Field of the Invention

The present invention relates to the combination therapy of anti-GPRC5D/anti-CD3 bispecific antibodies with immunomodulatory imide drugs (IMiDs). To the combination therapy, a glucocorticosteroid may be added.

Background

Affecting -75,000 new patients every year in the EU and US, multiple myeloma (MM) is one of the most common hematological malignancies, which remains a high unmet medical need. Multiple myeloma is characterized by terminally differentiated plasma cells that secrete nonfunctional monoclonal immunoglobulins. In the short-term, the immunomodulatory drugs such as lenalidomide and pomalidomide, and proteasome inhibitors such as carfilzomib or bortezomib may remain the backbone of 1 ^st line therapy for multiple myeloma (Moreau, P. and S. V. Rajkumar, multiple myeloma-translation of trial results into reality. Lancet, 2016. 388(10040): p. 111-3). However, these drugs do not target specifically the diseased tumor cells e.g. diseased plasma cells (PC). Efforts have been made towards selectively depleting the plasma cells in multiple myeloma. The lack of surface proteins that specifically mark plasma cells has hampered the development of antibodies or cellular therapies for multiple myeloma. So far, there are few cases of successful biologies, including daratumumab (anti-CD38) and elotuzumab (anti-CD319), with the caveat that these two molecules are not uniquely expressed by plasma cells. Therefore, novel targets from plasma cells in multiple myeloma were identified using RNA-sequencing, such as the G protein- coupled receptor class C group 5 member D (GPRC5D) that is differentially expressed by plasma cells in multiple myeloma versus plasma cells form healthy donors. It has been reported that GPRC5D is associated with prognosis and tumour load in multiple myeloma patients (Atamaniuk, J., et al., Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma. Eur J Clin Invest, 2012. 42(9): p. 953-60; and Cohen, Y ., et al., GPRC5D is a promising marker for monitoring the tumour load and to target multiple myeloma cells. Hematology, 2013. 18(6): p. 348-51).

GPRC5D is an orphan receptor with no known ligand and largely unknown biology in men in general and in cancer specifically. The GPRC5D encoding gene, which is mapped on chromosome 12p 13.3, contains three exons and spans about 9.6 kb (Brauner-Osborne, H., et al., Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochim Biophys Acta, 2001. 1518(3): p. 237-48). The large first exon encodes the seven- transmembrane domain. It has been shown that GPRC5D is involved in keratin formation in hair follicles in animals (Gao, Y., et al., Comparative Transcriptome Analysis of Fetal Skin Reveals Key Genes Related to Hair Follicle Morphogenesis in Cashmere Goats. PLoS One, 2016. 11(3): p. e0151118; and Inoue, S., T. Nambu, and T. Shimomura, The RAIG family member, GPRC5D, is associated with hard-keratinized structures. J Invest Dermatol, 2004. 122(3): p. 565-73).

WO 2018/017786 A2 and WO 2021/018859 Al disclose GPRC5D-specific antibodies or antigenbinding fragments that bind GPRC5D on target cells and an activating T-cell antigen such as CD3 on T-cells. Upon simultaneous binding of such an antibody to both of its targets, a T-cell synapse will be formed, leading to activation of the (cytotoxic) T cell and subsequent lysis of the target cell. T cell bispecific antibodies (TCBs) have become a novel therapeutic option for relapsed refractory myeloma (RRMM) patients based on their promising objective response rates (ORR), favorable safety profile and off-the-shelf availability as compared to CAR-T cell therapies (van de Donk, N.W.C.J. et al., T-cell redirecting bispecific and trispecific antibodies in multiple myeloma beyond BCMA. Curr Opin Oncol, 2023. 35:000-000). Although BCMA- and GPRC5D-targeted TCBs have been reported to induce deep clinical responses, antigen drift represents a tumor intrinsic resistance mechanism limiting durability of responses (Mailankody, S. et al., GPRC5D- Targeted CAR T Cells for Myeloma. N Engl J Med. 2022;387(13): 1196-1206).

Given that all standard-of-care treatments are not able to cure multiple myeloma patients, there is a clear need to develop potent and specific new therapies. Thus, the present invention provides the combination of anti-GPRC5D/anti-CD3 bispecific antibodies with IMiDs and optionally a glucocorticosteroid.

Summary of the Invention

In a first aspect, the present invention provides an anti-GPRC5D/anti-CD3 bispecific antibody in combination with an immunomodulatory imide drug (IMiD) for use as a combination therapy in the treatment of cancer. In a further aspect, the invention provides the use of an anti-GPRC5D/anti- CD3 bispecific antibody in combination with an immunomodulatory imide drug (IMiD) in the manufacture of a medicament for the treatment of cancer. In yet a further aspect, the invention provides a A method of treating cancer in an individual comprising administering to said individual anti-GPRC5D/anti-CD3 bispecific antibody in combination with an immunomodulatory imide drug (IMiD). In another aspect, the invention provides a 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 instruction for administration of the first medicament in combination with the second medicament for treating cancer in an individual.

In an embodiment of any one of the above aspects, the the anti-GPRC5D/anti-CD3 bispecific antibody comprises (i) a first antigen binding moiety 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, a HCDR 2 of SEQ ID NO: 13, and a 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, a LCDR 2 of SEQ ID NO: 16 and a LCDR 3 of SEQ ID NO: 17; and (ii) a second antigen binding moiety that specifically binds to CD3 and comprises heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, a HCDR 2 of SEQ ID NO: 19, and a 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, a LCDR 2 of SEQ ID NO: 22 and a LCDR 3 of SEQ ID NO: 23. In another embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody comprises (i) a first antigen binding moiety that specifically binds 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 a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24, and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the first and/or the second antigen binding moiety of the anti- GPRC5D/anti-CD3 bispecific antibody is a Fab molecule. In a further embodiment, second antigen binding moiety is a Fab molecule wherein the variable domains VL and VH or the constant domains CL and CHI, particularly the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are replaced by each other. In one embodiment, the first antigen binding moiety is a Fab molecule wherein in the constant domain the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). In another embodiment, the first and the second antigen binding moiety are fused to each other, optionally via a peptide linker. In yet another embodiment, the first and the second antigen binding moiety are each a Fab molecule and wherein either (i) the second antigen binding moiety is fused at the C -terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, or (ii) the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety.

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

In one embodiment of any one of the above aspects, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. In one embodiment, the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.

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

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

In another aspect, the combinations as described in any one of the above aspects further comprises a glucocorticosteroid. In one embodiment, the glucocorticosteroid is dexamethasone.

Brief Description of the Drawings

Figure 1A, IB, 1C: Results of an efficacy/PD experiment evaluating GPRC5D-TCB as single agent and in combination with Lenalidomide. (Fig. 1A) The 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 weekly and Lenalidomide was administered daily at 20 mg/kg using oral gavage and tumor growth was compared over a period of 18 days. (Fig. IB) Tumor load of individual mice was evaluated at the end of study (day 18). (Fig. 1C) Tumors of 5 scouts per group were harvested 48h after second GPRC5D-TCB injection and 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).

Figure 2A, 2B: Results of an efficacy experiment evaluating GPRC5D-TCB as single agent and in combination with Lenalidomide with or without additional Dexamethasone against KMS-12BM multiple myeloma tumors subcutaneously engrafted into stem cell humanized NSG mice. (Fig. 2A) GPRC5D-TCB was injected intravenously at 1 mg/kg once weekly and combined with daily administration of Lenalidomide at 20 mg/kg using oral gavage with or without additional oral Dexamethasone at 2 mg/kg. (Fig. 2B) Tumor load of individual mice was evaluated at day 35 (end of study). 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).

Figure 3A, 3B, 3C, 3D: Results of an efficacy experiment evaluating GPRC5D-TCB as single agent and in combination with Pomalidomide against NCI-H929 multiple myeloma tumors subcutaneously engrafted into stem cell humanized NSG mice. (Fig. 3 A) Growth of NCI-H929 xenograft tumors upon weekly intravenous administration of GPRC5D-TCB at 0.1 mg/kg and daily injection of Pomalidomide at 10 mg/kg using oral gavage. IFN-y (Fig. 3B), IL-2 (Fig. 3C) and TNF-a (Fig. 3D) levels in serum of mice were determined 48 hours after first GPRC5D-TCB and 24h after Pomalidomide injection using multiplex technology. 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).

Figure 4A, 4B, 4C, 4D: Results of an efficacy experiment evaluating GPRC5D-TCB as single agent and in combination with Iberdomide against NCI-H929 multiple myeloma tumors subcutaneously engrafted into stem cell humanized NSG mice. (Fig. 4A) Growth of NCI-H929 xenograft tumors upon weekly intravenous administration of GPRC5D-TCB at 0.1 mg/kg and daily injection of Iberdomide at 10 mg/kg using oral gavage. IFN-y (Fig. 4B), IL-2 (Fig. 4C) and TNF-a (Fig. 4D) levels in serum of mice were determined 48 hours after first GPRC5D-TCB and 24h after Iberodomide injection using multiplex technology. 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).

Figure 5A, 5B, 5C, 5D: Tumor growth kinetics of individual mice treated with vehicle (Fig. 5 A), GPRC5D-TCB monotherapry (Fig. 5B) or combination with Pomalidomide (Fig. 5C) or Iberdomide (Fig. 5D), as described in Figures 3 and 4, respectively.

Figure 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H: Results of an efficacy experiment evaluating GPRC5D- TCB as single agent and in combination with high or low dose mezigdomide against NCI-H929 multiple myeloma tumors subcutaneously engrafted into stem cell humanized NSG mice. NCI- 14929 tumor growth in individual mice upon weekly subcutaneous (s.c.) injection of vehicle (Fig. 6A), GPRC5D-TCB using step up dosing at 0.0005 - 0.002 - 0.04 mg/kg followed by maintenance dosing of 0.04 mg/kg (Fig. 6B) and GPRC5D-TCB administration in combination with mezigdomide at 3 mg/kg 5 days (5q7d; Fig. 6C), 3 days (3q7d; Fig. 6D), 1 day (lq7d; Fig. 6E) a week or 1 mg/kg 5 days (5q7d; Fig. 6F), 3 days (3q7d; Fig. 6G), 1 day (lq7d; Fig. 6H) a week. Figure 7 A, 7B, 7C, 7D: Results of cytokine analysis performed in blood NCI-H929 engrafted stem cell humanized NSG mice treated with GPRC5D-TCB as single agent and in combination with mezigdomide. Cytokines IL-2 (Fig. 7A), IP-10 (Fig. 7B), IL-10 (Fig. 7C) and MIP-la (Fig. 7D) were measured in serum of mice 48 hours after GPRC5D-TCB dosing at cycle 1 day 1 (C1D1, 0.0005 mg/kg), cycle 1 day 8 (C1D8, 0.002 mg/kg) and cycle 1 day 15 (CID 15, 0.04 mg/kg) and 24h after mezigdomide administration at 3mg/kg or 1 mg/kg using multiplex technology.

