TW202430211A - Combination therapy of a gprc5d tcb and imids - Google Patents

Abstract

The present invention relates to the combination therapy of anti-GPRC5D/anti-CD3 bispecific antibodies with IMiDs. The combination therapy may further comprise a glucocorticosteroid.

Description

GPRC5D TCB and IMID combination therapy

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

Multiple myeloma (MM) is one of the most common hematological malignancies, with approximately 75,000 new cases per year in the EU and the US, representing a significant unmet medical need. Multiple myeloma is characterized by the secretion of non-functional monoclonal immunoglobulins by terminally differentiated plasma cells. In the short term, immunomodulatory drugs such as lenalidomide and pomalidomide, and proteasome inhibitors such as carfilzomib or bortezomib are likely to remain the backbone of first-line treatment 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 specifically target diseased tumor cells, such as diseased plasma cells (PCs). Efforts have been made to selectively deplete PCs in multiple myeloma. The lack of surface proteins that specifically mark plasma cells has hampered the development of antibody or cell-based therapies for multiple myeloma. To date, there have been only a few successful examples of biologics, including daratumumab (anti-CD38) and elotuzumab (anti-CD319), but it is important to note that these two molecules are not exclusively expressed by plasma cells. Therefore, RNA sequencing from multiple myeloma plasma cells has been used to identify novel targets, such as G protein-coupled receptor class C group 5 member D (GPRC5D), which is differentially expressed in multiple myeloma plasma cells compared to plasma cells derived from healthy donors. It has been reported that GPRC5D is associated with the prognosis and tumor load of patients with multiple myeloma (Atamaniuk, J. et al., Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma. Eur J Clin Invest, 2012. 42(9): 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 ligands and unknown biological properties in males in general and in cancer in particular. The gene encoding GPRC5D is mapped to chromosome 12p13.3 and contains three exons spanning approximately 9.6 kb (Brauner-Osborne, H. et al., Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochim Biophys Acta, 2001. 1518(3): p. 237-48). The large first exon encodes seven transmembrane domains. GPRC5D has been shown to be involved in the formation of keratin in animal hair follicles (Gao, Y. et al., Comparative Transcriptome Analysis of Fetal Skin Reveals Key Genes Related to Hair Follicle Morphogenesis in Cashmere Goats. PLoS One, 2016. 11(3): p. e0151118; and Inoue, S., T. Nambu and T. Shimomura, The RAIG family member, GPRC5D, is associated with hard-keratinized structures. J Invest Dermatol, 2004. 122(3): p. 565-73).

WO 2018/017786 A2 and WO 2021/018859 A1 disclose GPRC5D-specific antibodies or antigen-binding fragments that bind to GPRC5D on target cells and to activated T cell antigens such as CD3 on T cells. When the antibody binds to both of its targets simultaneously, a T cell synapse is formed, leading to activation of (cytotoxic) T cells and subsequent lysis of target cells. Compared with CAR-T cell therapy, T-cell bispecific antibodies (TCBs) have become a new treatment option for patients with relapsed refractory myeloma (RRMM) based on their good objective response rate (ORR), favorable safety profile, and ready availability (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 TCBs targeting BCMA and GPRC5D have been reported to induce profound clinical responses, antigenic drift represents a tumor-intrinsic resistance mechanism that limits the durability of responses (Mailankody, S. et al., GPRC5D-Targeted CAR T Cells for Myeloma. N Engl J Med. 2022;387(13):1196-1206).

Given that all standard of care treatments have failed to cure multiple myeloma patients, there is a clear need to develop new, effective and specific therapies. Accordingly, the present invention provides a combination of an anti-GPRC5D/anti-CD3 bispecific antibody with an IMiD and, optionally, a glycocorticosteroid.

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

In an embodiment of any of the above aspects, the anti-GPRC5D/anti-CD3 bispecific antibody comprises (i) a first antigen-binding portion that specifically binds to GPRC5D and comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; and (ii) a second antigen-binding portion that specifically binds to CD3 and comprises: a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 18, LCDR 2 of SEQ ID NO: 19, and LCDR 3 of SEQ ID NO: 20. A heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 19, HCDR 2 of SEQ ID NO: 20, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23. In another embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody comprises (i) a first antigen-binding portion that specifically binds to GPRC5D, comprising a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11; and (ii) a second antigen-binding portion that specifically binds 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. 95%, 96%, 97%, 98%, 99% or 100% identical VL. In one embodiment, the first antigen-binding portion and/or the second antigen-binding portion of the anti-GPRC5D/anti-CD3 bispecific antibody is a Fab molecule. In another embodiment, the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain, in particular the variable domains VL and VH are replaced with each other. In one embodiment, the first antigen binding portion is a Fab molecule, wherein in the invariant domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the invariant domain CH1, the amino acid at position 147 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering). In another embodiment, the first antigen binding moiety and the second antigen binding moiety are fused to each other, optionally via a peptide linker. In yet another embodiment, the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule, and wherein (i) the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, or (ii) the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety.

In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody according to any of the above aspects comprises a third antigen binding moiety. In another embodiment, the third antigen binding moiety is the same as the first antigen binding moiety. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody comprises an Fc domain consisting of a first unit and a second unit. In one aspect, the first antigen binding moiety, the second antigen binding moiety, and the third antigen binding moiety, if present, are each a Fab molecule, and wherein 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, if present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. In one embodiment, the Fc domain is an IgG Fc domain. In one embodiment, the Fc domain is an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain.

In one embodiment of any of the above aspects, the amino acid residues in the CH3 domain of the first unit of the Fc domain are substituted with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first unit, and the protrusion can be placed in the cavity in the CH3 domain of the second unit, and the amino acid residues in the CH3 domain of the second unit of the Fc domain are substituted with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second unit, and the protrusion in the CH3 domain of the first unit can be placed in the cavity. In one embodiment, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector function.

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

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

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

Definition

Definitions Unless otherwise defined below, the terms used herein are those commonly used in the art.

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

The term "bispecific" means that the antigen binding molecule is capable of specifically binding to at least two different epitopes. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different epitope. In certain embodiments, the bispecific antigen binding molecule is capable of simultaneously binding to two epitopes, particularly two epitopes expressed on two different cells.

As used herein, the term "valent" refers to the presence of a specified number of antigen binding sites in an antigen binding molecule. Thus, the term "monovalent binding to an antigen" refers to the presence of one (and no more than one) antigen binding site specific for the antigen in an antigen binding molecule.

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

The term "antigen binding moiety" as used herein refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen binding moiety is capable of directing 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 carrying the antigenic determinant. In another embodiment, the antigen binding moiety is capable of activating signaling through its target antigen (e.g., a T cell receptor complex antigen). Antigen binding moieties include antibodies and fragments thereof as further defined herein. Specific antigen binding moieties include the antigen binding domain of an antibody, which comprises an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding moiety may include an antibody constant region as further defined herein and known in the art. Available heavy chain constant regions include any of five isotypes: α, δ, ε, γ, or μ. Available light chain constant regions include any of two isotypes: κ and λ.

