affinity to Fc receptors and / or reduced effector functions compared to the native IgG1 Fc domain. In one such embodiment, the Fc domain (or a bispecific antigen binding molecule comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% binding affinity to Fc receptors compared to the native IgG1 Fc domain (or a bispecific antigen binding molecule comprising the native IgG1 Fc domain), and / or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% effector functions compared to the native IgG1 Fc domain (or a bispecific antigen binding molecule comprising the native IgG1 Fc domain). In one embodiment, the Fc domain (or a bispecific antigen binding molecule comprising the Fc domain) does not substantially bind to the Fc receptor and / or induce effector functions. In a particular 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 activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more particularly human FcγRIIIa, FcγRI or FcγRIIa, most particularly human FcγRIIIa. In one embodiment, the effector function is one or more effector functions selected from the group of CDC, ADCC, ADCP and cytokine secretion. In a specific embodiment, the effector function is ADCC. In one embodiment, compared with the natural IgG1 Fc domain, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn). When the Fc domain (or the bispecific antigen binding molecule comprising the Fc domain) exhibits a binding affinity of the natural IgG1 Fc domain (or the bispecific antigen binding molecule comprising the natural IgG1 Fc domain) to FcRn greater than about 70%, particularly greater than about 80%, more particularly greater than about 90%, substantially similar binding to FcRn is achieved.

[0245] In certain embodiments, the Fc domain is engineered to have a reduced binding affinity to Fc receptors and / or reduced effector functions compared to a non-engineered Fc domain. In a specific embodiment, the Fc domain of a bispecific antigen binding molecule comprises one or more amino acid mutations, which reduce the binding affinity and / or effector functions of the Fc domain to Fc receptors. 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 mutation reduces the binding affinity of the Fc domain to Fc receptors. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to Fc receptors by at least 2 times, at least 5 times, or at least 10 times. In the embodiment where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to Fc receptors, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to Fc receptors by at least 10 times, at least 20 times, or even at least 50 times. In one embodiment, compared with the bispecific antigen binding molecule comprising a non-engineered Fc domain, the bispecific antigen binding molecule comprising an engineered Fc domain exhibits less than 20% of the binding affinity to the Fc receptor, particularly less than 10%, and more particularly less than 5%. 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 activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more particularly human FcγRIIIa, FcγRI or FcγRIIa, most particularly human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some embodiments, the binding affinity to complement components, particularly the binding affinity to C1q, is also reduced. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. When the Fc domain (or the bispecific antigen binding molecule comprising the Fc domain) exhibits greater than about 70% of the binding affinity of the non-engineered form of the Fc domain (or the bispecific antigen binding molecule comprising the non-engineered form of the Fc domain) to FcRn, substantially similar binding to FcRn is achieved, i.e., the binding affinity of the Fc domain to the receptor is maintained. The Fc domain or the bispecific antigen binding molecule of the present invention comprising the Fc domain may exhibit greater than about 80% or even greater than about 90% of this affinity. In certain embodiments, the Fc domain of the bispecific antigen binding molecule is engineered to have reduced effector function compared to a non-engineered Fc domain.The effector function of reduction may include but is not limited to one or more of the following items: reduced complement dependent cytotoxicity (CDC), reduced antibody dependent cell-mediated cytotoxicity (ADCC), reduced antibody dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen presenting cells to antigen uptake, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling induced apoptosis, reduced target binding antibody crosslinking, reduced dendritic cell maturation, or reduced T cell sensitization. In one embodiment, the effector function of reduction is selected from the group of reduced CDC, reduced ADCC, reduced ADCP and reduced cytokine secretion. In a specific embodiment, the effector function of reduction is reduced ADCC. In one embodiment, the ADCC reduced is less than 20% of the ADCC induced by non-engineered Fc domains (or bispecific antigen binding molecules comprising the non-engineered Fc domains).

[0246] In one embodiment, the amino acid mutation that reduces the binding affinity of Fc domain to Fc receptor and / or effector function is amino acid substitution.In one embodiment, Fc domain is included in the amino acid substitution (according to Kabat EU index numbering) at the position of the group selected from E233, L234, L235, N297, P331 and P329.In a more specific embodiment, Fc domain is included in the amino acid substitution (according to Kabat EU index numbering) at the position of the group selected from L234, L235 and P329.In certain embodiments, Fc domain includes amino acid substitution L234A and L235A (according to Kabat EU index numbering).In such an embodiment, Fc domain is IgG1 Fc domain, particularly human IgG1 Fc domain.In one embodiment, Fc domain is included in the amino acid substitution at position P329.In a more specific embodiment, amino acid substitution is P329A or P329G, particularly P329G (according to Kabat EU index numbering). In one embodiment, the Fc domains are included in the amino acid replacement at position P329, and further amino acid replacement (according to Kabat EU index numbering) at a position selected from E233, L234, L235, N297 and P331. In a more specific embodiment, the further amino acid replacement is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a specific embodiment, the Fc domains are included in the amino acid replacement (according to Kabat EU index numbering) at position P329, L234 and L235. In a more specific embodiment, the Fc domains include amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). Specifically, in a specific embodiment, each subunit of the Fc domain comprises amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e., in each of the first subunit and the second subunit of the Fc domain, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (according to the Kabat EU index numbering).

[0247] In one such embodiment, the Fc domain is an IgG1 Fc domain, in particular a human IgG1 Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely eliminates Fcγ receptor (and complement) binding of human IgG1 Fc domains, as described in PCT Publication No. WO 2012 / 130831, the entire contents of which are incorporated herein by reference. WO2012 / 130831 also describes methods for preparing such mutant Fc domains and methods for determining their properties (such as Fc receptor binding or effector function).

[0248] Compared with IgG1 antibody, IgG4 antibody shows reduced binding affinity to Fc receptors and reduced effector function.Therefore, in some embodiments, the Fc domain of bispecific antigen binding molecule is IgG4 Fc domain, particularly human IgG4 Fc domain.In one embodiment, IgG4 Fc domain is included in the amino acid replacement at position S228, particularly amino acid replacement S228P (according to Kabat EU index numbering).In order to further reduce its binding affinity to Fc receptors and / or its effector function, in one embodiment, IgG4 Fc domain includes amino acid replacement at position L235, particularly amino acid replacement L235E (according to Kabat EU index numbering).In another embodiment, IgG4 Fc domain includes amino acid replacement at position P329, particularly amino acid replacement P329G (according to Kabat EU index numbering). In a specific embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, particularly amino acid substitutions S228P, L235E and P329G (numbered according to the Kabat EU index). Such IgG4 Fc domain mutants and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012 / 130831, the entire contents of which are incorporated herein by reference.