Figure 8A, 8B, 8C, 8D, 8E: Presents the results of quantitative flow cytometry analysis performed in blood NCI-H929 engrafted stem cell humanized NSG mice treated with GPRC5D-TCB as single agent and in combination with mezigdomide. CD8a ⁺ T cells (Fig. 8A), regulatory T cells (Fig. 8B), B cells (Fig. 8C), conventional CD4 ⁺ cells (Fig. 8D) and NK cells (Fig. 8E) were quantified in blood of mice 164 hours after cycle 3 (= C4 predosing) and cycle 5 (= C6 predosing) administration of GPRC5D-TCB at 0.04 mg/kg and 48h (5q7d), 96h (3q7d) or 144h (lq7d) after mezigdomide administration at 3 mg/kg or 1 mg/kg using flow cytometry.

Figure 9A, 9B, 9C, 9D: Presents the results of phenotypic flow cytometry analysis performed in blood NCI-H929 engrafted stem cell humanized NSG mice treated with GPRC5D-TCB as single agent and in combination with mezigdomide. Phenotype of circulating immune cells was analyzed in blood of mice 164 hours after cycle 3 (= C4 predosing) and cycle 5 (= C6 predosing) administration of GPRC5D-TCB at 0.04 mg/kg and 48h (5q7d), 96h (3q7d) or 144h (lq7d) after mezigdomide administration at 3mg/kg or 1 mg/kg using flow cytometry. Percent of CD8a ⁺ T cells expressing TIGIT (Fig. 9A) and Lag3 (Fig. 9B) as well as conventional CD4 ⁺ T cells expressing TIGIT (Fig. 9C) and Lag3 (Fig. 9D) was assessed.

Detailed Description of the Invention

Definitions

Terms are used herein as generally used in the art, unless otherwise defined in the following.

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

The term “bispecific” means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.

The term “valent” as used herein denotes the presence of a specified number of antigen binding sites in an antigen binding molecule. As such, the term “monovalent binding to an antigen” denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the antigen binding molecule.

An “antigen binding site” refers to the site, i.e. one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites; a Fab molecule typically has a single antigen binding site.

As used herein, the term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen binding moiety is able to direct the entity to which it is attached (e.g. a second antigen binding moiety) to a target site, for example to a specific type of tumor cell bearing the antigenic determinant. In another embodiment an antigen binding moiety is able to activate signaling through its target antigen, for example a T cell receptor complex antigen. Antigen binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen binding moieties include an antigen binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding moieties may comprise antibody constant regions as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: a, 5, a, y, or p. Useful light chain constant regions include any of the two isotypes: K and X.

As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope", and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-anti gen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM). The proteins referred to as antigens herein (e.g. GPRC5D, CD3) can be any native form of the proteins from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. In a particular embodiment the antigen is a human protein. Where reference is made to a specific protein herein, the term encompasses the “full-length”, unprocessed protein as well as any form of the protein that results from processing in the cell. The term also encompasses naturally occurring variants of the protein, e.g. splice variants or allelic variants. An exemplary human protein useful as antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 185), NCBI RefSeq no. NP 000724.1, SEQ ID NO: 4 for the human sequence; or UniProt no. Q95LI5 (version 69), NCBI GenBank no. BAB71849.1, SEQ ID NO: 5 for the cynomolgus [Macaca fascicularis] sequence), or GPRC5D (see UniProt no. Q9NZD1 (version 115); NCBI RefSeq no. NP 061124.1, SEQ ID NO: 9 for the human sequence). In certain embodiments the bispecific antigen binding molecule binds to an epitope of CD3 or GPRC5D that is conserved among the CD3 or GPRC5D antigens from different species. In particular embodiments, the bispecific antigen binding molecule binds to human GPRC5D.

By "specific binding" is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed e.g. on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antigen binding moiety to an unrelated protein is less than about 10% of the binding of the antigen binding moiety to the antigen as measured, e.g., by SPR. In certain embodiments, an antigen binding moiety that binds to the antigen, or an antigen binding molecule comprising that antigen binding moiety, has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10' ⁸ M or less, e.g. from 10’ ⁸ M to 10’ ¹³ M, e.g., from 10’ ⁹ M to IO’ ¹³ M).

“Affinity” refers to the strength of the sum total 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 indicated otherwise, 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 represented by the dissociation constant (KD), which is the ratio of dissociation and association rate constants (k _o ff and k _on , respectively). Thus, equivalent affinities may comprise 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).

“Reduced binding”, for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity, the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.

An “activating T cell antigen” as used herein refers to an antigenic determinant expressed on the surface of a T lymphocyte, particularly a cytotoxic T lymphocyte, which is capable of inducing T cell activation upon interaction with an antigen binding molecule. Specifically, interaction of an antigen binding molecule with an activating T cell antigen may induce T cell activation by triggering the signaling cascade of the T cell receptor complex. In a particular embodiment the activating T cell antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 144), NCBIRefSeq no. NP_000724.1, SEQ ID NO: 4 for the human sequence; or UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, SEQ ID NO: 5 for the cynomolgus [Macaca fascicularis] sequence).

“T cell activation” as used herein refers to one or more cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays to measure T cell activation are known in the art and described herein.

A “target cell antigen” as used herein refers to an antigenic determinant presented on the surface of a target cell, for example a cell in a tumor such as a cancer cell or a cell of the tumor stroma. In a particular embodiment, the target cell antigen is GPRC5D, particularly human GPRC5D according to SEQ ID NO: 9.

As used herein, the terms “first”, “second” or “third” with respect to Fab molecules etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the bispecific antigen binding molecule unless explicitly so stated.

By “fused” is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

A “Fab molecule” refers to a protein consisting of the VH and CHI domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin. By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CHI (VL-CH1, in N- to C-terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in N- to C-terminal direction). For clarity, in a crossover Fab molecule wherein the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1 CHI is referred to herein as the “heavy chain” of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule wherein the constant domains of the Fab light chain and 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 (crossover) Fab molecule.

In contrast thereto, by a “conventional” Fab molecule is meant a Fab molecule in its natural format, i.e. comprising a heavy chain composed of the heavy chain variable and constant domains (VH- CH1, in N- to C-terminal direction), and a light chain composed of the light chain variable and constant domains (VL-CL, in N- to C-terminal direction).

The term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called a (IgA), 5 (IgD), 8 (IgE), y (IgG), or p (IgM), some of which may be further divided into subtypes, e.g. yi (IgGi), 72 (IgG?), 73 (IgGs), 74 (IgG4), ai (IgAi) and a? (IgA?). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.

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.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprised in the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

An "isolated" antibody is one which has been separated from a component of its natural environment, i.e. that is not in its natural milieu. No particular level of purification is required. For example, an isolated antibody can be removed from its native or natural environment. Recombinantly produced antibodies expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant antibodies which have been separated, fractionated, or partially or substantially purified by any suitable technique. As such, the antibodies and bispecific antigen binding molecules of the present invention are isolated. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds 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 e.g. Pliickthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo halflife, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, 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 comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see e.g. U.S. Patent No. 6,248,516 Bl). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.

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

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6 ^th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. As used herein in connection with variable region sequences, "Kabat numbering" refers to the numbering system set forth by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), referred to as “numbering according to Kabaf ’ or “Kabat numbering” herein. Specifically the Kabat numbering system (see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of kappa and lambda isotype and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CHI, Hinge, CH2 and CH3), which is herein further clarified by referring to “numbering according to Kabat EU index” in this case.

The term “hypervariable region” or “HVR”, as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”; CDRs of the heavy chain variable region/domain are abbreviated e.g. as HCDR1, HCDR2 and HCDR3; CDRs of the light chain variable region/domain are abbreviated e.g. as LCDR1, LCDR2 and LCDR3 ) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26- 32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (Hl), SO- 65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30- 35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (Hl), 26-35b (Hl), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

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

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH (or VL) : FR1 -H 1 (L 1 )-FR2-H2(L2)-FR3 -H3 (L3 )-FR4. A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from nonhuman HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Such variable domains are referred to herein as “humanized variable region”. A humanized antibody optionally may 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., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity. A “humanized form” of an antibody, e.g. of a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigenbinding residues. In certain embodiments, a human antibody is derived from a non-human transgenic mammal, for example a mouse, a rat, or a rabbit. In certain embodiments, a human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from human antibody libraries are also considered human antibodies or human antibody fragments herein.

The “class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG?, IgGs, IgG ₄ , IgAi, and IgA?. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively.

The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl -terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full- length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain (also referred to herein as a “cleaved variant heavy chain”). This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447), of the Fc region may or may not be present. Amino acid sequences of heavy chains including Fc domains (or a subunit of an Fc domain as defined herein) are denoted herein without C-terminal glycine-lysine dipeptide if not indicated otherwise. In one embodiment of the invention, a heavy chain including a subunit of an Fc domain as specified herein, comprised in an antibody or bispecific antigen binding molecule according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a heavy chain including a subunit of an Fc domain as specified herein, comprised in an antibody or bispecific antigen binding molecule according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). Compositions of the invention, such as the pharmaceutical compositions described herein, comprise a population of antibodies or bispecific antigen binding molecules of the invention. The population of antibodies or bispecific antigen binding molecules may comprise molecules having a full-length heavy chain and molecules having a cleaved variant heavy chain. The population of antibodies or bispecific antigen binding molecules may consist of a mixture of molecules having a full-length heavy chain and molecules having a cleaved variant heavy chain, 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 a cleaved variant heavy chain. In one embodiment of the invention a composition comprising a population of antibodies or bispecific antigen binding molecules of the invention comprises an antibody or bispecific antigen binding molecule comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention a composition comprising a population of antibodies or bispecific antigen binding molecules of the invention comprises an antibody or bispecific antigen binding molecule comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). In one embodiment of the invention such a composition comprises a population of antibodies or bispecific antigen binding molecules comprised of molecules comprising a heavy chain including a subunit of an Fc domain as specified herein; molecules comprising a heavy chain including a subunit of a Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat); and molecules comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). A “subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

A “modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, a modification promoting association may alter 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, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which might be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding moieties) are not the same. In some embodiments the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a particular embodiment, the modification promoting association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.

The term “effector functions” 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: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex -mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.

As used herein, the terms “engineer, engineered, engineering”, are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.

The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions include amino- and/or carboxyterminal deletions and insertions of amino acids. Particular amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an Fc region, nonconservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and/or chemical properties, are particularly preferred. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3- methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site- directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc domain to glycine can be indicated as 329G, G329, G329, P329G, or Pro329Gly.

“Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored 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, a public server accessible at http://fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used to compare the sequences, using the ggsearch (global protein: protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.

An “activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcyRIIIa (CD16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. As used herein, the term “reduced ADCC” is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example, the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006/082515 or PCT publication no. WO 2012/130831).

“Forimtamig” refers to a specific GPRC5D-TCB as listed in Recommended International Nonproprietary Names: List 89 (WHO Drug Information, Vol. 37, No. 1, 2023).

An "effective amount" of an agent refers to the amount that is necessary to result in a physiological change in the cell or tissue to which it is administered.

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 prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.

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

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

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies or bispecific antigen binding molecules of the invention are used to delay development of a disease or to slow the progression of a disease.

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.