As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope", and refers to the site on the polypeptide macromolecule to which the antigen-binding moiety binds that forms the antigen-binding moiety-antigen complex (e.g., a continuous stretch of amino acids or a conformational configuration composed of different regions of non-continuous amino acids). For example, available antigenic determinants may be present on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, not present in serum, and/or present in the extracellular matrix (ECM). Proteins referred to herein as antigens (e.g., GPRC5D, CD3) may be any native form of a protein derived from any vertebrate, including mammals, such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats), unless otherwise specified. In a particular embodiment, the antigen is a human protein. Where a particular protein is referred to herein, the term encompasses "full-length," unprocessed protein and any form of the protein resulting from processing in a cell. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants. Exemplary human proteins suitable for use as antigens are CD3, in particular the epsilon subunit of CD3 (see UniProt No. P07766 (version 185), NCBI RefSeq No. NP_000724.1, for human sequences, SEQ ID NO: 4; or UniProt No. Q95LI5 (version 69), NCBI GenBank No. BAB71849.1, for cynomolgus macaque sequences, SEQ ID NO: 5) or GPRC5D (see UniProt No. Q9NZD1 (version 115); NCBI RefSeq No. NP_061124.1, for human sequences, SEQ ID NO: 9). In certain embodiments, the bispecific antigen binding molecule binds to an epitope of CD3 or GPRC5D that is conserved among CD3 or GPRC5D antigens from different species. In a specific embodiment, the bispecific antigen binding molecule binds to human GPRC5D.

"Specific binding" means that the binding is selective for the antigen and can distinguish undesired or non-specific interactions. The ability of an antigen binding moiety to bind a specific antigenic determinant can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (e.g., analysis on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent to which the antigen binding moiety binds to an unrelated protein is less than about 10% of the extent to which the antigen binding moiety binds to the antigen, e.g., as determined by SPR. In certain embodiments, the antigen-binding portion, or an antigen-binding molecule comprising the antigen-binding portion, that binds an antigen has a dissociation constant (KD) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., ^10-8 M or less, e.g., ^10-8 M to ^10-13 M, e.g. _, ^10-9 M to ^10-13 M).

"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between the members of a binding pair (e.g., an antigen binding moiety and an antigen or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be expressed in terms of a dissociation constant ( _KD ), which is the ratio of the dissociation rate constant to the association rate constant ( _koff and _kon , respectively). Thus, equivalent affinities may include different rate constants as long as the ratio of the rate constants remains the same. Affinity may be determined by established methods known in the art, including those described herein. A particular method for determining affinity is surface plasmon resonance (SPR).

"Reduced binding", e.g. to an Fc receptor, means a decrease in the affinity of the respective interaction as measured, e.g., by SPR. For the sake of clarity, the term also includes a decrease in affinity to zero (or below the detection limit of the analytical method), i.e. complete abolition of the interaction. Conversely, "increased binding" means an increase in the binding affinity of the respective interaction.

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

As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes (particularly cytotoxic T lymphocytes) selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein.

As used herein, "target cell antigen" refers to an antigenic determinant present on the surface of a target cell (e.g., a cell in a tumor, such as a cancer cell or a cell of a tumor stroma). In a specific embodiment, the target cell antigen is GPRC5D, specifically 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 to distinguish when more than one of each type of moiety is present. Unless expressly stated, the use of these terms is not intended to confer a particular order or orientation on the bispecific antigen-binding molecules.

"Fusion" means that the components (e.g., a Fab molecule and an Fc domain subunit) are linked via a peptide bond, directly or via one or more peptide linkers.

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

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

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

The term "immunoglobulin molecule" refers to a protein with the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a heavy chain variable domain or a heavy chain variable region, followed by three constant domains (CH1, CH2 and CH3), also called heavy chain constant regions. Similarly, from N-terminus to C-terminus, each light chain has a variable domain (VL), also called a light chain variable domain or light chain variable region, followed by a light chain constant (CL) domain, also called a light chain constant region. The heavy chains of immunoglobulins can be classified into one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, such as γ ₁ (IgG ₁ ), γ ₂ (IgG ₂ ), γ ₃ (IgG ₃ ), γ ₄ (IgG ₄ ), α ₁ (IgA ₁ ) and α ₂ (IgA ₂ ). Based on the amino acid sequence of its homeodomain, the light chain of an immunoglobulin can be classified into one of two types, called kappa (κ) and lambda (λ). An immunoglobulin is basically composed of two Fab molecules and an Fc domain connected by an immunoglobulin hinge region.

The term "antibody" herein is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen-binding activity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies contained in the population are identical and/or bind to the same antigenic determinant, but excludes antibodies that contain, for example, naturally occurring mutations or possible variants that arise during the production of the monoclonal antibody preparation, which variants are usually present in small quantities. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (antigenic determinants), each individual antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody's characteristics are obtained from a substantially homogeneous antibody population, and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies intended for use in accordance with the present invention may be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals with genes comprising all or part of the human immunoglobulin loci, these methods and other exemplary methods for making monoclonal antibodies are described herein.

An "isolated" antibody is an isolated antibody that is separated from components of its natural environment, i.e., an isolated antibody that is not in its natural environment. No particular level of purification is required. For example, an isolated antibody may be removed from its natural or native environment. For purposes of the present invention, recombinantly produced antibodies expressed in host cells are considered isolated, as are natural or recombinant antibodies that have been separated, fractionated, or partially or substantially purified by any suitable technique. Thus, the antibodies and bispecific antigen-binding molecules of the present invention are isolated. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC). For a review of methods for assessing 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 interchangeably herein to refer to an antibody that has a structure substantially similar to that of a native antibody.

"Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , bifunctional antibodies, 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 general description of scFv fragments, see, e.g., Pluckthün, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments that contain antigen-binding residues that rescue receptors and have increased in vivo half-lives, see U.S. Pat. No. 5,869,046. Bifunctional antibodies are antibody fragments that have two antigen-binding sites (which may be bivalent or bispecific). See, e.g., EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Trifunctional antibodies and tetrafunctional antibodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that include all or part of the heavy chain variable domain of an antibody or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1). Antibody fragments can be produced by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies as described herein and production in recombinant host cells (eg, E. coli or bacteriophage).

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

The term "variable region" or "variable domain" refers to the domain of the antibody heavy chain or light chain that is involved in binding the antibody to the antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, and each domain comprises four conserved framework regions (FR) and three highly variable regions (HVR). See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. "Kabat numbering" as used herein in connection with variable region sequences refers to the numbering system described 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 chains 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 herein as "according to Kabat numbering" or "Kabat numbering"). Specifically, the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) pp. 647-660) is used for the light chain constant domain CL of the kappa and lambda isotypes and the Kabat and EU index numbering systems (see pp. 661-723) are used for the heavy chain constant domains (CH1, hinge, CH2 and CH3), which in this case are further clarified herein by reference to "numbering according to the Kabat EU index".