[0249] In a specific embodiment, 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 amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising amino acid substitutions S228P, L235E and optionally P329G (numbered according to the Kabat EU index).

[0250] In certain embodiments, N-glycosylation of the Fc domain has been eliminated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, in particular an amino acid substitution replacing asparagine with alanine (N297A) or aspartic acid (N297D) (numbering according to the Kabat EU index).

[0251] In addition to the Fc domains described above and in PCT Publication No. WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or reduced effector function also include those Fc domains with substitutions of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbered according to the KabatEU index). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including so-called "DANA" Fc mutants, in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0252] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods can include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The correct nucleotide changes can be verified, for example, by sequencing.

[0253] Binding to Fc receptors can be easily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare), and Fc receptors such as can be obtained by recombinant expression. Alternatively, cell lines known to express specific Fc receptors (such as human NK cells expressing FcγIIIa receptors) can be used to assess the binding affinity of the Fc domain or a bispecific antigen binding molecule comprising the Fc domain to the Fc receptor.

[0254] The effector function of the bispecific antigen binding molecule of the Fc domain or comprising the Fc domain can be measured by methods known in the art. The example of in vitro determination for evaluating the ADCC activity of the molecule of interest is 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 determination methods (see, for example, ACTI for flow cytometry) can be used. TM Nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0255] In some embodiments, the binding of the Fc domain to complement components, particularly C1q, is reduced. Therefore, in some embodiments, wherein the Fc domain is engineered to have reduced effector functions, the reduced effector functions including reduced CDC. A C1q binding assay can be performed to determine whether the Fc domain or the bispecific antigen binding molecule comprising the Fc domain is able to bind to C1q and therefore has CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. In order to assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0256] FcRn binding and in vivo clearance / half-life assays 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).

[0257] Composition, formulation and route of administration

[0258] In a further aspect, the present invention provides a pharmaceutical composition comprising any of the antibodies or bispecific antigen binding molecules provided herein, for example, for use in any of the following treatment methods. In one embodiment, the pharmaceutical composition comprises any of the antibodies or bispecific antigen binding molecules provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the antibodies or bispecific antigen binding molecules provided herein and at least one additional therapeutic agent as described below.

[0259] Also provided is 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, thereby formulating the antibody or bispecific antigen binding molecule preparation for in vivo administration.

[0260] The pharmaceutical composition of the present invention comprises a therapeutically effective amount of an antibody or bispecific antigen binding molecule dissolved or dispersed in a pharmaceutical carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to that molecular entities and compositions are generally nontoxic to recipients at the dosage and concentration adopted, i.e., when applied to animals (such as, for example, people) as appropriate, no adverse, allergic or other adverse reactions will be produced. The preparation of a pharmaceutical composition containing an antibody or bispecific antigen binding molecule and optionally additional active ingredients will be known to those skilled in the art in view of the present disclosure, as illustrated by Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, the document is incorporated herein by reference. In addition, for animal (e.g., people) administration, it should be understood that the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Biological Standards Office or other countries / regions corresponding authorities. Preferred compositions are lyophilized preparations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such and similar substances and combinations thereof, as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Except in the case where any conventional carrier is incompatible with the active ingredient, the use of such carrier in the therapeutic or pharmaceutical composition is contemplated.

[0261] The immunoconjugates of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic.

[0262] Parenteral compositions include those designed for use by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection). For injection, the antibody or bispecific antigen binding molecule of the present invention can be prepared in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks solution, Ringer's solution or saline. The solution may contain a formulation (formulatory agent), such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the antibody or bispecific antigen binding molecule can be in powder form for use with a suitable vehicle (e.g., sterile pyrogen-free water) before use. As required, a sterile injectable solution is prepared by incorporating the antibody or bispecific antigen binding molecule of the present invention into a suitable solvent with various other ingredients listed below in the desired amount. For example, sterility can be easily achieved by filtering with a sterile filtration membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile solvent containing a basic dispersion medium and / or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred method of preparation is vacuum drying or freeze drying techniques, which produce a powder of the active ingredient plus any additional required ingredients from a previously sterile filtered liquid medium. If necessary, the liquid medium should be appropriately buffered, and sufficient saline or glucose should first be used to make the liquid diluent isotonic before injection. The composition must be stable under manufacturing and storage conditions and is preserved to resist the contaminating effects of microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a minimum, for example, at a safety level below 0.5 ng / mg protein. Suitable pharmaceutical carriers include, but are not limited to, buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol 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; proteins such as Serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinyl pyrrolidone; 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., zinc protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, etc.Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound to allow the preparation of a highly concentrated solution. In addition, the suspension of the active compound may be prepared as an appropriate oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil; or synthetic fatty acid esters such as ethyl oleate or triglycerides; or liposomes.

[0263] The active ingredient can be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization; embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or embedded in coarse emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Printing Company, 1990). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing polypeptides in the form of molded articles such as films or microcapsules. In particular embodiments, extended absorption of injectable compositions can be achieved by using agents that delay absorption in the composition (such as, for example, aluminum monostearate, gelatin, or a combination thereof).

[0264] In addition to the compositions described previously, antibodies or bispecific antigen binding molecules can also be formulated into long-acting preparations. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular implantation) or by intramuscular injection. Therefore, for example, antibodies or bispecific antigen binding molecules can be formulated with suitable polymerization or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or with ion exchange resins, or formulated as slightly soluble derivatives, such as, for example, as slightly soluble salts.

[0265] Pharmaceutical compositions comprising antibodies or bispecific antigen binding molecules of the present invention can be produced by means of conventional mixing, dissolving, emulsifying, encapsulating, embedding or lyophilizing processes. Pharmaceutical compositions can be prepared in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants, which help process the protein into a pharmaceutically usable preparation. Appropriate formulations depend on the selected route of administration.