Bispecific antigen binding molecules that bind to GPRC5D and CD3

The anti-GPRC5D/anti-CD3 bispecific antigen binding molecule, also referred to herein as “GPRC5D TCB” used in the combination therapy described herein comprises at least two antigen binding moieties capable of specific binding to two distinct antigenic determinants (a first and a second antigen). Suitable bispecific antigen binding molecules that bind to GPRC5D and CD3 for use in the present invention are described e.g. in WO 2021/018859 Al, WO 2019/154890 Al and WO 2018017786 A2.

According to particular embodiments of the invention, the antigen binding moieties comprised in the bispecific antigen binding molecule are Fab molecules (i.e. antigen binding domains composed of a heavy and a light chain, each comprising a variable and a constant domain). In one embodiment, the first and/or the second antigen binding moiety is a Fab molecule. In one embodiment, said Fab molecule is human. In a particular embodiment, said Fab molecule is humanized. In yet another embodiment, said Fab molecule comprises human heavy and light chain constant domains.

Preferably, at least one of the antigen binding moieties is a crossover Fab molecule. Such modification reduces mispairing of heavy and light chains from different Fab molecules, thereby improving the yield and purity of the bispecific antigen binding molecule of the invention in recombinant production. In a particular crossover Fab molecule useful for the bispecific antigen binding molecule of the invention, the variable domains of the Fab light chain and the Fab heavy chain (VL and VH, respectively) are exchanged. Even with this domain exchange, however, the preparation of the bispecific antigen binding molecule may comprise certain side products due to a so-called Bence Jones-type interaction between misspaired heavy and light chains (see Schaefer et al, PNAS, 108 (2011) 11187-11191). To further reduce mispairing of heavy and light chains from different Fab molecules and thus increase the purity and yield of the desired bispecific antigen binding molecule, charged amino acids with opposite charges may be introduced at specific amino acid positions in the CHI and CL domains of either the Fab molecule(s) binding to the first antigen (GPRC5D), or the Fab molecule binding to the second antigen (CD3), as further described herein. Charge modifications are made either in the conventional Fab molecule(s) comprised in the bispecific antigen binding molecule, or in the VH/VL crossover Fab molecule(s) comprised in the bispecific antigen binding molecule (but not in both). In particular embodiments, the charge modifications are made in the conventional Fab molecule(s) comprised in the bispecific antigen binding molecule (which in particular embodiments bind(s) to the first antigen, i.e. GPRC5D). The bispecific antigen binding molecule is capable of simultaneous binding to the first antigen (i.e. GPRC5D), and the second antigen (i.e. CD3). The bispecific antigen binding molecule is capable of crosslinking a T cell and a target cell by simultaneous binding GPRC5D and an activating T cell antigen. Such simultaneous binding results in lysis of the target cell, particularly a GPRC5D expressing tumor cell, activation of the T cellsand in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.

In one embodiment, the bispecific antigen binding molecule is capable of re-directing cytotoxic activity of a T cell to a target cell. In a particular embodiment, said re-direction is independent of MHC-mediated peptide antigen presentation by the target cell and and/or specificity of the T cell. Particularly, a T cell according to any of the embodiments of the invention is a cytotoxic T cell. In some embodiments the T cell is a CD4 ⁺ or a CD8 ⁺ T cell, particularly a CD8 ⁺ T cell.

First moiety

The bispecific antigen binding molecule comprises at least one antigen binding moiety, particularly a Fab molecule, that binds to GPRC5D (first antigen). In certain embodiments, the bispecific antigen binding molecule comprises two antigen binding moieties, particularly Fab molecules, which bind to GPRC5D. In a particular such embodiment, each of these antigen binding moieties binds to the same antigenic determinant. In an even more particular embodiment, all of these antigen binding moieties are identical, i.e. they comprise the same amino acid sequences including the same amino acid substitutions in the CHI and CL domain as described herein (if any). In one embodiment, the bispecific antigen binding molecule comprises not more than two antigen binding moieties, particularly Fab molecules, which bind to GPRC5D.

In particular embodiments, the antigen binding moiety(ies) which bind to GPRC5D is/are a conventional Fab molecule. In such embodiments, the antigen binding moiety(ies) that binds to a second antigen is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CHI and CL of the Fab heavy and light chains are exchanged / replaced by each other.

In alternative embodiments, the antigen binding moiety(ies)which bind to GPRC5D is/are a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CHI and CL of the Fab heavy and light chains are exchanged / replaced by each other. In such embodiments, the antigen binding moiety(ies) that binds a second antigen is a conventional Fab molecule.

The GPRC5D binding moiety is able to direct the bispecific antigen binding molecule to a target site, for example to a specific type of tumor cell that expresses GPRC5D.

The first antigen binding moiety of the bispecific antigen binding molecule may incorporate any of the features, singly or in combination, described herein in relation to the antibody that binds GPRC5D, unless scientifically clearly unreasonable or impossible.

In one aspect, the bispecific antigen binding molecule comprises (a) a first antigen binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen binding moiety comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, a HCDR 2 of SEQ ID NO: 13, and a 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, a LCDR 2 of SEQ ID NO: 16 and a LCDR 3 of SEQ ID NO: 17, and (b) a second antigen binding moiety that binds to CD3. In some embodiments, the first antigen binding moiety is (derived from) a humanized antibody. In one embodiment, the VH is a humanized VH and/or the VL is a humanized VL. In one embodiment, the first antigen binding moiety comprises CDRs as in any of the above embodiments, and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.

In one embodiment, the VH of the first antigen binding moiety 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 moiety 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.

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

In one embodiment, the first antigen binding moiety comprises a VH comprising an amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 11. In one embodiment, the first antigen binding moiety comprises a VH sequence of SEQ ID NO: 10 and the VL sequence of SEQ ID NO: 11. In a particular 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 particular embodiment, the first antigen binding moiety comprises the VH sequence of SEQ ID NO: 48 and the VL sequence of SEQ ID NO: 11.

In one embodiment, the first antigen binding moiety comprises a human constant region. In one embodiment, the first antigen binding moiety is a Fab molecule comprising a human constant region, particularly a human CHI and/or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs 1 and 2 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 3 (human IgGi heavy chain constant domains CH1-CH2-CH3). In some embodiments, the first antigen binding moiety comprises a light chain constant region comprising 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: 1 or SEQ ID NO: 2, particularly the amino acid sequence of SEQ ID NO: 1. Particularly, the light chain constant region may comprise amino acid mutations as described herein under “charge modifications” and/or may comprise deletion or substitutions of one or more (particularly two) N-terminal amino acids if in a crossover Fab molecule. In some embodiments, the first antigen binding moiety 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 CHI domain sequence comprised in the amino acid sequence of SEQ ID NO: 3. Particularly, the heavy chain constant region (specifically CHI domain) may comprise amino acid mutations as described herein under “charge modifications”.

Second moiety

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

In particular embodiments, the antigen binding moiety that binds the second antigen is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CHI and CL of the Fab heavy and light chains are exchanged / replaced by each other. In such embodiments, the antigen binding moiety(ies) 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, particularly Fab molecule, that binds to GPRC5D comprised in the bispecific antigen binding molecule, the antigen binding moiety that binds to the second antigen preferably is a crossover Fab molecule and the antigen binding moieties that bind to GPRC5D are conventional Fab molecules. In alternative embodiments, the antigen binding moiety that binds to the second antigen is a conventional Fab molecule. In such embodiments, the antigen binding moiety(ies) that binds to the first antigen (i.e. GPRC5D) is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CHI and CL of the Fab heavy and light chains are exchanged / replaced by each other. In embodiments where there is more than one antigen binding moiety, particularly Fab molecule, that binds to a second antigen comprised in the bispecific antigen binding molecule, the antigen binding moiety that binds to GPRC5D preferably is a crossover Fab molecule and the antigen binding moieties that bind to the second antigen are conventional Fab molecules.

The second antigen, i.e. CD3, is an activating T cell antigen (also referred to herein as an “activating T cell antigen binding moiety, or activating T cell antigen binding Fab molecule”). In a particular embodiment, the bispecific antigen binding molecule comprises not more than one antigen binding moiety capable of specific binding to an activating T cell antigen. In one embodiment the bispecific antigen binding molecule provides monovalent binding to the activating T cell antigen.

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

In one embodiment, the second antigen binding moiety comprises a HCDR 1 of SEQ ID NO: 18, a HCDR 2 of SEQ ID NO: 19, a HCDR 3 of SEQ ID NO: 20, a LCDR 1 of SEQ ID NO: 21, a LCDR 2 of SEQ ID NO: 22 and a LCDR 3 of SEQ ID NO: 23. In one embodiment, the second antigen binding moiety comprises a VH comprising a HCDR 1 of SEQ ID NO: 18, a HCDR 2 of SEQ ID NO: 19, and a HCDR 3 of SEQ ID NO: 20, and a VL comprising a LCDR 1 of SEQ ID NO: 21, a LCDR 2 of SEQ ID NO: 22 and a LCDR 3 of SEQ ID NO: 23. In some embodiments, the second antigen binding moiety is (derived from) a humanized antibody. In one embodiment, the VH is a humanized VH and/or the VL is a humanized VL. In one embodiment, the second antigen binding moiety comprises CDRs as in any of the above embodiments, and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework. In one embodiment, the second antigen binding moiety 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 moiety 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 moiety 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 moiety 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 moiety 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 moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 24, and a VL comprising the amino acid sequence of SEQ ID NO: 25. In one embodiment, the second antigen binding moiety comprises the VH sequence of SEQ ID NO: 24, and the VL sequence of SEQ ID NO: 25.

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 CHI and/or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs 1 and 2 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 3 (human IgGi heavy chain constant domains CH1-CH2-CH3). In some embodiments, the second antigen binding moiety comprises a light chain constant region comprising 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: 1 or SEQ ID NO: 2, particularly the amino acid sequence of SEQ ID NO: 1. Particularly, the light chain constant region may comprise amino acid mutations as described herein under “charge modifications” and/or may comprise deletion or substitutions of one or more (particularly two) N-terminal amino acids if in a crossover Fab molecule. In some embodiments, the second antigen binding moiety 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 CHI domain sequence comprised in the amino acid sequence of SEQ ID NO: 3. Particularly, the heavy chain constant region (specifically CHI domain) may comprise amino acid mutations as described herein under “charge modifications”.

In some embodiments, the second antigen binding moiety is a Fab molecule wherein the variable domains VL and VH or the constant domains CL and CHI, particularly the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are replaced by each other (i.e. according to such embodiment, the second antigen binding moiety is a crossover Fab molecule wherein the variable or constant domains of the Fab light chain and the Fab heavy chain are exchanged). In one such embodiment, the first (and the third, if any) antigen binding moiety is a conventional Fab molecule.

In one embodiment, not more than one antigen binding moiety that binds to the second antigen (i.e. CD3) is present in the bispecific antigen binding molecule (i.e. the bispecific antigen binding molecule provides monovalent binding to the second antigen).