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is highly variable in sequence ("complementary determining region" or "CDR"; CDRs of heavy chain variable regions/domains are abbreviated as, e.g., HCDR1, HCDR2, and HCDR3; CDRs of light chain variable regions/domains are abbreviated as, e.g., LCDR1, LCDR2, and LCDR3) and/or form structurally defined loops ("hypervariable loops") and/or contain antigen contact residues ("antigen contacts"). Generally, an antibody comprises six HVRs; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). As used herein, exemplary HVRs include: (a) highly variable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest , 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts are present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) a combination of (a), (b) and/or (c) comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

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

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

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

A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using the human antibody repertoire or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues. In certain embodiments, human antibodies are derived from non-human transgenic mammals, such as mice, rats, or rabbits. In certain embodiments, human antibodies are derived from fusion tumor cell lines. Antibodies or antibody fragments isolated from a human antibody library 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 region of its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . The constant domains of the heavy chains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes 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 may vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from Cys226 or 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 at the C-terminus of the heavy chain. Thus, antibodies produced by host cells by expressing a particular nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or may include a cleavage variant of the full-length heavy chain (also referred to herein as a "cleavage variant heavy chain"). The last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. Unless otherwise specified, the amino acid sequence of a heavy chain including an Fc domain (or a subunit of an Fc domain as defined herein) is herein referred to as excluding the C-terminal glycine-lysine dipeptide. In one embodiment of the present invention, the heavy chain contained in the antibody or bispecific antigen-binding molecule according to the present invention (including the subunit of the Fc domain specified herein) comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of the present invention, the heavy chain contained in the antibody or bispecific antigen-binding molecule according to the present invention (including the subunit of the Fc domain specified herein) comprises an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index). The composition of the present invention, such as the pharmaceutical composition described herein, comprises the antibody or bispecific antigen-binding molecule population of the present invention. The population of antibodies or bispecific antigen-binding molecules may include molecules with full-length heavy chains and molecules with cleavage variant heavy chains. The population of antibodies or bispecific antigen-binding molecules may consist of a mixture of molecules with full-length heavy chains and molecules with cleavage variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or bispecific antigen-binding molecules have cleavage variant heavy chains. In one embodiment of the present invention, a composition comprising a population of antibodies or bispecific antigen-binding molecules of the present invention comprises an antibody or bispecific antigen-binding molecule comprising a heavy chain having a subunit of an Fc domain as specified herein and an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of the present invention, a composition comprising a population of antibodies or bispecific antigen-binding molecules of the present invention comprises an antibody or bispecific antigen-binding molecule comprising a heavy chain having a subunit of an Fc domain as specified herein and an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index). In one embodiment of the present invention, such compositions comprise a population of antibodies or bispecific antigen-binding molecules, which are composed of the following molecules: a molecule comprising a heavy chain comprising a subunit of an Fc domain as specified herein; a molecule comprising a heavy chain comprising a subunit of an Fc domain as specified herein and an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index); and a molecule comprising a heavy chain comprising a subunit of an Fc domain as specified herein and an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). Unless otherwise indicated herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system (also known as the EU index) as described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) (see also above). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., the polypeptide that comprises the C-terminal constant region that is capable of self-association and is stable to the immunoglobulin heavy chain. For example, the subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

"Modifications that promote the association of the first and second Fc domain subunits" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the association of a polypeptide comprising the Fc domain subunit with the same polypeptide to form a homodimer. As used herein, modifications that promote association specifically include individual modifications made to each of the two Fc domain subunits (i.e., the first and second Fc domain subunits) that are desired to be associated, wherein the modifications complement each other to promote the association of the two Fc domain subunits. For example, modifications that promote association may alter the structure or charge of one or both Fc domain subunits to make them sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may differ with respect to the components further fused to each subunit (e.g., antigen binding moiety). In some embodiments, the modification promoting association comprises an amino acid mutation, particularly an amino acid substitution, in the Fc domain. In a particular embodiment, the modification promoting association comprises a single amino acid mutation, particularly an amino acid substitution, in each of the two subunits of the Fc domain.

The term "effector function" refers to those biological activities attributed to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

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

As used herein, the term "amino acid mutation" is meant to encompass amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications may be performed to obtain a final construct, provided that the final construct has the desired characteristics, for example, reduced binding to an Fc receptor or increased binding to another peptide. Amino acid sequence deletions and insertions include deletions and insertions of the amino and/or carboxyl termini of an amino acid. A specific amino acid mutation is an amino acid substitution. To alter, for example, the binding characteristics of an Fc region, non-conservative amino acid substitutions are particularly preferred, i.e., replacing one amino acid with another amino acid having different structural and/or chemical properties. Amino acid substitutions include substitutions with non-natural amino acids or naturally occurring amino acid derivatives (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) of the twenty standard amino acids. Amino acid mutations can be produced using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is expected that methods other than genetic engineering such as chemical modification to change the side chain groups of amino acids may also be useful. Various names may be used herein to indicate the same amino acid mutation. For example, the proline at position 329 of the Fc domain is substituted with glycine, which can be represented as 329G, G329, G ₃₂₉ , P329G or Pro329Gly.

"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing differences (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage of amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program suite. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve maximum alignment over the full length of the sequences being compared. However, for the purposes of this article, % amino acid sequence identity values were generated using the ggsearch program in the FASTA package version 36.3.8c or later and the BLOSUM50 comparison matrix. The FASTA package was developed by W. R. Pearson and D. J. Lipman, (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227-258; and Pearson et al. (1997) (Genomics 46:24-36) and is publicly accessible at the following URL: http://fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml. Alternatively, sequences can be compared using a public server accessed through http://fasta.bioch.virginia.edu/fasta_www2/index.cgi using the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure that a global rather than a local alignment is performed. The percentage of amino acid identity is provided in the output alignment header.

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

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which antibodies or derivatives thereof contain an Fc region, which specifically bind to the Fc region, usually via a protein moiety as the N-terminus. The term "reducing ADCC" as used herein refers to a decrease in the number of target cells lysed in a given time by a given concentration of antibody in the culture medium surrounding the target cells via the ADCC mechanism defined above, and/or an increase in the concentration of antibody in the culture medium surrounding the target cells required to achieve the lysis of a given number of target cells in a given time via the ADCC mechanism. The reduction in ADCC is relative to the ADCC mediated by the same antibody (but not engineered) produced by the same type of host cells using the same standard production, purification, formulation and storage methods (methods known to those of ordinary skill in the art). For example, the reduction in ADCC mediated by an antibody comprising an amino acid substitution in the Fc domain that reduces ADCC is relative to the ADCC mediated by the same antibody without such amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT Publication No. WO 2006/082515 or PCT Publication No. WO 2012/130831).