[0266] The antibody or bispecific antigen binding molecule can be formulated into a composition in the form of a free acid or base, neutral or salt. A pharmaceutically acceptable salt is a salt that substantially retains the biological activity of a free acid or free base. These pharmaceutically acceptable salts include acid addition salts, such as acid addition salts formed with the free amino groups of a protein composition, or acid addition salts formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases, such as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0267] Immunomodulatory imide drugs (IMiDs)

[0268] The term "immunomodulatory imide drugs (IMiD)" refers to a class of immunomodulatory drugs (drugs that regulate immune responses) containing an imide group. IMiDs include both first-generation IMiDs and Cereblon E3 ligase modulators (CELMoDs; also known as next-generation IMiDs), both of which contain a conserved glutarimide ring. The first and next-generation IMiDs bind to Cereblon (CRBN), a receptor for the Cullin ring 4 ubiquitin ligase (CRL4) complex, and regulate ubiquitin ligase activity. Ligase specificity is redirected to non-physiological protein targets, which are also known as "new substrates", which are subsequently ubiquitinated and / or degraded. The conserved glutarimide ring binds to CRBN, while the variable side groups interact with CRBN and new substrates. The term "first-generation IMiD" refers to thalidomide and thalidomide derivatives lenalidomide and pomalidomide. The term "CELMoD" or "next generation IMiD" refers to thalidomide derivatives with extended side groups that are capable of improving interactions with CRBN and / or new substrates, and include but are not limited to ibrolimumab (also known as CC-220), avadomide (also known as CC-122), mezigimide (also known as CC-92480), CC885, CC647, CC-90009, and CC3060.

[0269] The term "lenalidomide" refers to the compound with the following chemical structure:

[0270]

[0271] The empirical formula of lenalidomide is C 13 H 13 N3O3, CAS registration number 191732-72-6, and molecular weight 259.3. Lenalidomide is a thalidomide analog, sold under the trade name Sale.

[0272] The term "pomalidomide" refers to the compound with the following chemical structure:

[0273]

[0274] The empirical formula of pomalidomide is C 13 H 11 N3O4, CAS Registry Number 19171-19-8, and Molecular Weight 273.24. Pomalidomide is a thalidomide analog, sold under the trade name Imnovid in the EU and Pomalyst in the US.

[0275] The term "ibolidomide" refers to a compound with the following chemical structure:

[0276]

[0277] The empirical formula of ibolidomide is C 25 H 27 N3O5, CAS registration number is 1323403-33-3, and the molecular weight is 449.5.

[0278] The term "mezigimide" refers to a compound having the following chemical structure:

[0279]

[0280] The empirical formula of Mezigmet is C 32 H 30 FN5O4, CAS registration number is 2259648-80-9, and the molecular weight is 567.6.

[0281] The present invention provides a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD). The IMiD for the combination therapy described herein may 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 ibrolimumide, avalimumide, mezigamide (CC-92480), CC885, CC647, CC-90009 and CC3060. In one embodiment, the IMiD is selected from the group of lenalidomide, pomalidomide, ibrolimumide and mezigamide.

[0282] Glucocorticosteroids

[0283] The present invention further provides a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody with an immunomodulatory imide drug (IMiD) and a glucocorticoid.

[0284] As used herein, "glucocorticoid" or "glucocorticoid" refers to a class of corticosteroid compounds that participate in the metabolism of carbohydrates, proteins and fats and have anti-inflammatory activity. Glucocorticoids are mainly used in therapy for their anti-inflammatory and immunosuppressive effects. Glucocorticoids include, but are not limited to, dexamethasone, prednisone, prednisolone, methylprednisolone and substitutes.

[0285] The term "dexamethasone" refers to the compound with the following chemical structure:

[0286]

[0287] The empirical formula of dexamethasone is C 22 H 29 FO5, CAS registry number is 50-02-2, and the molecular weight is 392.46.

[0288] The present invention further provides a combination therapy of an anti-GPRC5D / anti-CD3 bispecific antibody, an immunomodulatory imide drug (IMiD) and a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone.

[0289] Methods of treatment and compositions

[0290] The present invention comprises a combination therapy comprising a combination of an anti-GPRC5D / anti-CD3 bispecific antibody and an IMiD. Optionally, the combination therapy described herein may further comprise a glucocorticoid.

[0291] The present invention comprises a method for treating a patient in need of treatment, characterized in that a therapeutically effective amount of an anti-GPRC5D / anti-CD3 bispecific antibody and a combination therapy of an IMiD are administered to the patient. The present invention comprises a method for treating a patient in need of treatment, characterized in that a therapeutically effective amount of an anti-GPRC5D / anti-CD3 bispecific antibody and a combination therapy of an IMiD and a glucocorticoid are administered to the patient.

[0292] A preferred embodiment of the present invention is a combination therapy of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for treating cancer or tumor. Another preferred embodiment of the present invention is a combination therapy of anti-GPRC5D / anti-CD3 bispecific antibody, IMiD and glucocorticoid for treating cancer or tumor.

[0293] One embodiment of the present invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody described herein with an IMiD as described herein for treating a tumor or cancer. One embodiment of the present invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody described herein with an IMiD as described herein and a glucocorticoid as described herein for treating a tumor or cancer.

[0294] Another embodiment of the present invention is a combination of an IMiD described herein with an anti-GPRC5D / anti-CD3 bispecific antibody as described herein for use in treating cancer or tumors. Another embodiment of the present invention is a combination of an IMiD described herein with an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and a glucocorticoid as described herein for use in treating cancer or tumors.

[0295] A further embodiment is the combination of a glucocorticoid as described herein with an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and an IMiD as described herein for use in the treatment of cancer or tumors.

[0296] 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, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferation disorders that can be treated with the combination therapy of the present invention include, but are not limited to, tumors located in the following parts: abdomen, bones, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary glands, testicles, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral nervous system), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital system. Precancerous conditions or lesions and cancer metastasis are also included. In certain embodiments, 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, cancer is a cancer expressing GPRC5D. In one embodiment, the cancer is multiple myeloma.

[0297] An embodiment of the invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein with an IMiD as described herein for treating any of the cancers or tumors described above. An embodiment of the invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein with an IMiD and a glucocorticoid as described herein for treating any of the cancers or tumors described above.

[0298] An embodiment of the invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein with an IMiD as described herein for treating multiple myeloma. An embodiment of the invention is a combination of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein with an IMiD and a glucocorticoid as described herein for treating multiple myeloma.

[0299] The present invention comprises a method for treating a patient in need of treatment, characterized in that a therapeutically effective amount of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and a combination therapy of an IMiD as described herein are administered to the patient. The present invention further comprises a method for treating a patient in need of treatment, characterized in that a therapeutically effective amount of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and a combination therapy of an IMiD as described herein and a glucocorticoid as described herein are administered to the patient.