Charge modifications

The bispecific antigen binding molecules may comprise amino acid substitutions in Fab molecules comprised therein which are particularly efficient in reducing mispairing of light chains with nonmatching heavy chains (Bence-Jones-type side products), which can occur in the production of Fab-based bi-/multispecific antigen binding molecules with a VH/VL exchange in one (or more, in case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see also PCT publication no. WO 2015/150447, particularly the examples therein, incorporated herein by reference in its entirety). The ratio of a desired bispecific antigen binding molecule compared to undesired side products, in particular Bence Jones-type side products occurring in bispecific antigen binding molecules with a VH/VL domain exchange in one of their binding arms, can be improved by the introduction of charged amino acids with opposite charges at specific amino acid positions in the CHI and CL domains (sometimes referred to herein as “charge modifications”).

Accordingly, in some embodiments wherein the first and the second antigen binding moiety of the bispecific antigen binding molecule are both Fab molecules, and in one of the antigen binding moieties (particularly the second antigen binding moiety) the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other, i) in the constant domain CL of the first antigen binding moiety the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CHI 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 (numbering according to Kabat EU index); or ii) in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CHI of the second antigen binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index). The bispecific antigen binding molecule does not comprise both modifications mentioned under i) and ii). The constant domains CL and CHI of the antigen binding moiety having the VH/VL exchange are not replaced by each other (i.e. remain unexchanged).

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

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

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

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

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

In particular embodiments, if amino acid substitutions according to the above embodiments are made in the constant domain CL and the constant domain CHI of the first antigen binding moiety, the constant domain CL of the first antigen binding moiety is of kappa isotype.

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

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

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

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

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

In a particular embodiment, the bispecific antigen binding molecule comprises

(a) a first antigen binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, a HCDR 2 of SEQ ID NO: 13, and a 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, a LCDR 2 of SEQ ID NO: 16 and a LCDR 3 of SEQ ID NO: 17, and

(b) a second antigen binding moiety that binds to a second antigen, wherein the second antigen is CD3 and the second antigen binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, a HCDR 2 of SEQ ID NO: 19, and a 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, a LCDR 2 of SEQ ID NO: 22 and a 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 by each other; wherein in the constant domain CL of the first antigen binding moiety the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in a particular embodiment independently by lysine (K) or arginine (R)) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in a particular embodiment independently by lysine (K) or arginine (R)), and in the constant domain CHI of the first antigen binding moiety the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

Bispecific antigen binding molecule formats

In certain embodiments, an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, the multispecific antibody has three or more binding specificities. In certain embodiments, bispecific antibodies may bind to two (or more) different epitopes of GPRC5D. Multispecific (e.g., bispecific) antibodies may also be used to localize cytotoxic agents to cells which express GPRC5D. Multispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc- heterodimeric molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547- 1553 (1992) and WO 2011/034605); using the common light chain technology for circumventing the light chain miss-pairing problem (see, e.g., WO 98/50431); using "diabody" technology for making 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, e.g., in Tutt et al. J. Immunol. 147: 60 (1991). Engineered antibodies with three or more antigen binding sites, including for example, “Octopus antibodies,” or DVD-Ig are also included herein (see, e.g. WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO2010/145792, and WO 2013/026831. The bispecific antibody or antigen binding fragment thereof also includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to GPRC5D as well as CD3 (see, e.g., US 2008/0069820 and WO 2015/095539).

Multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging the VH/VL domains (see e.g., WO 2009/080252 and WO 2015/150447), the CH1/CL domains (see e.g., WO 2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab pairing. See e.g., WO 2016/172485.

Various further molecular formats for multispecific antibodies are known in the art and are included herein (see e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, the so-called “BiTE” (bispecific T cell engager) molecules wherein two scFv molecules are fused by a flexible linker (see, e.g., W02004/106381, W02005/061547, W02007/042261, and W02008/119567, Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (dual affinity retargeting) molecules which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which are whole hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Particular T cell bispecific antibody formats included herein are described in WO 2013/026833, WO2013/026839, WO 2016/020309; Bacac et al., Oncoimmunology 5(8) (2016) el203498.

The components of the bi specific antigen binding molecule can be fused to each other in a variety of configurations.

In particular embodiments, the antigen binding moieties comprised in the bispecific antigen binding molecule are Fab molecules. In such embodiments, the first, second, third etc. antigen binding moiety may be referred to herein as first, second, third etc. Fab molecule, respectively. In one embodiment, the first and the second antigen binding moiety of the bispecific antigen binding molecule are fused to each other, optionally via a peptide linker. In particular embodiments, the first and the second antigen binding moiety are each a Fab molecule. In one such embodiment, the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the first antigen binding moiety. In another such embodiment, 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. In embodiments wherein either (i) the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the first antigen binding moiety or (ii) the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety, additionally the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may be fused to each other, optionally via a peptide linker.

A bispecific antigen binding molecule with a single antigen binding moiety (such as a Fab molecule) capable of specific binding to a target cell antigen such as GPRC5D is useful, particularly in cases where internalization of the target cell antigen is to be expected following binding of a high affinity antigen binding moiety. In such cases, the presence of more than one antigen binding moiety specific for the target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availability.

In other cases, however, it will be advantageous to have a bispecific antigen binding molecule comprising two or more antigen binding moieties (such as Fab molecules) specific for a target cell antigen for example to optimize targeting to the target site or to allow crosslinking of target cell antigens.

Accordingly, in particular embodiments, the bispecific antigen binding molecule comprises a third antigen binding moiety.

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

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

The third antigen binding moiety of the bispecific antigen binding molecule may incorporate any of the features, singly or in combination, described herein in relation to the first antigen binding moiety and/or the antibody that binds GPRC5D, unless scientifically clearly unreasonable or impossible. In one embodiment, the third antigen binding moiety comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, a HCDR 2 of SEQ ID NO: 13, and a 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, a LCDR 2 of SEQ ID NO: 16 and a LCDR 3 of SEQ ID NO: 17.

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

In one embodiment, the VH of the third antigen binding moiety 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 moiety 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.

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

In one embodiment, the third antigen binding moiety comprises a VH comprising an amino acid sequence of SEQ ID NO: 10, and a VL comprising the amino acid sequence of SEQ ID NO: 11.

In one embodiment, the third antigen binding moiety comprises a VH sequence of SEQ ID NO: 10 and the VL sequence of SEQ ID NO: 11.

In a particular 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 particular embodiment, the third antigen binding moiety comprises the VH sequence of SEQ ID NO: 48 and the VL sequence of SEQ ID NO: 11.

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 CHI and/or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs 1 and 2 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 3 (human IgGi heavy chain constant domains CH1-CH2-CH3). In some embodiments, the third antigen binding moiety comprises a light chain constant region comprising 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: 1 or SEQ ID NO: 2, particularly the amino acid sequence of SEQ ID NO: 1. Particularly, the light chain constant region may comprise amino acid mutations as described herein under “charge modifications” and/or may comprise deletion or substitutions of one or more (particularly two) N-terminal amino acids if in a crossover Fab molecule. In some embodiments, the third antigen binding moiety 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 CHI domain sequence comprised in the amino acid sequence of SEQ ID NO: 3. Particularly, the heavy chain constant region (specifically CHI domain) may comprise amino acid mutations as described herein under “charge modifications”.

In particular embodiments, the third and the first antigen binding moiety are each a Fab molecule and the third antigen binding moiety is identical to the first antigen binding moiety. Thus, in these embodiments the first and the third antigen binding moiety comprise the same heavy and light chain amino acid sequences and have the same arrangement of domains (i.e. conventional or crossover)). Furthermore, 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 modifications” will be made in the constant domain CL and the constant domain CHI of each of the first antigen binding moiety and the third antigen binding moiety. Alternatively, said amino acid substitutions may be made in the constant domain CL and the constant domain CHI of the second antigen binding moiety (which in particular embodiments is also a Fab molecule), but not in the constant domain CL and the constant domain CHI of the first antigen binding moiety and the third antigen binding moiety.

Like the first antigen binding moiety, the third antigen binding moiety particularly is a conventional Fab molecule. Embodiments wherein the first and the third antigen binding moieties are crossover Fab molecules (and the second antigen binding moiety is a conventional Fab molecule) are, however, also contemplated. Thus, in particular embodiments, the first and the third antigen binding moieties are each a conventional Fab molecule, and the second antigen binding moiety is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CL and CHI of the Fab heavy and light chains are exchanged / replaced by each other. In other embodiments, the first and the third antigen binding moieties are each a crossover Fab molecule and the second antigen binding moiety is a conventional Fab molecule. If a third antigen binding moiety is present, in a particular embodiment the first and the third antigen moiety bind to GPRC5D, and the second antigen binding moiety binds to CD3, particularly CD3 epsilon.

In particular embodiments, the bispecific antigen binding molecule comprises an Fc domain composed of a first and a second subunit. The first and the second subunit of the Fc domain are capable of stable association.

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

In some embodiments, the first and the second antigen binding moiety are each a Fab molecule and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N- terminus of the first or the second subunit of the Fc domain. In such embodiments, the first antigen binding moiety may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety or to the N-terminus of the other one of the subunits of the Fc domain. In particular such embodiments, said first antigen binding moiety is a conventional Fab molecule, and the second antigen binding moiety is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CL and CHI of the Fab heavy and light chains are exchanged / replaced by each other. In other such embodiments, said first Fab molecule is a crossover Fab molecule and the second Fab molecule is a conventional Fab molecule.

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

In another embodiment, the first and the second antigen binding moiety are each a Fab molecule and the first and the second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. In a specific embodiment, the bispecific antigen binding molecule essentially consists of the first and the second Fab molecule, the Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first and the second Fab molecule 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. The first and the second Fab molecule may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment the first and the second Fab molecule are each fused to the Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgGi hinge region, particularly where the Fc domain is an IgGi Fc domain.

In some embodiments, the first and the second antigen binding moiety are each a Fab molecule and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N- terminus of the first or the second subunit of the Fc domain. In such embodiments, the second antigen binding moiety may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety or (as described above) to the N- terminus of the other one of the subunits of the Fc domain. In particular such embodiments, said first antigen binding moiety is a conventional Fab molecule, and the second antigen binding moiety is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CL and CHI of the Fab heavy and light chains are exchanged / replaced by each other. In other such embodiments, said first Fab molecule is a crossover Fab molecule and the second Fab molecule is a conventional Fab molecule.

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

In some embodiments, a third antigen binding moiety, particularly a third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In particular such embodiments, said first and third Fab molecules are each a conventional Fab molecule, and the second Fab molecule is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CL and CHI of the Fab heavy and light chains are exchanged / replaced by each other. In other such embodiments, said first and third Fab molecules are each a crossover Fab molecule and the second Fab molecule is a conventional Fab molecule.

In a particular such embodiment, the second and the third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the second Fab molecule. In a specific embodiment, the bispecific antigen binding molecule essentially consists of the first, the second and the third Fab molecule, the Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The second and the third Fab molecule may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment the second and the third Fab molecule are each fused to the Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgGi hinge region, particularly where the Fc domain is an IgGi 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.

In another such embodiment, the first and the third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N- terminus of the Fab heavy chain of the first antigen binding moiety. In a specific embodiment, the bispecific antigen binding molecule essentially consists of the first, the second and the third Fab molecule, the Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The first and the third Fab molecule may be fused to the Fc domain directly or through a peptide linker. In a particular embodiment the first and the third Fab molecule are each fused to the Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgGi hinge region, particularly where the Fc domain is an IgGi 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.