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

An "effective amount" of a drug refers to the amount required to induce a physiological change in the cells or tissues to which it is administered.

A "therapeutically effective amount" of a drug, such as a pharmaceutical composition, is an amount effective to achieve the desired therapeutic or preventive effect, in the amount and time period required. A therapeutically effective amount of a drug, for example, eliminates, reduces, delays, minimizes or prevents the adverse effects of a disease.

A "subject" or "individual" 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). Specifically, the subject or individual is a human.

The term "pharmaceutical composition" refers to a preparation which is in a form which permits the biological activity of the active ingredient contained therein to be effective and which contains no other components which are unacceptably toxic to the subject to which the composition is to be administered.

"Pharmaceutically acceptable carriers" refer to ingredients in pharmaceutical compositions other than active ingredients that are non-toxic to individuals. Pharmaceutically acceptable carriers include but are not limited to buffers, excipients, stabilizers or preservatives.

As used herein, "treatment" (and grammatical variants such as "treatment process" or "treating") refers to clinical intervention that attempts to alter the natural course of a disease in the individual being treated, and can be performed preventively or during the course of clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or reducing the disease state, and relieving or improving prognosis. In some embodiments, the antibodies or bispecific antigen-binding molecules of the present invention are used to delay the development of a disease or slow the progression of a disease.

The term "product leaflet" is used to refer to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, routes of administration, combination therapy, contraindications and/or warnings for the use of such therapeutic products.

and GPRC5D and CD3 Bispecific antigen binding molecules

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

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

Preferably, at least one of the antigen binding moieties is a crossover Fab molecule. Such modifications reduce the mispairing of heavy and light chains from different Fab molecules, thereby increasing the yield and purity of the recombinant production of the bispecific antigen binding molecules of the present invention. In certain crossover Fab molecules used in the bispecific antigen binding molecules of the present invention, the variable domains of the Fab light chain and the Fab heavy chain (VL and VH, respectively) are exchanged. However, even with this domain exchange, the preparation of the bispecific antigen binding molecules may also contain certain byproducts due to the so-called Bence Jones type interactions between the mispaired heavy and light chains (see Schaefer et al., PNAS, 108 (2011) 11187-11191). To further reduce the mispairing of the heavy and light chains from different Fab molecules, and thus increase the purity and yield of the desired bispecific antigen-binding molecules, amino acids with opposite charges can be introduced at specific amino acid positions in the CH1 and CL domains of the Fab molecules that bind to the first antigen (GPRC5D) or the Fab molecules that bind to the second antigen (CD3), as further described herein. Charge modification can be performed in conventional Fab molecules contained in the bispecific antigen-binding molecules or in conventional Fab molecules contained in the VH/VL crossover Fab molecules (but not both). In a specific embodiment, charge modification is performed in a known Fab molecule contained in a bispecific antigen-binding molecule (in a specific embodiment, it binds to a first antigen, i.e., GPRC5D).

The bispecific antigen binding molecule is capable of binding to a first antigen (i.e., GPRC5D) and a second antigen (i.e., CD3) simultaneously. The bispecific antigen binding molecule is capable of crosslinking T cells to target cells by simultaneously binding to GPRC5D and an activating T cell antigen. Such simultaneous binding results in cytolysis of target cells (specifically, tumor cells expressing GPRC5D), activation of T cells, and cellular responses of T lymphocytes (specifically, cytotoxic T lymphocytes), wherein the cellular responses are selected from the group consisting of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers.

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

In particular, the T cell according to any embodiment of the present invention is a cytotoxic T cell. In some embodiments, the T cell is a CD4 ⁺ or CD8 ⁺ cell, in particular a CD8 ⁺ T cell.

First antigen binding moiety

The bispecific antigen-binding molecule comprises at least one antigen-binding moiety, in particular a Fab molecule, that binds to GPRC5D (a first antigen). In certain embodiments, the bispecific antigen-binding molecule comprises two antigen-binding moieties, in particular a Fab molecule that binds to GPRC5D. In a particular such embodiment, each of these antigen-binding moieties binds to the same antigenic determinant. In a more specific embodiment, all of these antigen-binding moieties are identical, i.e., they comprise the same amino acid sequence, which includes the same amino acid substitutions (if any) in the CH1 and CL domains as described herein. In one embodiment, the bispecific antigen-binding molecule comprises no more than two antigen-binding moieties, in particular a Fab molecule that binds to GPRC5D.

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

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

The GPRC5D binding moiety is able to direct the bispecific antigen binding molecule to a target site, such as a specific type of tumor cell expressing GPRC5D.

Unless it is scientifically unreasonable or impossible, the first antigen-binding portion of the bispecific antigen-binding molecule may bind any of the features described herein with respect to antibodies that bind to GPRC5D, either alone or in combination.

In one aspect, the bispecific antigen-binding molecule comprises (a) a first antigen-binding portion that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; and (b) a second antigen-binding portion that binds to CD3.

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

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

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

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

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

In a specific embodiment, the first antigen binding moiety comprises: a VH comprising the amino acid sequence of SEQ ID NO: 10; and a VL comprising the amino acid sequence of SEQ ID NO: 53. In a specific embodiment, the first antigen binding moiety comprises 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, which comprises a human constant region, particularly a human CH1 and/or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs 1 and 2 (human κ and λ CL domains, respectively) and SEQ ID NO: 3 (human IgG ₁ heavy chain constant domain CH1-CH2-CH3). In some embodiments, the first antigen binding moiety comprises a light chain constant region, which 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: 1 or SEQ ID NO: 2, particularly the amino acid sequence of SEQ ID NO: 1. In particular, the light chain constant region may comprise amino acid mutations as described herein under "charge modification", and/or may comprise a deletion or substitution of one or more (particularly two) N-terminal amino acids in the crossover Fab molecule. In some embodiments, the first antigen binding portion comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 3. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations as described herein under "charge modification".

Second antigen binding moiety

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

In a specific embodiment, the antigen-binding moiety that binds to the second antigen is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy chain and light chain are exchanged/replaced with each other. In such embodiments, the antigen-binding moiety that binds to the first antigen (i.e., GPRC5D) is preferably a conventional Fab molecule. In embodiments in which there is more than one antigen-binding moiety that binds to GPRC5D contained in the bispecific antigen-binding molecule, in particular a Fab molecule, the antigen-binding moiety that binds to the second antigen is preferably a crossover Fab molecule, and the antigen-binding moiety that binds to GPRC5D is a conventional Fab molecule.

In alternative embodiments, the antigen binding moiety that binds to the second antigen is a conventional Fab molecule. In these embodiments, the antigen binding moiety that binds to the first antigen (i.e., GPRC5D) is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged/replaced with each other. In embodiments in which there is more than one antigen binding moiety, particularly a Fab molecule, that binds to the second antigen contained in the bispecific antigen binding molecule, the antigen binding moiety that binds to GPRC5D is preferably a crossover Fab molecule, and the antigen binding moiety that binds to the second antigen is a conventional Fab molecule.