[0300] The present invention comprises a method of treating cancer in an individual, the method comprising administering to the individual a combination of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and an IMiD as described herein. The present invention further comprises a method of treating cancer in an individual, the method comprising administering to the individual a combination of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and an IMiD as described herein and a glucocorticoid as described herein.

[0301] The present invention comprises a method for preventing or treating tumor metastasis in a patient in need of treatment, characterized in that a therapeutically effective amount of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and a combination therapy of an IMiD as described herein are administered to the patient. The present invention further comprises a method for preventing or treating tumor metastasis in a patient in need of treatment, characterized in that a therapeutically effective amount of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and a combination therapy of an IMiD as described herein and a glucocorticoid as described herein are administered to the patient.

[0302] The present invention comprises the use of the anti-GPRC5D / anti-CD3 bispecific antibody according to the present invention together with IMiD for the described combination therapy. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined therapy and medical use described above 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 for the combined therapy and medical use described above is selected from the group of lenalidomide, pomalidomide, ibolidomide and mezigamide. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined therapy and medical use described above 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, ibolidomide and mezigamide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for combined therapy and medical use described above 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 lenalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for combined therapy and medical use described above 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 pomalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for combined therapy and medical use described above 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 ibolidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined treatment and medical use described above 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 mezigamide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined treatment and medical use described above is voritumimab, and the IMiD is lenalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined treatment and medical use described above is voritumimab, and the IMiD is pomalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined treatment and medical use described above is voritumimab, and the IMiD is ibolidomide.In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined therapy and medical use described above is voritumimab, and the IMiD is mezigimib.

[0303] The present invention comprises the use of an anti-GPRC5D / anti-CD3 bispecific antibody according to the present invention together with an IMiD and a glucocorticoid for the described combination therapy.

[0304] In a preferred embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined treatment and medical use described above 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 for the combined treatment and medical use described above is selected from the group of lenalidomide, pomalidomide, ibolidomide and mezigamide. In a further embodiment, the glucocorticoid for the combined treatment and medical use described above is dexamethasone. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined treatment and medical use described above comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQID NO: 29, and the IMiD for the combined treatment and medical use described above is selected from the group of lenalidomide, pomalidomide, ibolidomide and mezigamide. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined treatment and medical use described above comprises the polypeptide sequence 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, ibrolidomide and mezigamide, and the glucocorticoid is dexamethasone. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined treatment and medical use described above comprises the polypeptide sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQID NO: 29, and the IMiD is lenalidomide, and the glucocorticoid is dexamethasone. In another embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the combined treatment and medical use described above is voritumimab, and the IMiD is lenalidomide, and the glucocorticoid is dexamethasone.

[0305] The present invention comprises a combination of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and an IMiD as described herein, for use in the manufacture of a medicament for the treatment of cancer. The present invention comprises a combination of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and an IMiD as described herein and a glucocorticoid as described herein, for use in the manufacture of a medicament for the treatment of cancer.

[0306] In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, comprising an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and an IMiD as described herein, formulated with a pharmaceutical carrier. In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, comprising an anti-GPRC5D / anti-CD3 bispecific antibody as described herein, an IMiD as described herein, and a glucocorticoid as described herein, formulated with a pharmaceutical carrier.

[0307] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption / resorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for injection or infusion.

[0308] The compositions of the present invention can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results.

[0309] Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions. Such media and agents are known in the art for use in pharmaceutically active substances. In addition to water, the carrier can also be, for example, an isotonic buffered saline solution.

[0310] Regardless of the administration route selected, the compound of the present invention and / or the pharmaceutical composition of the present invention, which can be used in a suitable hydrated form, can be formulated into a pharmaceutical dosage form by conventional methods known to those skilled in the art.

[0311] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition and mode of administration without causing toxicity to the patient (effective amount). The dosage may include an increasing dosing cycle of the active ingredient. The term "dose" refers to the amount (i.e., dosage) and frequency of administration of the active ingredient. The term "increasing dosing cycle" refers to a treatment period during which the dosage of the active ingredient gradually increases during the treatment period. This can be achieved by increasing the dosage of the active ingredient and / or increasing the frequency of administration. Therefore, in one embodiment, the dosage of the combination of anti-GPRC5D / anti-CD3 bispecific antibodies and IMiDs as described herein includes at least one increasing dosing cycle. In one embodiment, the dosage of the combination of anti-GPRC5D / anti-CD3 bispecific antibodies and IMiDs as described herein includes at least one increasing dosing cycle of anti-GPRC5D / anti-CD3 bispecific antibodies. In one embodiment, the dosage of the combination of anti-GPRC5D / anti-CD3 bispecific antibodies and IMiDs as described herein includes at least one increasing dosing cycle of anti-GPRC5D / anti-CD3 bispecific antibodies. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for combined therapy and medical use as described herein is administered in an effective amount, wherein the dose comprises at least one ascending dosing cycle. In one embodiment, voritumimab for combined therapy and medical use as described herein is administered in an effective amount, wherein the dose comprises at least one ascending dosing cycle. The selected dose level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the invention or its ester, salt or amide, the route of administration, the time of administration, the excretion rate of the specific compound employed, other drugs, compounds and / or materials used in combination with the specific composition employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0312] The present invention comprises a combination of an IMiD as described herein and an anti-GPRC5D / anti-CD3 bispecific antibody as described herein, which is used to manufacture a medicament for treating cancer. The present invention comprises a combination of an IMiD as described herein and an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and a glucocorticoid as described herein, which is used to manufacture a medicament for treating cancer. The present invention comprises a combination of a glucocorticoid as described herein and an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and an IMiD as described herein. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention is voritumimab. In one embodiment, the IMiD for the manufacture of a medicament for treating cancer according to the present invention is selected from the group of lenalidomide, pomalidomide, ibolidomide and mezigamide. In one embodiment, the glucocorticoid for the manufacture of a medicament for treating cancer according to the present invention is dexamethasone. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is selected from the group of lenalidomide, pomalidomide, ibrolidomide and mezigamide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is lenalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is pomalidomide.In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is ibrolimumab. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is mezigamide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention is voritumimab, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is lenalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention is voritumimab, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is pomalidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention is voritumimab, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is ibolidomide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention is voritumimab, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is mezigamide. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is selected from the group of lenalidomide, pomalidomide, ibrolidomide and mezigamide, and the glucocorticoid for the manufacture of a medicament for treating cancer according to the present invention is dexamethasone. In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is lenalidomide, and the glucocorticoid for the manufacture of a medicament for treating cancer according to the present invention is dexamethasone.In one embodiment, the anti-GPRC5D / anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention is voritumimab, and the IMiD for the manufacture of a medicament for treating cancer according to the present invention is lenalidomide, and the glucocorticoid for the manufacture of a medicament for treating cancer according to the present invention is dexamethasone.