In configurations of the bispecific antigen binding molecule wherein a Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of each of the subunits of the Fc domain through an immunoglobulin hinge regions, the two Fab molecules, the hinge regions and the Fc domain essentially form an immunoglobulin molecule. In a particular embodiment the immunoglobulin molecule is an IgG class immunoglobulin. In an even more particular embodiment the immunoglobulin is an IgGi subclass immunoglobulin. In another embodiment the immunoglobulin is an IgG4 subclass immunoglobulin. In a further particular 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.

In some of the bispecific antigen binding molecules, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are fused to each other, optionally via a peptide linker. Depending on the configuration of the first and the second Fab molecule, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. Fusion of the Fab light chains of the first and the second Fab molecule further reduces mispairing of unmatched Fab heavy and light chains, and also reduces the number of plasmids needed for expression of some of the bispecific antigen binding molecules.

The antigen binding moieties may 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 are described herein. Suitable, non-immunogenic peptide linkers include, for example, (G4S) _n , (SG4)n, (G4S) _n or G4(SG4) _n peptide linkers, “n” is generally an integer from 1 to 10, typically from 2 to 4. In one embodiment said peptide linker has a length of at least 5 amino acids, in one embodiment a length of 5 to 100, in a further embodiment of 10 to 50 amino acids. In one embodiment said peptide linker is (GxS) _n or (GxS) _n G _m with 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 a further embodiment x=4 and n=2. In one embodiment said peptide linker is (G4S)2. A particularly suitable peptide linker for fusing the Fab light chains of the first 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 and the second Fab fragments comprises the sequence (D)-(G4S)2 (SEQ ID NOS 7 and 8). Another suitable such linker comprises the sequence (G4S)4. Additionally, linkers may comprise (a portion of) an immunoglobulin hinge region. Particularly where a Fab molecule is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

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

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

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

In some of these embodiments the bispecific antigen binding molecule further comprises a crossover Fab light chain polypeptide of the second Fab molecule, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH(2)-CL(2)), and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In others of these embodiments the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule (VH(2)-CL(2)-VL(i)-CL(i)}, or a polypeptide wherein the Fab light chain polypeptide of the first Fab molecule shares a carboxy- terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VL(i)-CL(i)-VH(2)-CL(2)}, as appropriate.

The bispecific antigen binding molecule according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(3)-CH1(3)-CH2-CH3(-CH4)) and the Fab light chain polypeptide of a third Fab molecule (VL(3)-CL(3)). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond.

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

In some of these embodiments the bispecific antigen binding molecule further comprises a crossover Fab light chain polypeptide of the second Fab molecule, wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL(2)-CH1(2)), and the Fab light chain polypeptide of the first Fab molecule (VL(i)-CL(i)). In others of these embodiments the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule (VL(2)-CHl(2)-VL(i)-CL(i)}, or a polypeptide wherein the Fab light chain polypeptide of the first Fab molecule shares a carboxy- terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VL(i)-CL(i)-VL(2)-CHl(2)}, as appropriate.

The bispecific antigen binding molecule according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(3)-CH1(3)-CH2-CH3(-CH4)) and the Fab light chain polypeptide of a third Fab molecule (VL(3)-CL(3)). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond.

In certain embodiments, the bispecific antigen binding molecule does not comprise an Fc domain. In particular such embodiments, said first and, if present third Fab molecules are each a conventional Fab molecule, and the second Fab molecule is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CL and CHI of the Fab heavy and light chains are exchanged / replaced by each other. In other such embodiments, said first and, if present third Fab molecules are each a crossover Fab molecule and the second Fab molecule is a conventional Fab molecule.

In one such embodiment, the bispecific antigen binding molecule essentially consists of the first and the 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 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.

In another such embodiment, the bispecific antigen binding molecule essentially consists of the first and the 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.

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

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

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

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

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

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

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

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

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

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

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

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

In a particular embodiment, the 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 comprising a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, a HCDR 2 of SEQ ID NO: 13, and a 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, a LCDR 2 of SEQ ID NO: 16 and a 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 or the constant domains CL and CHI of the Fab light chain and the Fab heavy chain are replaced by each other, and wherein the Fab molecule comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, a HCDR 2 of SEQ ID NO: 19, and a 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, a LCDR 2 of SEQ ID NO: 22 and a LCDR 3 of SEQ ID NO: 23; 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 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). In another embodiment, the 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 comprising a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, a HCDR 2 of SEQ ID NO: 13, and a 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, a LCDR 2 of SEQ ID NO: 16 and a 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 or the constant domains CL and CHI of the Fab light chain and the Fab heavy chain are replaced by each other, and wherein the Fab molecule comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, a HCDR 2 of SEQ ID NO: 19, and a 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, a LCDR 2 of SEQ ID NO: 22 and a LCDR 3 of SEQ ID NO: 23; c) an Fc domain composed of a first and a second subunit; wherein (i) 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).

In all of the different configurations of the bispecific antigen binding molecule, the amino acid substitutions described herein, if present, may either be in the CHI and CL domains of the first and (if present) the third antigen binding moiety/Fab molecule, or in the CHI and CL domains of the second antigen binding moiety/Fab molecule. Preferably, they are in the CHI and CL domains of the first and (if present) the third antigen binding moiety/Fab molecule. In accordance with the concept of the invention, if amino acid substitutions as described herein are made in the first (and, if present, the third) antigen binding moiety/Fab molecule, no such amino acid substitutions are made in the second antigen binding moiety/Fab molecule. Conversely, if amino acid substitutions as described herein are made in the second antigen binding moiety/Fab molecule, no such amino acid substitutions are made in the first (and, if present, the third) antigen binding moiety/Fab molecule. Amino acid substitutions are particularly made in bispecific antigen binding molecules comprising a Fab molecule wherein the variable domains VL and VH1 of the Fab light chain and the Fab heavy chain are replaced by each other.

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

In one embodiment, the 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 comprising a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, a HCDR 2 of SEQ ID NO: 13, and a 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, a LCDR 2 of SEQ ID NO: 16 and a 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 by each other, and wherein the Fab molecule comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, a HCDR 2 of SEQ ID NO: 19, and a 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, a LCDR 2 of SEQ ID NO: 22 and a LCDR 3 of SEQ ID NO: 23; c) an Fc domain composed of a first and a second subunit; wherein in the constant domain CL of the first antigen binding moiety under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein in the constant domain CHI of the first antigen binding moiety under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); 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).

In a particular embodiment, the 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 comprising a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, a HCDR 2 of SEQ ID NO: 13, and a 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, a LCDR 2 of SEQ ID NO: 16 and a 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 by each other, and wherein the Fab molecule comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, a HCDR 2 of SEQ ID NO: 19, and a 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, a LCDR 2 of SEQ ID NO: 22 and a LCDR 3 of SEQ ID NO: 23; 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 and a second subunit; wherein in the constant domain CL of the first antigen binding moiety under a) and the third antigen binding moiety under c) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein in the constant domain CHI of the first antigen binding moiety under a) and the third antigen binding moiety under c) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); 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).

In another embodiment, the 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 comprising a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, a HCDR 2 of SEQ ID NO: 13, and a 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, a LCDR 2 of SEQ ID NO: 16 and a 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 by each other, and wherein the Fab molecule comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, a HCDR 2 of SEQ ID NO: 19, and a 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, a LCDR 2 of SEQ ID NO: 22 and a LCDR 3 of SEQ ID NO: 23; c) an Fc domain composed of a first and a second subunit; wherein in the constant domain CL of the first antigen binding moiety under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein in the constant domain CHI of the first antigen binding moiety under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); 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).

According to any of the above embodiments, components of the bispecific antigen binding molecule (e.g. Fab molecules, Fc domain) may be fused directly or through various linkers, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, that are described herein or are known in the art. Suitable, non-immunogenic peptide linkers include, for example, (G4S) _n , (SGQn, (G4S) _n or G4(SG4) _n peptide linkers, wherein n is generally an integer from 1 to 10, typically from 2 to 4. In a particular aspect, the bispecific antigen binding molecule comprising 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 comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; 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 Fab molecule wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25; c) an Fc domain composed of a first and a second subunit; wherein in the constant domain CL of the first and the third antigen binding moiety under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat) (most particularly by arginine (R)), and wherein in the constant domain CHI of the first and the third antigen binding moiety under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); 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).

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 with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368 A) (numberings according to Kabat EU index).

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 with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with 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) (numberings according to Kabat EU index).

In still a further embodiment, in each of the first and the second subunit of the Fc domain of the bispecific antigen binding molecule, 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 by a glycine residue (P329G) (numbering according to Kabat EU index).

In still a further embodiment, the Fc domain is a human IgGl Fc domain.

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

Fc domain

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

The Fc domain of the bispecific antigen binding molecule consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment, the bispecific antigen binding molecule of the invention comprises not more than one Fc domain.

In one embodiment, the Fc domain of the bispecific antigen binding molecule is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgGi Fc domain. In another embodiment the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG ₄ antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment, the Fc domain is a human Fc domain. In an even more particular embodiment, the Fc domain is a human IgGi Fc domain. An exemplary sequence of a human IgGi Fc region is given in SEQ ID NO: 6.

Fc domain modifications promoting heterodimerization

Bispecific antigen binding molecules comprise different antigen binding moieties, which may be fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of bispecific antigen binding molecules in recombinant production, it will thus be advantageous to introduce in the Fc domain of the bi specific antigen binding molecule a modification promoting the association of the desired polypeptides.

Accordingly, in particular embodiments, the Fc domain of the bi specific antigen binding molecule comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment said modification is in the CH3 domain of the Fc domain.

There exist several approaches for modifications in the CH3 domain of the Fc domain in order to enforce heterodimerization, which are well described e.g. 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 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed). These different approaches for improved heavy chain heterodimerization are contemplated as different alternatives in combination with the heavy-light chain modifications (e.g. VH and VL exchange/replacement in one binding arm and the introduction of substitutions of charged amino acids with opposite charges in the CH1/CL interface) in the bispecific antigen binding molecule which reduce heavy/light chain mispairing and Bence Jones-type side products.

In a specific embodiment said modification promoting the association of the first and the second subunit of the Fc domain is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in 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). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen binding molecule an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

Preferably said 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).

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

The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.

In a specific embodiment, in (the CH3 domain of) the first subunit of the Fc domain (the “knobs” subunit) the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in (the CH3 domain of) the second subunit of the Fc domain (the “hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).

In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with 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) (numberings according to Kabat EU index). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In a particular embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index). In a particular embodiment the antigen binding moiety that binds to the second antigen (e.g. an activating T cell antigen) is fused (optionally via the first antigen binding moiety, which binds to GPRC5D, and/or a peptide linker) to the first subunit of the Fc domain (comprising the “knob” modification). Without wishing to be bound by theory, fusion of the antigen binding moiety that binds a second antigen, such as an activating T cell antigen, to the knob-containing subunit of the Fc domain will (further) minimize the generation of antigen binding molecules comprising two antigen binding moieties that bind to an activating T cell antigen (steric clash of two knobcontaining polypeptides).