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 an activating T cell antigen binding Fab molecule"). In a particular embodiment, the bispecific antigen binding molecule comprises no more than one antigen binding moiety capable of specifically binding to an activating T cell antigen. In one embodiment, the bispecific antigen binding molecule provides monovalent binding to an activating T cell antigen.

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

In one embodiment, the second antigen binding moiety comprises HCDR 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, HCDR 3 of SEQ ID NO: 20, LCDR 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23. In one embodiment, the second antigen binding moiety comprises: VH comprising HCDR 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20; and VL comprising LCDR 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23. In some embodiments, the second antigen binding moiety is (or 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 the CDRs as described in any of the above embodiments, and further comprises a human receptor framework, such as 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 wherein 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 CH1 and/or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs 1 and 2 (human κ and λ CL domains, respectively) and SEQ ID NO: 3 (human IgG ₁ heavy chain constant domain CH1-CH2-CH3). In some embodiments, the second antigen binding moiety comprises a light chain constant region, which 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: 1 or SEQ ID NO: 2, particularly the amino acid sequence of SEQ ID NO: 1. In particular, the light chain constant region may comprise amino acid mutations as described herein under "charge modification", and/or may comprise a deletion or substitution of one or more (particularly two) N-terminal amino acids in the 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 CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 3. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations as described herein under "charge modification".

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 CH1 of the Fab light chain and the Fab heavy chain, in particular the variable domains VL and VH are substituted for each other (i.e., according to these embodiments, the second antigen binding moiety is a crossover Fab molecule, wherein the variable domains or the constant domains of the Fab light chain and the Fab heavy chain are exchanged). In one of these embodiments, the first (and third, if any) antigen binding moiety is a conventional Fab molecule.

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

Charge modification

The bispecific antigen-binding molecule may comprise amino acid substitutions in the Fab molecules comprised therein that are particularly effective in reducing mispairing of the light chain with unmatched heavy chains (Bence-Jones type byproducts) that may occur in the preparation of Fab-based bi/multispecific antigen-binding molecules in which VH/VL exchange occurs in one (or more, in the case where the molecule comprises more than two antigen-binding Fab molecules) of its binding arms (see also PCT Publication No. WO 2015/150447, in particular for examples therein, which is incorporated herein by reference in its entirety). The ratio of desired bispecific antigen-binding molecules to undesired byproducts, particularly Bence Jones-type byproducts occurring in bispecific antigen-binding molecules having a VH/VL domain exchange in one of their binding arms, can be improved by introducing amino acids with opposite charges at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

Thus, in some embodiments, the first antigen-binding moiety 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 substituted for each other.

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

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

The bispecific antigen-binding molecule does not contain the modifications described under i) and ii). The constant domains CL and CH1 of the antigen-binding portion having VH/VL exchange are not substituted for each other (i.e., remain in an unexchanged state).

In a more specific embodiment,

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

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

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

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

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

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

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

In a specific embodiment, if the amino acid substitution according to the above embodiment occurs in the constant domain CL and the constant domain CH1 of the first antigen-binding moiety, the constant domain CL of the first antigen-binding moiety is of the κ isotype.

Alternatively, the amino acid substitutions according to the above embodiments may occur in the constant domain CL and constant domain CH1 of the second antigen binding moiety instead of the constant domain CL and constant domain CH1 of the first antigen binding moiety. In specific such embodiments, the constant domain CL of the second antigen binding moiety is of the κ isotype.

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

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

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

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

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

In a specific embodiment, the bispecific antigen-binding molecule comprises (a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding moiety is a Fab molecule, wherein the Fab molecule comprises a heavy chain variable region (VH), wherein the VH comprises a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and a light chain variable region (VL), wherein the VL comprises a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; and (b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3, and the second antigen binding part is a Fab molecule, the Fab molecule comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19 and HCDR 3 of SEQ ID NO: 20, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22 and LCDR 3 of SEQ ID NO: 23, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other; wherein, in the constant domain CL of the first antigen binding part, the amino acid at position 124 is replaced by lysine (K), arginine (R), (R) or histidine (H) (according to Kabat numbering) (in a specific embodiment, lysine (K) or arginine (R) is independently substituted), and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) (in a specific embodiment, substituted by lysine (K) or arginine (R)), and in the constant domain CH1 of the first antigen-binding portion, the amino acid at position 147 is independently substituted by glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering).

Bispecific antigen binding molecule format

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

Techniques for preparing multispecific antibodies include, but are not limited to, recombining immunoglobulin heavy chain-light chain pairs that co-express two 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)). Multispecific antibodies can also be prepared by the following methods: engineering electrostatic steering effects for preparing antibody Fc-heterodimer molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980; and Brennan et al., Science , 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. , 148(5):1547-1553 (1992); and WO 2011/034605); using common light chain technology to circumvent the light chain mispairing problem (see, e.g., WO 98/50431); using "diabody" technology to prepare 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 according to the method described in, e.g., Tutt et al. , J. Immunol. 147:60 (1991).

Also included herein are engineered antibodies with three or more antigen binding sites, including, for example, "Octopus antibodies" or DVD-Ig (see, for example, WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792 and WO 2013/026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual-acting FAbs" or "DAFs," which contain antigen-binding sites that bind to GPRC5D and CD3 (see, e.g., US 2008/0069820 and WO 2015/095539).

Multispecific antibodies can also be provided in an asymmetric format comprising domains that are crossed in one or more binding arms with the same antigenic specificity, i.e. by exchanging VH/VL domains (see, e.g., WO 2009/080252 and WO 2015/150447), CH1/CL domains (see, e.g., WO 2009/080253) or complete Fab arms (see, e.g., WO 2009/080251, WO 2016/016299, see also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). Asymmetric Fab arms can also be designed by introducing charged or uncharged amino acid mutations into the domain interface to guide correct Fab pairing. See, for example, WO 2016/172485.

Various other 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).

Bispecific antibody formats that can be used for this purpose include, but are not limited to, so-called "BiTE" (bispecific T cell engager) molecules, in which two scFv molecules are fused via a flexible linker (see, e.g., WO2004/106381, WO2005/061547, WO2007/042261, and WO2008/119567; Nagorsen and Bäuerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAb"; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); "DART" (dual affinity repositioning) molecules, which are based on the diabody format but have a C-terminal disulfide bond for further stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and the so-called triomabs, which are complete mouse/rat IgG hybrid molecules (see Seimetz et al. for a review: Cancer Treat Rev 36, 458-467 (2010)). Specific T cell bispecific antibody formats included herein are described in: WO 2013/026833; WO2013/026839; WO 2016/020309; and Bacac et al. Oncoimmunology 5(8) (2016) e1203498.