[0313] The present invention further provides the use of an anti-GPRC5D / anti-CD3 bispecific antibody according to the present invention as described herein and an IMiD according to the present invention as described herein in the manufacture of a pharmaceutical medicament for treating a patient suffering from cancer, preferably together with a pharmaceutically acceptable carrier. The present invention further provides the use of an anti-GPRC5D / anti-CD3 bispecific antibody according to the present invention as described herein and an IMiD according to the present invention as described herein and a glucocorticoid according to the present invention as described herein in the manufacture of a pharmaceutical medicament for treating a patient suffering from cancer, preferably together with a pharmaceutically acceptable carrier.

[0314] In one aspect, the present 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 as described herein, and (b) an IMiD as described herein, and 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 present 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 as described herein, and (b) an IMiD as described herein, (c) a glucocorticoid as described herein, and optionally further comprising (d) a package insert comprising printed instructions directing the use of the combination therapy as a method for treating the disease.

[0315] In addition, the kit may include (a) a first container containing a composition, wherein the composition comprises an anti-GPRC5D / anti-CD3 bispecific antibody as described herein; (b) a second container containing a composition, wherein the composition comprises an IMiD as described herein; and optionally (c) a third container containing a composition, wherein the composition comprises a further cytotoxic agent or other therapeutic agent. The kit in this embodiment of the invention may also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the kit may further include a fourth container, which contains a pharmaceutical buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0316] In addition, the kit may include (a) a first container containing a composition, wherein the composition comprises an anti-GPRC5D / anti-CD3 bispecific antibody as described herein; (b) a second container containing a composition, wherein the composition comprises an IMiD as described herein; (c) a third container containing a composition, wherein the composition comprises a glucocorticoid as described herein, and optionally (d) a fourth container containing a composition, wherein the composition comprises a further cytotoxic agent or other therapeutic agent. The kit in this embodiment of the invention may also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the kit may further include a fifth container, which contains a pharmaceutical buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0317] In one aspect, the present invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising an anti-GPRC5D / anti-CD3 bispecific antibody as described herein, and (b) a package insert comprising instructions for the use of the anti-GPRC5D / anti-CD3 bispecific antibody in combination therapy with an IMiD as described herein as a method for treating the disease. In one aspect, the present invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising an anti-GPRC5D / anti-CD3 bispecific antibody as described herein, and (b) a package insert comprising instructions for the use of the anti-GPRC5D / anti-CD3 bispecific antibody in combination therapy with an IMiD and a glucocorticoid as described herein as a method for treating the disease.

[0318] In another aspect, the present invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising an IMiD as described herein, and (b) a package insert comprising instructions for the use of the IMiD in a combination therapy with an anti-GPRC5D / anti-CD3 bispecific antibody as described herein as a method for treating the disease. In another aspect, the present invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising an IMiD as described herein, and (b) a package insert comprising instructions for the use of the IMiD in a combination therapy with an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and a glucocorticoid as a method for treating the disease.

[0319] In another aspect, the present invention provides a kit intended for use in treating a disease, comprising (a) a container comprising a glucocorticoid as described herein, and (b) a package insert comprising instructions directing the use of the glucocorticoid in combination therapy with an anti-GPRC5D / anti-CD3 bispecific antibody and IMiD as described herein as a method of treating the disease.

[0320] In a further aspect, the present invention provides a medicament intended for use in the treatment of a disease, comprising an anti-GPRC5D / anti-CD3 bispecific antibody as described herein, wherein the medicament is used in combination therapy with an IMiD as described herein and optionally comprises a package insert comprising printed instructions directing the use of the combination therapy as a method for treating the disease. In a further aspect, the present invention provides a medicament intended for use in the treatment of a disease, comprising an anti-GPRC5D / anti-CD3 bispecific antibody as described herein, wherein the medicament is used in combination therapy with an IMiD and a glucocorticoid as described herein and optionally comprises a package insert comprising printed instructions directing the use of the combination therapy as a method for treating the disease.

[0321] The term "treatment" or its equivalent, when applied to, for example, cancer, refers to a procedure or course of action intended to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of cancer. A "treatment" of cancer or another proliferative disease does not necessarily mean that the cancer cells or other disease will actually be eliminated, that the number of cells or the disease will actually be reduced, or that the cancer or other disease will actually be in remission. In general, a treatment for cancer may have a low probability of success but is still considered to induce an overall beneficial course of action, given the patient's medical history and estimated survival expectancy.

[0322] The term "combination administration" or "co-administration", "co-administration", "combination therapy" or "combination treatment" refers to the administration of an anti-GPRC5D / anti-CD3 bispecific antibody as described herein and an IMiD as described herein and optionally a glucocorticoid, for example as separate formulations / applications (or as a single formulation / application). Co-administration can be performed simultaneously or sequentially in any order, wherein preferably there is a time period during which all active agents exert their biological activities simultaneously. The active agents are co-administered simultaneously or sequentially by continuous infusion or oral administration (e.g., intravenously (iv)). When all therapeutic agents are co-administered sequentially, they can be administered in two separate administrations on the same day, or one of the agents can be administered on day 1, and the second agent can be co-administered on day 2 to day 7, preferably day 2 to day 4. Therefore, in one embodiment, the term "sequentially" means within 7 days after administration of the first component, preferably 4 days after administration of the first component; and the term "simultaneously" means preferably at the same time. The term "co-administered" with respect to maintenance doses of anti-GPRC5D / anti-CD3 bispecific antibodies and / or IMiDs and / or (where applicable) glucocorticoids means that if the treatment cycle is applicable for all drugs, e.g. weekly, the maintenance doses can be co-administered simultaneously.

[0323] It is understood that the antibody is administered to a patient in a "therapeutically effective amount" (or simply "effective amount"), which is the amount of the corresponding compound or combination that will elicit the biological or medical response of a tissue, system, animal or human that the researcher, veterinarian, medical doctor or other clinician is seeking.