Other techniques of CH3 -modification for enforcing the heterodimerization are contemplated as alternatives according to the invention and are described e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

In one embodiment, the heterodimerization approach described in EP 1870459, is used alternatively. This approach 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. One preferred embodiment for the bi specific antigen binding molecule of the invention are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (numbering according to Kabat EU index).

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

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

In one embodiment, the heterodimerization approach described in WO 2013/157953 is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L35 ID (numberings according to Kabat EU index). In a further embodiment, the first CH3 domain comprises further amino acid mutation L351K. In a further embodiment, the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numberings according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO 2012/058768 is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g. selected from 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 (numberings according to Kabat EU index). In a further embodiment a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F. In a further embodiment, a first CH3 domain comprises amino acid mutation Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (numberings according to Kabat EU index).

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

In one embodiment, the heterodimerization approach described in WO 2011/090762, which also uses the knobs-into-holes technology described above, is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A. In one embodiment, a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (numberings according to Kabat EU index).

In one embodiment, the bispecific antigen binding molecule or its Fc domain is of IgG? subclass and the heterodimerization approach described in WO 2010/129304 is used alternatively.

In an alternative embodiment, a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In one such embodiment, a first CH3 domain comprises amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K392D or N392D) and a second CH3 domain comprises amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K, D356K, or E357K, and more preferably D399K and E356K). In a further embodiment, the first CH3 domain further comprises amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). In a further embodiment the first CH3 domain further or alternatively comprises amino acid substitution of K439 and/or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to Kabat EU index). In yet a further embodiment, the heterodimerization approach described in WO 2007/147901 is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (numberings according to Kabat EU index).

In still another embodiment, the heterodimerization approach described in WO 2007/110205 can be used alternatively.

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 Kabat EU index).

Fc domain modifications reducing Fc and/or effector function

The Fc domain confers to the bispecific antigen binding molecule favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time, it may, however, lead to undesirable targeting of the bispecific antigen binding molecule to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with the T cell activating properties (e.g. in embodiments of the bispecific antigen binding molecule wherein the second antigen binding moiety binds to an activating T cell antigen) and the long half-life of the bispecific antigen binding molecule, results in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce efficacy of the bispecific antigen binding molecule (particularly a bispecific antigen binding molecule wherein the second antigen binding moiety binds to an activating T cell antigen) due to the potential destruction of T cells e.g. by NK cells.

Accordingly, in particular embodiments, the Fc domain of the bi specific antigen binding molecule exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain. In one such embodiment the Fc domain (or the bispecific antigen binding molecule comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGi Fc domain (or a bispecific antigen binding molecule comprising a native IgGi Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgGi Fc domain (or a bispecific antigen binding molecule comprising a native IgGi Fc domain). In one embodiment, the Fc domain (or the bispecific antigen binding molecule comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function. In a particular embodiment the Fc receptor is an Fey receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. In one embodiment the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGi Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the bispecific antigen binding molecule comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGi Fc domain (or the bispecific antigen binding molecule comprising a native IgGi Fc domain) to FcRn.

In certain embodiments the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In particular embodiments, the Fc domain of the bispecific antigen binding molecule comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutation is 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 an Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment the bispecific antigen binding molecule comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to a bispecific antigen binding molecule comprising a non-engineered Fc domain. In a particular embodiment, the Fc receptor is an Fey receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to a complement component, specifically binding affinity to Clq, is also reduced. In one embodiment, binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or the bispecific antigen binding molecule comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or the bispecific antigen binding molecule comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or bispecific antigen binding molecules of the invention comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments, the Fc domain of the bispecific antigen binding molecule is engineered to have reduced effector function, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment, the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a nonengineered Fc domain (or a bispecific antigen binding molecule comprising a non-engineered Fc domain).

In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). In more particular embodiments, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”). Specifically, in particular embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and 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 by a glycine residue (P329G) (numbering according to Kabat EU index).

In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. The “P329G LALA” combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGi Fc domain, as described in PCT publication no. WO 2012/130831, which is incorporated herein by reference in its entirety. WO 2012/130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.

IgG ₄ antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgGi antibodies. Hence, in some embodiments, the Fc domain of the bispecific antigen binding moleculeis an IgG ₄ Fc domain, particularly a human IgG ₄ Fc domain. In one embodiment, the IgG ₄ Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P (numberings according to Kabat EU index). To further reduce its binding affinity to an Fc receptor and/or its effector function, in one embodiment, the IgG ₄ Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numberings according to Kabat EU index). In another embodiment, the IgG ₄ Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numberings according to Kabat EU index). In a particular embodiment, the IgG ₄ Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numberings according to Kabat EU index). Such IgG ₄ Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety.

In a particular embodiment, the Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain, is a human IgGi Fc domain comprising the amino acid substitutions L234A, L235 A and optionally P329G, or a human IgG ₄ Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numberings according to Kabat EU index).

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, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) (numberings according to Kabat EU index).

In addition to the Fc domains described hereinabove and in PCT publication no. WO 2012/130831, Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) (numberings according to Kabat EU index). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include sitespecific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. Alternatively, binding affinity of Fc domains orbispecific antigen binding molecules comprising anFc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.

Effector function of an Fc domain, or a bispecific antigen binding molecule comprising an Fc domain, can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component, specifically to Clq, is reduced. Accordingly, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. Clq binding assays may be carried out to determine whether the Fc domain, or the bispecific antigen binding molecule comprising the Fc domain, is able to bind Clq and hence has CDC activity. See e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may 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)).

FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006); WO 2013/120929).

Compositions, Formulations, and Routes of Administration

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

Further provided is a method of producing an antibody or bispecific antigen binding molecule of the invention in a form suitable for administration in vivo, the method comprising (a) obtaining an antibody or bispecific antigen binding molecule according to the invention, and (b) formulating the antibody or bispecific antigen binding molecule with at least one pharmaceutically acceptable carrier, whereby a preparation of antibody or bispecific antigen binding molecule is formulated for administration in vivo.

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

An immunoconjugate of the invention (and any additional therapeutic agent) 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. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

Parenteral compositions include those designed for administration by injection, e.g. subcutaneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal or intraperitoneal injection. For injection, the antibodies or bispecific antigen binding molecules of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the antibodies or bispecific antigen binding molecules may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the antibodies or bispecific antigen binding molecules of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, the antibodies or bispecific antigen binding molecules may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the antibodies or bispecific antigen binding molecules may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the antibodies or bispecific antigen binding molecules of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. The antibodies or bispecific antigen binding molecules may be formulated into a composition in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.

Immunomodulatory imide drugs (IMiDs)

The term “immunomodulatory imide drugs (IMiDs)” refers to a class of immunomodulatory drugs (drugs that adjust immune responses) containing an imide group. IMiDs include both first generation IMiDs and Cereblon E3 ligase modulators (CELMoDs; also termed next generation IMiDs) which both comprise a conserved glutarimide ring. First and next generation IMiDs bind Cereblon (CRBN), a receptor for the Cullin-ring 4 ubiquitin-ligase (CRL4) complex, and modulate the ubiquitin ligase activity. The ligase specificity is redirected towards non-physiological proteins targets, also termed “neo-substrates”, which are subsequently ubiquitinated and/or degraded. The conserved glutarimide ring binds to CRBN while the variable side groups interact with CRBN and neo-substrates. The term “first generation IMiDs” refers to thalidomide and the thalidomide derivatives lenalidomide and pomalidomide. The term “CELMoDs” or “next generation IMiDs” refers to thalidomide derivatives with extended side groups enabling improved interaction with CRBN and/or neo-substrates and include but are not limited to iberdomide (also known as CC- 220), avadomide (also known as CC-122), mezigdomide (also known as CC-92480), CC885, CC647, CC-90009 and CC3060.

The term “lenalidomide” refers to the compound with the following chemical structure:

The empirical formula for lenalidomide is C13H13N3O3, CAS registry number 191732-72-6 and the gram molecular weight is 259.3. Lenalidomide is a thalidomide analogue marketed under the tradename REVLIMID " . The term “pomalidomide” refers to the compound with the following chemical structure:

The empirical formula for pomalidomide is C13H11N3O4, CAS Registry Number 19171-19-8 and the gram molecular weight is 273.24. Pomalidomide is a thalidomide analogue marketed under the tradename Imnovid in the EU and Pomalyst in the US.

The term “iberdomide” refers to the compound with the following chemical structure:

The empirical formula for iberdomide is C25H27N3O5, CAS Registry Number 1323403-33-3 and the gram molecular weight is 449.5.

The term “mezigdomide” refers to the compound with the following chemical structure:

The empirical formula for mezigdomide is C32H30FN5O4, CAS Registry Number is 2259648-80-9 and the gram molecular weight is 567.6.

The present invention provides a combination therapy of an anti-GPRC5D/anti-CD3 bispecific antibody with an immunomodulatory imide drug (IMiD). The IMiD used in 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 iberdomide, avadomide, mezigdomide (CC-92480), CC885, CC647, CC-90009 and CC3060. In one embodiment, the IMiD is selected from the group of of lenalidomide, pomalidomide, iberdomide and mezigdomide.

Gucocorticosteroids

The present invention further provides a combination therapy of an anti-GPRC5D/anti-CD3 bispecific antibody with an immunomodulatory imide drug (IMiD) and a glucocorticosteroid. “Glucocorticosteroids” or “glucocorticoids” as used herein refer to compounds of a class of corticosteroids which are involved in metabolism of carbohydrates, proteins, and fats and have anti-inflammatory activity. Glucocorticosteroids are therapeutically mainly used for their antiinflammatory and immunosuppressive effects. Glucocorticosteroids include, but are not limited to dexamethasone, prednisone, prednisolone, methylprednisolone and alternatives. The term “dexamethasone” refers to the compound with the following chemical structure:

The empirical formula for dexamethasone is C22H29FO5, CAS Registry Number 50-02-2 and the gram molecular weight is 392.46.

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. In one embodiment, the glucocorticosteroid is dexamethasone.

Therapeutic Methods and Compositions

The present invention comprises a combination therapy comprising an anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD. Optionally, the combination therapy described herein may further comprise a glucocorticosteroid.

The invention comprises a method for the treatment of a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of the combination therapy of an anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD. The invention comprises a method for the treatment of a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of the combination therapy of an anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD and a glucocorticosteroid.

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

One embodiment of the invention is an anti-GPRC5D/anti-CD3 bispecific antibody described herein for use in the treatment of cancer or a tumor in combination with an IMiD as described herein. One embodiment of the invention is an anti-GPRC5D/anti-CD3 bispecific antibody described herein for use in the treatment of cancer or a tumor in combination with an IMiD as described herein and a glucocorticosteroid as described herein.

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

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

Non-limiting examples of cancers 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 may be treated using the combination therapy of the present invention including, but not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. In certain embodiments the cancer is chosen from the group consisting of kidney cancer, bladder cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer and prostate cancer. In one embodiment, the cancer is a GPRC5D-expressing cancer. In one embodiment, the cancer is multiple myeloma.