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

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

In one embodiment, the first antigen binding moiety and the second antigen binding moiety of the bispecific antigen binding molecule are fused to each other, optionally via a peptide linker. In a specific embodiment, the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule. In one such embodiment, the second antigen binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen binding moiety at the C-terminus of the Fab heavy chain. In another such embodiment, the first antigen binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen binding moiety at the C-terminus of the Fab heavy chain. In addition, in embodiments where (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 Fab light chain of the first antigen-binding moiety may be fused to the Fab light chain of the second antigen-binding moiety, optionally via a peptide linker.

Bispecific antigen binding molecules with a single antigen binding moiety (e.g., a Fab molecule) that is capable of specifically binding to a target cell antigen (e.g., GPRC5D) may be used, particularly in cases where internalization of the target cell antigen is expected to occur after binding of the high affinity antigen binding moiety. In such cases, the presence of more than one antigen binding moiety for a particular target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availability.

However, in other cases it will be advantageous to have a bispecific antigen binding molecule comprising two or more antigen binding moieties (such as Fab molecules) that are specific for a target cell antigen, for example to optimize targeting to a target site or to cross-link a target cell antigen.

Accordingly, in a particular embodiment, 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 antigenic moiety is the same as the first antigenic binding moiety.

Unless it is scientifically unreasonable or impossible, the third antigen-binding moiety of the bispecific antigen-binding molecule may bind, alone or in combination, any of the features described herein with respect to the first antigen-binding moiety and/or antibody that binds to GPRC5D.

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

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

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

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

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

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

In a specific embodiment, the third antigen binding moiety comprises: a VH comprising the amino acid sequence of SEQ ID NO: 10; and a VL comprising the amino acid sequence of SEQ ID NO: 53. In a specific embodiment, the third antigen binding moiety comprises 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 CH1 and/or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs 1 and 2 (human κ and λ CL domains, respectively) and SEQ ID NO: 3 (human IgG ₁ heavy chain constant domain CH1-CH2-CH3). In some embodiments, the third antigen binding moiety comprises a light chain constant region, which 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: 1 or SEQ ID NO: 2, particularly the amino acid sequence of SEQ ID NO: 1. In particular, the light chain constant region may comprise amino acid mutations as described herein under "charge modification", and/or may comprise a deletion or substitution of one or more (particularly two) N-terminal amino acids in the 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 CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 3. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations as described herein under "charge modification".

In certain embodiments, the third and first antigen binding moieties are each Fab molecules, and the third antigen binding moiety is identical to the first antigen binding moiety. Thus, in these embodiments, the first antigen binding moiety 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., known or crossed). In addition, in these embodiments, the third antigen binding moiety comprises the same amino acid substitutions (if any) as the first antigen binding moiety. For example, the "charge modification" amino acid substitutions described herein will be performed in the constant domain CL and the constant domain CH1 of each of the first antigen binding moiety and the third antigen binding moiety. Alternatively, the amino acid substitutions may be made in the constant domain CL and constant domain CH1 of the second antigen-binding moiety (which is also a Fab molecule in a specific embodiment), but not in the constant domain CL and constant domain CH1 of the first antigen-binding moiety and the third antigen-binding moiety.

Similar to the first antigen binding moiety, the third antigen binding moiety is in particular a known Fab molecule. However, embodiments can also be envisioned in which the first antigen binding moiety and the third antigen binding moiety are crossover Fab molecules (and the second antigen binding moiety is a known Fab molecule). Therefore, in a specific embodiment, the first antigen binding moiety and the third antigen binding moiety are each a known Fab molecule, and the second antigen binding moiety is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy chain and light chain are exchanged/replaced with each other. In other embodiments, the first antigen binding moiety and the third antigen binding moiety are each a crossover Fab molecule, and the second antigen binding moiety is a known Fab molecule.

If a third antigen binding moiety is present, in a particular embodiment, the first antigenic moiety and the third antigenic moiety bind to GPRC5D, and the second antigen binding moiety binds to CD3, particularly CD3ε.

In certain embodiments, the bispecific antigen-binding molecule comprises an Fc domain, wherein the Fc domain is composed of a first unit and a second unit. The first unit and the second unit of the Fc domain are capable of stably binding.

The bispecific antigen binding molecule may have different configurations, i.e. the first antigen binding moiety, the second antigen binding moiety (and optionally the third antigen binding moiety) may be fused to each other and to the Fc domain in different ways. These components may be fused directly to each other or preferably via one or more suitable peptide linkers. In the case of Fab molecules fused to the N-terminus of the subunit of the Fc domain, they are usually fused via the immunoglobulin hinge region.

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

In one embodiment, the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule, the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second unit 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 consists essentially of a first Fab molecule and a second Fab molecule, the Fc domain consists of a first unit and a second unit 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 second unit of the Fc domain. In addition, as appropriate, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

In another embodiment, the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule, and the first antigen binding moiety 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 consists essentially of a first Fab molecule and a second Fab molecule, the Fc domain consists of a first unit and a second unit and, optionally, one or more peptide linkers, wherein the first Fab molecule and the second Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. The first Fab molecule and the second Fab molecule can be fused to the Fc domain directly or through a peptide linker. In a specific embodiment, the first Fab molecule and the second Fab molecule are each fused to the Fc domain through an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human _IgG1 hinge region, particularly, wherein the Fc domain is an _IgG1 Fc domain.

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

In one embodiment, the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second unit of the Fc domain, and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In a specific embodiment, the bispecific antigen binding molecule consists essentially of a first Fab molecule and a second Fab molecule, the Fc domain consists of the first and second units 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 second unit of the Fc domain. In addition, as appropriate, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

In some embodiments, the third antigen binding moiety, in particular the third Fab molecule, is fused to the N-terminus of the first or second unit of the Fc domain at the C-terminus of the Fab heavy chain. In specific embodiments, each of the first and third Fab molecules is a known Fab molecule, and the second Fab molecule is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged/replaced with each other. In other embodiments, each of the first and third Fab molecules is a crossover Fab molecule, and the second Fab molecule is a known Fab molecule.

In a specific such embodiment, the second antigen binding moiety 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 one embodiment, the bispecific antigen-binding molecule is essentially composed of a first Fab molecule, a second Fab molecule and a third Fab molecule, the Fc domain is composed of a first unit and a second unit 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 unit 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 unit of the Fc domain. The second Fab molecule and the third Fab molecule may be fused to the Fc domain directly or through a peptide linker. In a specific embodiment, the second Fab molecule 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 IgG ₁ hinge region, and in particular, the Fc domain is an IgG ₁ Fc domain. In addition, as appropriate, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

In another of these embodiments, the first antigen binding moiety 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 one embodiment, the bispecific antigen-binding molecule is essentially composed of a first Fab molecule, a second Fab molecule and a third Fab molecule, the Fc domain is composed of a first unit and a second unit and, optionally, one or more peptide linkers, wherein the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the first Fab molecule is fused to the N-terminus of the first unit of the Fc domain at the C-terminus of the Fab heavy chain, and wherein the third Fab molecule is fused to the N-terminus of the second unit of the Fc domain at the C-terminus of the Fab heavy chain. The first Fab molecule and the third Fab molecule may be fused to the Fc domain directly or through a peptide linker. In a specific embodiment, the first Fab molecule 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 IgG ₁ hinge region, and in particular, the Fc domain is an IgG ₁ Fc domain. In addition, as appropriate, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other.