[0324] The amount of co-administration and the timing of co-administration will depend on the type (species, sex, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated. The anti-GPRC5D / anti-CD3 bispecific antibody and / or IMiD and / or (where applicable) glucocorticoid are suitably co-administered to the patient at one time or in a series of treatments, e.g., on the same day or a subsequent day or weekly treatment.

[0325] The skilled artisan will readily recognize that in many cases, combination therapy may not provide a cure, but may only provide a partial benefit. In some embodiments, physiological changes that have some benefit are also considered therapeutically beneficial. Therefore, in some embodiments, the amount of the therapeutic combination that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount."

[0326] Aspects of the invention:

[0327] 1. A combination of an anti-GPRC5D / anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD) for use as a combination therapy for treating cancer.

[0328] 2. Use of a combination of an anti-GPRC5D / anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD) in the manufacture of a medicament for treating cancer.

[0329] 3. A method of treating cancer in an individual, the method comprising administering to the individual an anti-GPRC5D / anti-CD3 bispecific antibody in combination with an immunomodulatory imide drug (IMiD).

[0330] 4. A drug kit comprising a first medicament comprising an anti-GPRC5D / anti-CD3 bispecific antibody and a second medicament comprising an immunomodulatory imide drug (IMiD), and optionally further comprising a package insert comprising instructions for administering the combination of the first medicament and the second medicament for treating cancer in an individual.

[0331] 5. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody and an IMiD for use according to any one of the preceding aspects, wherein the anti-GPRC5D / anti-CD3 bispecific antibody comprises

[0332] (i) a first antigen binding portion that specifically binds to GPRC5D and comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; and

[0333] (ii) a second antigen binding portion, which specifically binds to CD3 and comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and HCDR3 of SEQ ID NO: 20; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23.

[0334] 6. A combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody and an IMiD for use according to any one of the preceding aspects, wherein the anti-GPRC5D / anti-CD3 bispecific antibody comprises

[0335] (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

[0336] (ii) a second antigen binding portion that specifically binds to CD3, which comprises 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.

[0337] 7. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to aspect 5 or 6, wherein the first antigen binding portion and / or the second antigen binding portion of the anti-GPRC5D / anti-CD3 bispecific antibody is a Fab molecule.

[0338] 8. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to any one of methods 5 to 7, wherein the second antigen binding moiety is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are replaced with each other.

[0339] 9. An anti-GPRC5D / anti-CD3 bispecific antibody for use, a combination, a use, a method or a kit of anti-IMiDs and IMiDs according to any one of aspects 5 to 8, wherein the first antigen binding moiety is a Fab molecule, wherein in the constant domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to 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) (according to the Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0340] 10. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to any one of aspects 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.

[0341] 11. A combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibodies and IMiDs for use according to any one of aspects 5 to 10, wherein the first antigen binding moiety and the second antigen binding moiety are each Fab molecules, and 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, 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.

[0342] 12. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody for use and IMiD according to aspects 1 to 11, wherein the anti-GPRC5D / anti-CD3 bispecific antibody comprises a third antigen binding portion.

[0343] 13. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to aspect 12, wherein the third antigenic moiety is the same as the first antigenic binding moiety.

[0344] 14. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody for use and an IMiD according to any one of the preceding aspects, wherein the anti-GPRC5D / anti-CD3 bispecific antibody comprises an Fc domain consisting of a first subunit and a second subunit.

[0345] 15. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to aspects 5 to 14, wherein the first antigen binding moiety, the second antigen binding moiety and, if present, the third antigen binding moiety are each Fab molecules;

[0346] and 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;

[0347] And wherein said third antigen binding moiety, when present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of said second subunit of said Fc domain.

[0348] 16. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to aspect 14 or 15, wherein the Fc domain is an IgG Fc domain.

[0349] 17. The combination, use, method or kit for use of an anti-GPRC5D / anti-CD3 bispecific antibody and an IMiD according to any one of aspects 14 to 16, wherein the Fc domain is an IgG1 Fc domain.

[0350] 18. The combination, use, method or kit for use of an anti-GPRC5D / anti-CD3 bispecific antibody and an IMiD according to any one of aspects 14 to 17, wherein the Fc domain is a human Fc domain.

[0351] 19. An anti-GPRC5D / anti-CD3 bispecific antibody for use with an IMiD according to any one of aspects 14 to 18, wherein amino acid residues in the CH3 domain of the first subunit of the Fc domain are replaced by amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion can be positioned in a cavity in the CH3 domain of the second subunit, and amino acid residues in the CH3 domain of the second subunit of the Fc domain are replaced by amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit can be positioned in the cavity.

[0352] 20. An anti-GPRC5D / anti-CD3 bispecific antibody for use, a combination, a use, a method or a kit with an IMiD according to any one of aspects 14 to 19, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0353] 21. An anti-GPRC5D / anti-CD3 bispecific antibody for use, a combination, a use, a method or a kit according to any one of aspects 1 to 19 and an IMiD, 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.

[0354] 22. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody for use and an IMiD according to any one of aspects 1 to 21, wherein the IMiD is a first generation IMiD or a Cereblon E3 ligase modulator (CELMoD).

[0355] 23. The combination, use, method or kit for use of an anti-GPRC5D / anti-CD3 bispecific antibody and an IMiD according to any one of aspects 1 to 22, wherein the IMiD is selected from the group of lenalidomide, pomalidomide and ibolidomide.

[0356] 24. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody for use and an IMiD according to any one of aspects 1 to 23, wherein the combination further comprises a glucocorticoid.

[0357] 25. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to aspect 24, wherein the glucocorticoid is dexamethasone.

[0358] Amino acid sequence

[0359]

[0360]

[0361]

[0362]

[0363] Examples

[0364] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced given the general description provided above.