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

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

The invention comprises a method for the treatment of a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of the combination therapy of an anti-GPRC5D/anti-CD3 bispecific antibody as described herein with an IMiD as described herein. The invention further comprises a method for the treatment of a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of the combination therapy of an anti-GPRC5D/anti-CD3 bispecific antibody as described herein with an IMiD as described herein and a glucocorticosteroid as described herein.

The invention comprises a method of treating cancer in an individual comprising administering to said individual an anti-GPRC5D/anti-CD3 bispecific antibody as described herein in combination with an IMiD as described herein. The invention further comprises a method of treating cancer in an individual comprising administering to said individual an anti-GPRC5D/anti-CD3 bispecific antibody as described herein in combination with an IMiD as described herein and a glucocorticosteroid as described herein.

The invention comprises a method for the prevention or treatment of metastasis a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of the combination therapy of an anti-GPRC5D/anti-CD3 bispecific antibody as described herein with an IMiD as described herein. The invention further comprises a method for the prevention or treatment of metastasis patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of the combination therapy of an anti-GPRC5D/anti-CD3 bispecific antibody as described herein with an IMiD as described herein and a glucocorticosteroid as described herein.

The invention comprises the use of an anti-GPRC5D/anti-CD3 bispecific antibody with an IMiD according to the invention for the described combination therapy. In one embodiment, the anti- GPRC5D/anti-CD3 bispecific antibody used in above described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29. In a further embodiment, the IMiD used in above described combination treatments and medical uses is selected from group of lenalidomide, pomalidomide, iberdomide and mezigdomide. In another embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD is selected from the group of lenalidomide, pomalidomide, iberdomide and mezigdomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD is lenalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD is pomalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD is iberdomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD is mezigdomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses is forimtamig and the IMiD is lenalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses is forimtamig and the IMiD is pomalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses is forimtamig and the IMiD is iberdomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses is forimtamig and the IMiD is mezigdomide.

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

In a preferred embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29. In a further embodiment, the IMiD used in the above described combination treatments and medical uses is selected from group of lenalidomide, pomalidomide, iberdomide and mezigdomide. In a further embodiment, the glucocorticoid used in the above described combination treatments and medical uses is dexamethasone. In another embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD used in the above described combination treatments and medical uses is selected from group of lenalidomide, pomalidomide, iberdomide and mezigdomide. In another embodiment, the anti- -n-

GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD is selected from the group of lenalidomide, pomalidomide, iberdomide and mezigdomide and the glucocorticosteroid is dexamethasone. In another embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD is lenalidomide and the glucocorticosteroid is dexamethasone. In another embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the above described combination treatments and medical uses is forimtamig, and the IMiD is lenalidomide and the glucocorticosteroid is dexamethasone.

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

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

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

A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. In addition to water, the carrier can be, for example, an isotonic buffered saline solution. Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient (effective amount). The dosage may comprise a step-up dosing cycle of an active ingredient. The term “dosage” refers to the amount, i.e. dose, and frequency of administration of an active ingredient. The term “step-up dosing cycle” refers to a treatment period wherein the dosage of the active ingredient is gradually increased over said treatment period. This may be achieved by increasing the dose of the active ingredient and/or increasing the frequency of administration. Thus, in one embodiment, the dosage of an anti- GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD as described herein comprises at least one step-up dosing cycle. In one embodiment, the dosage of an anti- GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD as described herein comprises at leat one step-up dosing cycle of the anti-GPRC5D/anti-CD3 bispecific antibody. In one embodiment, the dosage of an anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD as described herein comprises at least one step-up dosing cycle of the anti- GPRC5D/anti-CD3 bispecific antibody. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody used in the combination treatments and medical uses as described herein is dosed at an effective amount, wherein the dosage comprises at least one step-up dosing cycle. In one embodiment, forimtamig used in the combination treatments and medical uses as described herein is dosed at an effective amount, wherein the dosage comprises at least one step-up dosing cycle. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The invention comprises an IMiD as described herein in combination with an anti-GPRC5D/anti- CD3 bispecific antibody as described herein for use in the manufacture of a medicament for the treatment of cancer. The invention comprises an IMiD as described herein in combination with an anti-GPRC5D/anti-CD3 bispecific antibody as described herein and a glucocorticoid as described herein for use in the manufacture of a medicament for the treatment of cancer. The invention comprises glucocorticoid as described herein in combination with 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 use in the manufacture of a medicament for the treatment of cancer according to the invention comprises the 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 use in the manufacture of a medicament for the treatment of cancer according to the invention is forimtamig. In one embodiment the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is selected from the group of lenalidomide, pomalidomide, iberdomide and mezigdomide. In one embodiment the glucocorticoid for use in the manufacture of a medicament for the treatment of cancer according to the invention is dexamethasone. In one embodiment the anti-GPRC5D/anti- CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention comprises the the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is selected from the group of lenalidomide, pomalidomide, iberdomide and mezigdomide. In one embodiment the anti- GPRC5D/anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is lenalidomide. In one embodiment the anti-GPRC5D/anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention comprises the the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is pomalidomide. In one embodiment the anti-GPRC5D/anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention comprises the the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is iberdomide. In one embodiment the anti- GPRC5D/anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention comprises the the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is mezigdomide. In one embodiment the anti-GPRC5D/anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention is forimtamig and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is lenalidomide. In one embodiment the anti-GPRC5D/anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention is forimtamig and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is pomalidomide. In one embodiment the anti-GPRC5D/anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention is forimtamig and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is iberdomide. In one embodiment the anti- GPRC5D/anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention is forimtamig and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is mezigdomide. In one embodiment the anti-GPRC5D/anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention comprises the the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is selected from the group of lenalidomide, pomalidomide, iberdomide and mezigdomide and the glucocorticoid for use in the manufacture of a medicament for the treatment of cancer according to the invention is dexamethasone. In one embodiment the anti-GPRC5D/anti- CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention comprises the the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is lenalidomide and the glucocorticoid for use in the manufacture of a medicament for the treatment of cancer according to the invention is dexamethasone. In one embodiment the anti-GPRC5D/anti-CD3 bispecific antibody for use in the manufacture of a medicament for the treatment of cancer according to the invention is forimtamig and the IMiD for use in the manufacture of a medicament for the treatment of cancer according to the invention is lenalidomide and the glucocorticoid for use in the manufacture of a medicament for the treatment of cancer according to the invention is dexamethasone.

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

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

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

Moreover, the kit may comprise (a) a first container with a composition contained therein, wherein the composition comprises an anti-GPRC5D/anti-CD3 bispecific antibody as described herein; (b) a second container with a composition contained therein, wherein the composition comprises an IMiD as described herein; (c) a third container with a composition contained therein, wherein the composition comprises a glucocorticosteroid as described herein, and optionally (d) a fourth container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.. The kit in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the kit may further comprise a fifth container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

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

In another aspect the 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 directing 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 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 directing the use of the IMiD in a combination therapy with an anti-GPRC5D/anti-CD3 bispecific antibody and a glucocorticoid as described herein as a method for treating the disease.

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

The term "a method of treating" or its equivalent, when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of a cancer. "A method of treating" cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will, in fact, be eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated. Often, a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of a patient, is nevertheless deemed to induce an overall beneficial course of action.

The terms “administered in combination with” or “co-administration”, “co-administering”, “combination therapy“ or “combination treatment” refer to the administration of the anti- GPRC5D/anti-CD3 bispecific antibody as described herein and the IMiD and optionally a glucocorticosteroid as described herein e.g. as separate formulations/applications (or as one single formulation/application). The co-administration can be simultaneous or sequential in either order, wherein preferably there is a time period while or all active agents simultaneously exert their biological activities. Said active agents are co-administered either simultaneously or sequentially (e.g. intravenous (i.v.)) through a continuous infusion or given orally. When all therapeutic agents are co-administered sequentially the dose is administered either on the same day in two separate administrations, or one of the agents is administered on day 1 and the second is co-administered on day 2 to day 7, preferably on day 2 to 4. Thus in one embodiment the term “sequentially” means within 7 days after the dose of the first component, preferably within 4 days after the dose of the first component; and the term “simultaneously” means at the same time. The term “co- administration” with respect to the maintenance doses of the anti-GPRC5D/anti-CD3 bispecific antibody and/or the IMiD and/or where applicable the glucocorticosteroid means that the maintenance doses can be either co-administered simultaneously, if the treatment cycle is appropriate for all drugs, e.g. every week.

It is self-evident that the antibodies are administered to the patient in a “therapeutically effective amount” (or simply “effective amount”) which is the amount of the respective compound or combination that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

The amount of co-administration and the timing of co-administration will depend on the type (species, gender, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated. Said anti-GPRC5D/anti-CD3 bispecific antibody and/or IMiD and/or glucocorticosteroid where applicable are suitably co-administered to the patient at one time or over a series of treatments e.g. on the same day or on the day after or at weekly intervals. A skilled artisan readily recognizes that in many cases the combination therapy may not provide a cure but may only provide partial benefit. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of the therapeutic combination that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount".

Aspects of the invention:

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

2. The use of an anti-GPRC5D/anti-CD3 bispecific antibody in combination with an immunomodulatory imide drug (IMiD) in the manufacture of a medicament for the treatment of cancer.

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

4. A 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 instruction for administration of the first medicament in combination with the second medicament for treating cancer in an individual. 5. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of the preceding aspect, wherein the anti- GPRC5D/anti-CD3 bispecific antibody comprises

(i) a first antigen binding moiety 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, a HCDR 2 of SEQ ID NO: 13, and a 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, a LCDR 2 of SEQ ID NO: 16 and a LCDR 3 of SEQ ID NO: 17; and

(ii) a second antigen binding moiety that specifically binds to CD3 and comprises heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, a HCDR 2 of SEQ ID NO: 19, and a 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, a LCDR 2 of SEQ ID NO: 22 and a LCDR 3 of SEQ ID NO: 23.

6. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of the preceding aspects, wherein the anti- GPRC5D/anti-CD3 bispecific antibody comprises

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

8. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of aspects 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 CHI, particularly the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are replaced by each other. he anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit 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 substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of aspects 5 to 9, wherein the first and the second antigen binding moiety are fused to each other, optionally via a peptide linker. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of aspects 5 to 10, wherein the first and the second antigen binding moiety are each a Fab molecule and wherein either (i) the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, or (ii) the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to aspects 1 to 11, wherein the anti-GPRC5D/anti-CD3 bispecific antibody comprises a third antigen binding moiety. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to aspects 12, wherein the third antigen moiety is identical to the first antigen binding moiety. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of the previous aspects, wherein the anti- GPRC5D/anti-CD3 bispecific antibody comprises an Fc domain composed of a first and a second subunit. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to aspects 5 to 14, wherein the first, the second and, where present, the third antigen binding moiety are each a Fab molecule; and wherein either (i) the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety 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, where present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to aspects 14 or 15, wherein the Fc domain is an IgG Fc domain. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of aspects 14 to 16, wherein the Fc domain is an IgGl Fc domain. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of aspects 14 to 17, wherein the Fc domain is a human Fc domain. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of aspects 14 to 18, wherein an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of aspects 14 to 19, wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and/or effector function.

21. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of aspects 1 to 19, wherein the anti- GPRC5D/anti-CD3 bispecific antibody comprises a polypeptide sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29.

22. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of aspects 1 to 21, wherein the IMiD is a first generation IMiD or a Cereblon E3 ligase modulator (CELMoD). 23. The anti-GPRC5D/anti-CD3 bispecific antibody in combination with an IMiD for use, the use, the method or the kit according to any one of aspects 1 to 22, wherein the IMiD is selected from the group of lenalidomide, pomalidomide and iberdomide.

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

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

Amino Acid Sequences

Examples

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.

Materials and methods

All in vivo efficacy and PD experiments were performed in humanized NSG mice carrying subcutaneous multiple myeloma xenograft tumors. Female humanized NSG female mice were purchase from by Jackson Laboratories and delivered to the animal facility at Roche Innovation Center Munich 14-20 weeks after engraftment with human CD34 ⁺ hematopoietic stem cells. After arrival, animals were maintained for one week to get accustomed to new environment and for observation. Mice were maintained under specific-pathogen-free condition with daily cycles of 12 h light /12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). Continuous health monitoring was carried out on regular basis. Experimental study protocol was reviewed and approved by local government (ROB-55.2-2532. Vet_03-16-10 or ROB-55.2- 2532.Vet_03-20-170). To evaluate therapeutic effects against established multiple myeloma tumors, humanized NSG mice were engrafted subcutaneously with human tumor cell lines. Tumor cell lines were obtained from different providers and after expansion deposited in the Roche Munich internal cell bank (Table 1). All tumor cells were culture at 37 °C in a water-saturated atmosphere at 5 % CO2 and were co-injected with 50 pl Matrigel subcutaneously in the flank of anaesthetized, humanized NSG mice at different cell numbers and a viability of > 90% into the right flank of the animals (Table 1). When subcutaneous tumors reached an average volume of 200-300 mm ³ , humanized mice were randomized into different treatment groups based on tumor volume and body weight. For the evaluation of GPRC5D-TCB in combination with mezigdomide, animals were randomized into eight different treatment groups when subcutaneaous tumors reached an average volume of 180 mm ³ (Table 3). Upon randomization, animals were treated with GPRC5D-TCB (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29; as disclosed in WO 2021/018859 Al; RO7425781, forimtamig or “forim”) in monotherapy or in combination with an IMiD, which are standard of care (SoC) agents for the treatment of multiple myeloma. Moreover, addition of dexamethasone (Dex) to the combination therapy of GPRC5D- TCB and Lenalidomide was investigated. Treatment schedule, dose and route of administration for each therapy have been summarized in Table 2 and Table 3. All therapies were prepared freshly before injection. Animals were controlled daily for clinical symptoms and detection of adverse effects. Termination criteria for animals were visible sickness (scruffy fur, arched back, breathing problems, impaired locomotion), body weight loss >20% or tumor size. Tumors growth was monitored twice weekly using Caliper measurements. In order to quantify tumor infiltrating lymphocytes, in some experiments investigating GPRC5D-TCB in combination with Lenalidomide (Len), Pomalidomide and Iberdomide, tumors of scout animals were harvested and single cell suspensions were subjected to flow cytometry using FACSFortessa device and FlowJo software. To quantify and characterize peripheral immune cells in animals treated with GPRC5D- TCB in combination with mezigdomide (Mezi), whole blood was collected and processed for flow cytometry including erythrocyte lysis and flow cytometry was performed using the Cytek Aurora spectral analyzer and FlowJo software. Cytokine in serum of treated mice were analysed using the Bio-Plex Multiplex Immunoassay System from BioRad in combination with the Bio-Plex Pro Human Cytokine 27-plex Assay. Statistical analysis was performed using the Graph Pad Prism software. To compare results for ImmunoPD, tumor volumes or cytokine levels between different treatment groups, data were subjected to one-way ANOVA analysis corrected for multiple comparison (Tukey Test).

Table 1: Tumor cell lines

Table 2: Summary of treatment schedule, dose and route of administration.

Table 3: Experimental groups for the evaluation of GPRC5D-TCB in combination with mezigdomide.

Results Combination of GPRC5D-TCB with immunomodulatory drugs (IMIDs)

First generation IMIDs such as Lenalidomide and Pomalidomide are approved first line therapies in multiple myeloma patients ¹ . When combined with low dose GPRC5D-TCB therapy against OPM-2 xenografts in humanized mice, Lenalidomide was found to exhibit strong synergistic anti- tumoral activity as confirmed by tumor growth control and statistically significant reduction in tumor load as compared to monotherapies (Figure 1A and B). Moreover, combination with

Lenalidomide significantly boosted intratumoral T cell numbers as compare to GPRC5D-TCB monotherapy underpinning the synergistic mode of action of both drugs (Figure 1C). IMIDs are frequently combined with Dexamethasone for the treatment of multiple myeloma. Using a less response multiple myeloma tumor model, KMS-12BM, GPRC5D-TCB was combined with Lenalidomide and with Lenalidomide plus Dexamethasone. As compared to the control group, combination of Lenalidomide and GPRC5D-TCB induced statistically significant tumor growth inhibition against a hard to treat multiple myeloma xenograft model (Figure 2A and B). Strikingly, an even more robust efficacy was observed when Dexamethasone was added to the GPRC5D-TCB and Lenalidomide combination supporting the clinical development of the GPRC5D-TCB with this SoC backbone (Figure 2A and B). Despite very strong monotherapy response, high percentage of NCI-H929 xenograft tumors relapsed under GPRC5D-TCB monotherapy (Figure 3A and Figure 5B). When combined with Pomalidomide, frequency of mice with relapse was drastically reduced (Figure 3A and Figure 5C). Improved efficacy was correlated with increase cytokine levels detected in serum of mice 48 hours after first TCB and 24 after first Pomalidomide injection indicating enhanced immune activation in combination (Figure 3B, 3C and 3D). Interestingly, Pomalidomide combination did not induce complete tumor eradication as seen by onset of tumor growth in individual animals when treatment was stopped (Figure 3 A and Figure 5C).

Iberdomide belongs to a new class of IMIDs, called “cereblon E3 ligase modulators” (CELMoDs), currently being evaluated in early clinical development. Iberdomide was found to also prevent relapse of NCI-H929 tumors to GPRC5D-TCB monotherapy (Figure 4A and Figure 5D) and to induce even stronger boost T cell response as compared to Pomalidomide as confirmed by higher cytokine levels in combination (Figure 4B, 4C and 4D). Iberdomide but not Pomalidomide combination induced complete tumor response in humanized mice as highlighted by lack of tumor regrowth upon therapy withdrawal (Figure 5D).

In order to evaluate the combination of GPRC5D-TCB with mezigdomide in a clinical relevant in vivo setting, NCI-H929 tumor bearing humanized mice were treated once weekly (lq7d or q7d) with fixed duration GPRC5D-TCB using step up dosing (cycle 1, Cl) of 0.0005 mg/kg (step up dose 1 at day 1; C1D1), 0.002 mg/kg (step up dose 2 at day 8; C1D8) and 0.04 mg/kg (step up dose 3 at day 15; C1D15) followed by 5 cycles (C2-C6) maintenance dosing at 0.04 mg/kg and a treatment-free follow up of more than 2 weeks (Figure 6). Mezigdomide was administered 24h after each GPRC5D-TCB injection at 3 mg/kg or 1 mg/kg once weekly (q7d), thrice weekly (3q7d) or five time a week (5q7d). Although GPRC5D-TCB induced transient tumor regressions after step up dose 1 (C1D1) and 3 (C1D15), mice showed progressive disease at the end of cycle 1 (Cl) and the progression free survival (PFS) rate ate end of study was only 20% (Figure 6B). In contrast, combination with mezigdomide at 3q7d and 5q7d dosing led to rapid onset of tumor regression during Cl correlating with significantly improved PFS rates of 80% (3q7d; Figure 6D) and 100% (5q7d; Figure 6C) at 3mg/kg dose and of 60% (3q7d; Figure 6G) and 90% (5q7d; Figure 6F) at 1 mg/kg dose, respectively. Once weekly (lq7d) dosing of mezigdomide did not improve efficacy of GPRC5D-TCB during step up doses 1 (C1D1) and 2 (C1D8) but achieved deepening of responses especially at 3 mg/kg after target dose administration at cycle 1 day 15 (C1D15; Figure 6H). In correlation with less depth of early response, PFS rates for lq7d mezigdomide combination were not improved as compared to GPRC5D-TCB monotherapy (Figure 6E). In order to explore the impact of mezigdomide combination on immune activation, cytokine release was measured in serum of all mice 48h after each GPRC5D-TCB step up injection at CID 1, C1D8 and CID 15 and 24h after mezigdomide administration (Figure 7). When mezigdomide was administered 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 timepoints (Figure 7A). Interestingly, a different trend was observed for MIP- 1 a as serum levels decreased in correlation with the number of mezigdomide administrations (Figure 7D). In contrast to 5q7d and 3q7d schedules, less frequent dosing of mezigdomide (lq7d) induced a strong increase of IL-2, IP-10 and MIP-la after target dose administration at CID 15 (Figure 7A, 7B and 7D). In order to perform quantitative and phenotypic analysis on circulating immune cells, we took blood from all animals at the end of cycle 3 (C4 predosing) and cycle 5 (C6 predosing) and performed spectral flow cytometry. As compared to monotherapy, we observed a drop in peripheral CD8a ⁺ (CD8a-positive cells) and conventional CD4 ⁺ T cell counts when mezigdomide was dosed 3q7d at 3 mkg/kg and 5q7d at 1 mg/kg and an increase upon lq7d dosing at 1 mg/kg (CD8a ⁺ and CD4 ⁺ ) and 3q7d dosing at 1 mg/kg (CD4 ⁺ ) (Figure 8A and 8D). Numbers of regulatory T cells (Treg) slightly decreased upon 3q7d dosing at 3 mg/kg and strongly increased when mezigdomide was dosed 3q7d at 1 mg/kg (Figure 8B). Peripheral B cell numbers clearly dropped at 3q7d and 5q7d dosing irrespective of the dose level of mezigdomide (Figure 8C). In contrast, less frequent dosing of mezigdomide induced strong increase of not only B cell but also NK cell counts at C4 predosing but especially at C6 predosing (Figure 8C and 8E). We next evaluated if combination of GPRC5D-TCB with mezigdomide induces changes in exhaustion status of in circulating T lymphocytes. As compared to GPRC5D-TCB monotherapy, we observed a higher frequency of LAG3 and TIGIT positive CD4 ⁺ and CD8a ⁺ T cells when mice were treated 5q7d and 3q7d but not lq7d with mezigdomide (Figure 9A, 9B, 9C and 9D) indicating that repetitive treatment with mezigdomide induces T cell exhaustion.

Taken together, our data suggest that combination with mezigdomide can significantly improve PFS rates in multiple myeloma patients. Addition of mezigdomide to GPRC5D-TCB induced deep responses at early timepoints and allowed to overcome tumor relapse at later timepoints. Cytokine data indicate the combination of GPRC5D-TCB step up dosing with mezigdomide to not represent a major risk factor for developing or exacerbating cytokine release syndrome (CRS). References:

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

* * * Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the 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 in their entirety by reference.