In the configuration of a bispecific antigen-binding molecule in which the Fab molecule is fused at the C-terminus of the Fab heavy chain through the immunoglobulin hinge region to the N-terminus of the subunit of the Fc domain, the hinge region and the Fc domain essentially form an immunoglobulin molecule. In a particular embodiment, the immunoglobulin molecule is an IgG class immunoglobulin. In a more specific embodiment, the immunoglobulin is an IgG ₁ subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG ₄ subclass immunoglobulin. In another specific embodiment, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the immunoglobulin comprises a human constant region, particularly a human Fc region.

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 configurations of the first Fab molecule 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 chain of the first Fab molecule and the second Fab molecule further reduces mispairing of mismatched Fab heavy and light chains, and also reduces the amount of plasmids required to express some of the bispecific antigen-binding molecules.

The antigen binding portion can be fused directly to the Fc domain or to each other, or fused to the Fc domain or to each other through a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are well known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, ( _G4S ) _n , ( _SG4 ) _n , ( _G4S ) _n or _G4 ( _SG4 ) _n peptide linkers. "N" is typically an integer from 1 to 10, particularly from 2 to 4. In one embodiment, the peptide linker is at least 5 amino acids long; in one embodiment, the length is 5 to 100 amino acids; in a further embodiment, the length is 10 to 50 amino acids. In one embodiment, the peptide linker is (GxS) _n or (GxS) _{nGm wherein} G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5, and m=0, 1, 2 or 3), in one embodiment, x=4 and n=2 or 3, in another embodiment, x=4 and n=2. In one embodiment, the peptide linker is ( _G4S ) ₂ . A particularly suitable peptide linker for fusing the Fab light chains of the first Fab molecule and the second Fab molecule to each other is ( _G4S ) ₂ . An exemplary peptide linker suitable for linking the Fab heavy chain of the first Fab fragment and the second Fab fragment comprises the sequence (D)-(G ₄ S) ₂ (SEQ ID NOs 7 and 8). Another suitable such linker comprises the sequence (G ₄ S) ₄ . In addition, the linker may comprise (a portion of) an immunoglobulin hinge region. In particular, in the case where the Fab molecule is fused to the N-terminus of the Fc domain subunit, the fusion may be via an immunoglobulin hinge region or a portion thereof with or without an additional peptide linker.

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

In some of these embodiments, the bispecific antigen-binding molecule further comprises: a crossover Fab light chain polypeptide of a second Fab molecule (VH ₍₂₎ -CL ₍₂₎ ), 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; and a Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ). In other 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 carboxyl-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule (VH ₍₂₎ -CL ₍₂₎ -VL ₍₁₎ -CL ₍₁₎ ); or a polypeptide wherein the Fab light chain polypeptide of the first Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VL ₍₁₎ -CL ₍₁₎ -VH ₍₂₎ -CL ₍₂₎ ) (where appropriate).

The bispecific antigen-binding molecules according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH ₍₃₎ -CH1 ₍₃₎ -CH2-CH3(-CH4)), and a Fab light chain polypeptide of a third Fab molecule (VL ₍₃₎ -CL ₍₃₎ ). In certain embodiments, the polypeptides are covalently linked, for example, 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 carboxyl-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fc domain subunit (VH ₍₂₎ -CL ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ -CH2-CH3(-CH4)). In other embodiments, the bispecific antigen-binding molecule comprises a polypeptide wherein the Fab heavy chain of a first Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxyl-terminal peptide bond with an Fc domain subunit (VH ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CL ₍₂₎ -CH2-CH3(-CH4)).

In some of these embodiments, the bispecific antigen-binding molecule further comprises: a crossover Fab light chain polypeptide of a second Fab molecule (VL ₍ 2)-CH1 ₍₂₎ ), 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; and a Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ). In other 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 carboxyl-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule (VL ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CL ₍₁₎ ); or a polypeptide wherein the Fab light chain polypeptide of the first Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VL ₍₁₎ -CL ₍₁₎ -VL ₍₂₎ -CH1 ₍₂₎ ) (where appropriate).

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

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

In one such embodiment, the bispecific antigen-binding molecule consists essentially of a first antigen-binding moiety and a second antigen-binding moiety, and optionally comprises one or more peptide linkers, wherein the first antigen-binding moiety 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 of these embodiments, the bispecific antigen binding molecule consists essentially of a first antigen binding moiety and a second antigen binding moiety, and optionally comprises one or more peptide linkers, wherein the first antigen binding moiety 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 portion, particularly a third Fab molecule, wherein 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 certain of these embodiments, the bispecific antigen-binding molecule consists essentially of a first Fab molecule, a second Fab molecule, and a third Fab molecule, and optionally comprises 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 portion, particularly a third Fab molecule, wherein 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 of these embodiments, the bispecific antigen-binding molecule consists essentially of a first Fab molecule, a second Fab molecule, and a third Fab molecule, and optionally comprises 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 second 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 a first Fab molecule shares a carboxyl-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region) (VH ₍₁₎ -CH1 ₍₁₎ -VL ₍₂₎ -CH1 ₍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 (VH ₍₂₎ -CL ₍₂₎ ) shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the second Fab molecule; and a Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ).

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

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

In certain embodiments, the bispecific antigen-binding molecules according to the present invention comprise a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VL ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ ). In some embodiments, the bispecific antigen-binding molecule further comprises: a polypeptide, wherein the Fab heavy chain variable region of the second Fab molecule (VH ₍₂₎ -CL ₍₂₎ ) shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the second Fab molecule; and a Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ).

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

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

In certain embodiments, the bispecific antigen-binding molecules according to the present invention comprise a polypeptide wherein the Fab light chain variable region of the third Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain of the second Fab molecule (VL ₍₃₎ -CH1 ₍₃₎ -VL ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ ). In some embodiments, the bispecific antigen-binding molecule further comprises: a polypeptide, wherein the Fab heavy chain variable region of the first Fab molecule (VH ₍₁₎ -CL ₍₁₎ ) shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the first Fab molecule; and a 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 the third Fab molecule (VH ₍₃₎ -CL ₍₃₎ ) shares a carboxyl-terminal peptide bond with the Fab light chain constant region of the third Fab molecule.