[0365] Materials and methods

[0366] 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 implanted with human CD34 + Hematopoietic stem cells were delivered to the animal facility at Roche Innovation Center Munich 14-20 weeks later. After arrival, the animals were maintained for one week to adapt to the new environment and observed. According to the prescribed guidelines (GV-Solas; Felasa; TierschG), mice were maintained under conditions without specific pathogens, with a daily cycle of 12 hours of light / 12 hours of darkness. Continuous health status monitoring was performed regularly. The experimental research program was reviewed and approved by the local government (ROB-55.2-2532.Vet_03-16-10 or ROB-55.2-2532.Vet_03-20-170). In order to evaluate the therapeutic effect of confirmed multiple myeloma tumors, humanized NSG mice were subcutaneously implanted with human tumor cell lines. Tumor cell lines were obtained from different suppliers and stored in Roche Munich internal cell bank (Roche Munichinternal cell bank) after expansion (Table 1). All tumor cells were cultured at 37°C in a water-saturated atmosphere of 5% CO2 and injected subcutaneously with 50 μl of Matrigel, while the same amount of Matrigel was injected subcutaneously into the right abdomen of anesthetized humanized NSG mice at different cell numbers and >90% survival rate (Table 1). When the average volume of subcutaneous tumors reached about 200-300 mm 3 At 4 s, humanized mice were randomized into different treatment groups based on tumor volume and body weight. To evaluate the combination of GPRC5D-TCB and mezigimide, when the mean volume of subcutaneous tumors reached 180 mm 3Animals were randomly divided into eight different treatment groups (Table 3). After randomization, animals were treated with GPRC5D-TCB (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29; as disclosed in WO 2021 / 018859A1; RO7425781, voritumumab or "forim") as monotherapy or in combination with IMiD, which is a standard of care (SoC) agent for the treatment of multiple myeloma. In addition, the addition of dexamethasone (Dex) to the combination therapy of GPRC5D-TCB and lenalidomide was also studied. The treatment regimen, dose and route of administration for each therapy are summarized in Tables 2 and 3. All therapies were freshly prepared before injection. Animals were controlled for clinical symptoms and detection of adverse reactions every day. The termination criteria for animals were obvious disease (rough fur, arched back, respiratory problems, impaired motor ability), weight loss> 20% or tumor size. Tumor growth was monitored twice a week using caliper measurements. In order to quantify tumor infiltrating lymphocytes, in some experiments of the combination of GPRC5D-TCB and lenalidomide (Len), pomalidomide and ibrolimumab, the tumor of the scout animal was harvested, and single cell suspension was subjected to flow cytometry using FACSFortessa equipment and FlowJo software. In order to quantify and characterize the peripheral immune cells of animals treated with the combination of GPRC5D-TCB and Mezigmit (Mezi), whole blood was collected and flow cytometry was processed (including erythrocyte lysis), and flow cytometry was performed using Cytek Aurora spectrometer and FlowJo software. Using the combination of the Bio-Plex multiple immunoassay system from BioRad and the Bio-Plex Pro human cytokine 27-plex assay, the cytokines in the serum of treated mice were analyzed. Graph Pad Prism software was used for statistical analysis. In order to compare the results of immune PD, tumor volume or cytokine levels between different treatment groups, one-way ANOVA analysis was performed on the data to correct for multiple comparisons (Tukey test).

[0367] Table 1: Tumor cell lines

[0368]

[0369]

[0370] Table 2: Summary of treatment regimens, dosages and routes of administration.

[0371]

[0372] Table 3: Experimental groups used to evaluate the combination of GPRC5D-TCB and mezigimide.

[0373]

[0374]

[0375] result

[0376] Combination of GPRC5D-TCB with immunomodulatory drugs (IMIDs)

[0377] First-generation IMIDs (such as lenalidomide and pomalidomide) have been approved for first-line treatment of patients with multiple myeloma 1 When combined with low-dose GPRC5D-TCB therapy against OPM-2 xenografts in humanized mice, lenalidomide was found to exhibit potent synergistic antitumor activity compared with monotherapy, as evidenced by tumor growth control and statistically significant reductions in tumor burden ( Figure 1A Moreover, the combination with lenalidomide significantly increased the number of intratumoral T cells compared with GPRC5D-TCB monotherapy, supporting a synergistic mode of action of the two drugs ( Figure 1C IMIDs are often combined with dexamethasone to treat multiple myeloma. Using the less responsive multiple myeloma tumor model KMS-12BM, GPRC5D-TCB was combined with lenalidomide and with lenalidomide plus dexamethasone. The combination of lenalidomide and GPRC5D-TCB induced statistically significant tumor growth inhibition in a difficult-to-treat multiple myeloma xenograft model compared to the control group ( Figure 2A and B). Interestingly, even more robust efficacy was observed when dexamethasone was added to the GPRC5D-TCB and lenalidomide combination, supporting the clinical development of GPRC5D-TCB with this SoC backbone ( Figure 2A Despite very strong monotherapy responses, the recurrence rate of NCI-H929 xenograft tumors was high under GPRC5D-TCB monotherapy ( Figure 3A and Figure 5B When combined with pomalidomide, the mice relapsed much less frequently ( Figure 3A and Figure 5C The improvement in efficacy was associated with increased cytokine levels detected in mouse sera 48 hours after the first TCB and 24 hours after the first pomalidomide injection, suggesting enhanced immune activation in the combination ( Figure 3B , Figure 3C and Figure 3D Interestingly, the pomalidomide combination did not induce complete tumor eradication, as evidenced by tumor growth in individual animals upon cessation of treatment ( Figure 3A and Figure 5C ).

[0378] Ibolidomide belongs to a new class of IMIDs, called "cereblon E3 ligase modulators" (CELMoDs), which are currently being evaluated in early clinical development. Ibolidomide has been found to prevent recurrence of NCI-H929 tumors treated with GPRC5D-TCB monotherapy compared to pomalidomide ( Figure 4A and Figure 5D ), and induced an even stronger booster T cell response, as evidenced by higher cytokine levels in the combination ( Figure 4B , Figure 4C and Figure 4D The ibolidomide combination, but not the pomalidomide combination, induced a complete tumor response in humanized mice, as highlighted by the lack of tumor regrowth after cessation of treatment ( Figure 5D ).

[0379] To evaluate the combination of GPRC5D-TCB and mezigamide in a clinically relevant in vivo setting, humanized mice bearing NCI-H929 tumors were treated with fixed-duration GPRC5D-TCB once a week (1q7d or q7d), using ascending dosing: (1st cycle, C1) 0.0005 mg / kg (1st day ascending dose 1; C1D1), 0.002 mg / kg (8th day ascending dose 2; C1D8) and 0.04 mg / kg (15th day ascending dose 3; C1D15), followed by 5 cycles (C2-C6) of 0.04 mg / kg maintenance dosing, and more than 2 weeks of treatment-free follow-up (Figure 6). 3 mg / kg or 1 mg / kg of mezigamide was administered 24 hours after each GPRC5D-TCB injection, once a week (q7d), three times a week (3q7d) or five times a week (5q7d). Although GPRC5D-TCB induced transient tumor regression after escalating doses of 1 (C1D1) and 3 (C1D15), mice showed progressive disease at the end of cycle 1 (C1), and the progression-free survival (PFS) rate at the end of the study was only 20% ( Figure 6B In contrast, combination with mezigimide at 3q7d and 5q7d dosing resulted in rapid tumor regression during C1, which was associated with significantly improved PFS rates of 80% at the 3 mg / kg dose (3q7d; Fig.6D ) and 100% (5q7d; Figure 6C ), at a dose of 1 mg / kg, the PFS rates were 60% (3q7d; Figure 6G ) and 90% (5q7d; Fig. 6FOnce-weekly (1q7d) dosing of mezigimide did not improve the efficacy of GPRC5D-TCB during escalating doses 1 (C1D1) and 2 (C1D8), but a deepening of the response was achieved after the target dose on day 15 of cycle 1, especially at 3 mg / kg (C1D15; Figure 6H ). Associated with a lower depth of early response, the 1q7d mezigimide combination did not improve PFS rates compared with GPRC5D-TCB monotherapy ( Fig. 6E To explore the effect of mezigmitide combination on immune activation, cytokine release was measured in the serum of all mice 48 hours after each GPRC5D-TCB incremental injection at C1D1, C1D8, and C1D15 and 24 hours after mezigmitide administration (Figure 7). When mezigmitide was administered at 5q7d or 3q7d, serum levels of IL-10 and IP-10 were comparable to GPRC5D-TCB monotherapy ( Figure 7B and 7C ), while IL-2 levels slightly increased at all three time points ( Fig. 7A Interestingly, a different trend was observed for MIP-1a, as serum levels decreased in correlation with the number of mezigamide administrations ( Fig.7D In contrast to the 5q7d and 3q7d regimens, less frequent mezigimide dosing (1q7d) induced a robust increase in IL-2, IP-10, and MIP-1a following target dosing at C1D15 ( Fig. 7A , Figure 7B and Fig.7D ). To quantify and phenotype circulating immune cells, the inventors collected blood from all animals at the end of cycle 3 (C4 pre-dose) and cycle 5 (C6 pre-dose) and performed spectral flow cytometry. Compared to monotherapy, the inventors observed that peripheral CD8a + (CD8α positive cells) and conventional CD4 + T cell counts decreased, and when 1 mg / kg (CD8a + and CD4 + ) and 1 mg / kg (CD4 + ) of mezigimide, the cell count increased ( Fig. 8A and Fig.8D When 3 mg / kg of mezigamide was administered on 3q7d, the number of regulatory T cells (Treg) was slightly decreased, and when 1 mg / kg of mezigamide was administered on 3q7d, the number of regulatory T cells (Treg) was greatly increased ( Figure 8BRegardless of the dose level of mezigimide, the number of peripheral B cells decreased significantly when it was administered at 3q7d and 5q7d ( Figure 8C ). In contrast, less frequent mezigimide administration induced a strong increase not only in B cell counts but also in NK cell counts when pre-dosed with C4, but especially with C6 ( Figure 8C and Fig. 8E Next, the inventors evaluated whether the combination of GPRC5D-TCB and mezigamide would induce changes in the exhaustion status of circulating T lymphocytes. Compared with GPRC5D-TCB monotherapy, the inventors observed that LAG3 and TIGIT-positive CD4 + and CD8a + A higher frequency of T cells ( Fig.9A , Fig. 9B , Fig. 9C and Fig.9D ), indicating that repeated treatment with mezigimid induced T cell exhaustion.

[0380] In summary, the inventors' data suggest that the combination with mezigamide can significantly improve the PFS rate in patients with multiple myeloma. The addition of mezigamide to GPRC5D-TCB induced profound responses at early time points and overcame tumor recurrence at later time points. Cytokine data indicate that the combination of GPRC5D-TCB escalation dosing with mezigamide does not represent a major risk factor for the development or exacerbation of cytokine release syndrome (CRS).

[0381] References:

[0382] 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.

[0383] ***

[0384] Although the present invention has been previously described in considerable detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. A combination of an anti-GPRC5D / anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD) for use as a combination therapy for treating cancer.

2. Use of a combination of an anti-GPRC5D / anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD) in the manufacture of a medicament for treating cancer.

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

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

5. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to any one of the preceding claims, wherein 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, HCDR 2 of SEQ ID NO: 13, and HCDR 3 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, LCDR 2 of SEQ ID NO: 16, and LCDR 3 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, HCDR 2 of SEQ ID NO: 19, and HCDR 3 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, LCDR 2 of SEQ ID NO:22, and LCDR 3 of SEQ ID NO:

23.

6. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to any one of the preceding claims, wherein 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 moiety that specifically binds to CD3, comprising the residue corresponding to SEQ ID NO: The invention further comprises a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24 and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

25.

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

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

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

10. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use 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. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to any one of claims 5 to 10, wherein the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule, and 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, 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. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to claims 1 to 11, wherein the anti-GPRC5D / anti-CD3 bispecific antibody comprises a third antigen binding moiety.

13. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to claim 12, wherein the third antigenic moiety is the same as the first antigenic binding moiety.

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

15. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to claims 5 to 14, wherein the first antigen binding moiety, the second antigen binding moiety and the third antigen binding moiety, when present, are each a Fab molecule; and 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 wherein said third antigen binding moiety, when present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of said second subunit of said Fc domain.

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

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

18. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody and an IMiD for use according to any one of claims 14 to 17, wherein the Fc domain is a human Fc domain.

19. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to any one of claims 14 to 18, wherein amino acid residues in the CH3 domain of the first subunit of the Fc domain are replaced by amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion can be positioned in a cavity in the CH3 domain of the second subunit, and amino acid residues in the CH3 domain of the second subunit of the Fc domain are replaced by amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit can be positioned in the cavity.

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

21. An anti-GPRC5D / anti-CD3 bispecific antibody for use, a combination, a use, a method or a kit of parts for use with an IMiD 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. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody for use and an IMiD according to any one of claims 1 to 20, wherein the anti-GPRC5D / anti-CD3 bispecific antibody is voritumimab.

23. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody for use and an IMiD 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. The combination, use, method or kit of an anti-GPRC5D / anti-CD3 bispecific antibody for use and an IMiD according to any one of claims 1 to 23, wherein the IMiD is selected from the group consisting of lenalidomide, pomalidomide, ibolidomide and mezigamide.

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

26. The combination, use, method or kit of anti-GPRC5D / anti-CD3 bispecific antibody and IMiD for use according to claim 25, wherein the glucocorticoid is dexamethasone.

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