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

In a specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; 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 Fab light chain and the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain are substituted with 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, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23; c) a third antigen-binding moiety that binds to the first antigen, and the third antigen-binding moiety is the same as the first antigen-binding moiety; and d) an Fc domain consisting of a first unit and a second unit; wherein (i) the first antigen-binding moiety described 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 described under b), and the second antigen-binding moiety described under b) and the third antigen-binding moiety described 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 described under d); or (ii) the second antigen-binding moiety described 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 described under a), and the first antigen-binding moiety described under a) and the third antigen-binding moiety described 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 described under d). Fusion to the N-terminus of one of the subunits of the Fc domain.

In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3 The second antigen-binding portion is a Fab molecule, wherein the Fab light chain and the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain are replaced with 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, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20; and a light chain variable region (Vl) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23; c) an Fc domain consisting of a first unit and a second unit; wherein (i) in a) The first antigen binding moiety described under b) and the second antigen binding moiety described under b) are each fused to the C-terminus of the Fab heavy chain and to the N-terminus of one of the subunits of the Fc domain described under c).

In all the different configurations of the bispecific antigen-binding molecule, the amino acid substitutions described herein (if any) may be in the CH1 and CL domains of the first antigen-binding moiety/Fab molecule and (if any) the third antigen-binding moiety/Fab molecule or in the CH1 and CL domains of the second antigen-binding moiety/Fab molecule. Preferably, they are in the CH1 and CL domains of the first antigen-binding moiety and (if any) the third antigen-binding moiety/Fab molecule. According to the concept of the present invention, if the amino acid substitutions described herein are made in the first antigen-binding moiety (and (if any) the third) antigen-binding moiety/Fab molecule, such amino acid substitutions are not present in the second antigen-binding moiety/Fab molecule. Conversely, if the amino acid substitutions described herein are made in the second antigen-binding moiety/Fab molecule, such amino acid substitutions are not present in the first (and (if any) the third) antigen-binding moiety/Fab molecule. The amino acid substitutions are particularly performed in bispecific antigen-binding molecules comprising Fab molecules in which the variable domains VL and VH1 of the Fab light chain and the Fab heavy chain are substituted for each other.

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

In one embodiment, the bispecific antigen-binding molecule comprises: a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; 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 mutually replaced, 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, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20, and a light chain variable region (Vl) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23; c) an Fc domain consisting of a first unit and a second unit; wherein in the constant domain CL of the first antigen binding portion under a), at position 124 wherein the amino acid at position 147 is substituted by glutamine (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamine (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 the Fab heavy chain of the second antigen-binding moiety under b). 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 Fc domain subunits under c).

In a specific embodiment, the bispecific antigen-binding molecule comprises: a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3 and the second antigen-binding moiety is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20, and a light chain variable region (Vl) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23; c) a third antigen-binding portion that binds to a first antigen, and the third antigen-binding portion is the same as the first antigen-binding portion; and d) an Fc domain consisting of a first unit and a second unit; wherein in 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) (according to Kabat numbering), and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (according to Kabat numbering), most particularly by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety under a) and the third antigen-binding moiety under c), the amino acid at position 147 is substituted by glutamine (E) (according to Kabat EU index numbering), and the amino acid at position 213 is substituted by glutamine (E) (according to Kabat EU index numbering); and wherein (i) the first antigen-binding moiety under a) is in The Fab heavy chain is fused at the C-terminus of the Fab heavy chain to the N-terminus 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 comprises: a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3 and the second antigen-binding moiety is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20, and a light chain variable region (Vl) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23; c) an Fc domain consisting of a first unit and a second unit; wherein in the constant domain CL of the first antigen binding portion under a), at position 124 substituted with lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (according to Kabat numbering), most particularly with arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety under a), the amino acid at position 147 is substituted with glutamine (E) (according to Kabat EU index numbering) and the amino acid at position 213 is substituted with glutamine (E) (according to Kabat EU index numbering); and wherein the first antigen-binding moiety under a) and the second antigen-binding moiety under b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under c).

According to any of the above embodiments, the components of the bispecific antigen-binding molecule (e.g., Fab molecules, Fc domains) can be fused directly or through various linkers, in particular, through a peptide linker comprising one or more amino acids (usually about 2-20 amino acids) as described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, ( _G4S ) _n , ( _SG4 ) _n , ( _G4S ) _n or _G4 ( _SG4 ) _n peptide linkers, wherein n is generally an integer from 1 to 10, in particular from 2 to 4.

In a particular embodiment, the bispecific antigen-binding molecule comprises: a) a first antigen-binding moiety and a third antigen-binding moiety that binds to the first antigen; wherein the first antigen is GPRC5D, and wherein the first antigen-binding moiety and the second antigen-binding moiety are each (commonly used) Fab molecules, the Fab molecules comprising: a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 11; b) a second antigen-binding moiety that binds to a second antigen; wherein the second antigen is CD3, and wherein the second antigen-binding moiety is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, the Fab molecule comprising: a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising SEQ ID NO: 25 c) an Fc domain consisting of a first unit and a second unit; wherein in the constant domain CL of the first antigen-binding portion and the third antigen-binding portion under a), the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (according to Kabat numbering) (most specifically substituted by arginine (R)), and wherein in the constant domain CH1 of the first antigen-binding portion and the third antigen-binding portion under a), the amino acid at position 147 is substituted by glutamine (E) (according to Kabat EU index numbering), and the amino acid at position 213 is substituted by glutamine (E) (according to Kabat EU index numbering). Index number); and wherein the first antigen-binding moiety under a) is further 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 unit of the Fc domain of the bispecific antigen-binding molecule, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second unit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index).

In a further embodiment, in the first unit of the Fc domain of the bispecific antigen-binding molecule, additionally, the serine residue at position 354 is replaced by a cysteine residue (S354C) or the glutamine residue at position 356 is replaced by a cysteine residue (E356C) (specifically the serine residue at position 354 is replaced by a cysteine residue), and in the second unit of the Fc domain, additionally, the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numbering according to the Kabat EU index).

In yet another embodiment, in each of the first and second units of the Fc domain of the bispecific antigen-binding molecule, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to the Kabat EU index).

In yet another embodiment, the Fc domain is a human IgG1 Fc domain.

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

Fc domain

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

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

In one embodiment, the Fc domain of the bispecific antigen binding molecule is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG ₁ Fc domain. In another embodiment, the Fc domain is an IgG _{4 Fc domain. In a more specific embodiment, the Fc domain is an IgG 4 Fc} domain, which comprises an amino acid substitution at position S228 (numbered according to the Kabat EU index), in particular the amino acid substitution S228P. The amino acid substitution reduces the Fab arm exchange of IgG ₄ antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In another specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG ₁ Fc domain. An exemplary sequence of a human IgG ₁ Fc region is given in SEQ ID NO: 6.

Fc domain modifications that promote heterologous dimerization

The bispecific antigen-binding molecule comprises different antigen-binding moieties which may be fused to one or the other of the two subunits of the Fc domain, which are therefore usually contained in two different polypeptide chains. The recombinant co-expression of these polypeptides and the subsequent dimerization results in several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific antigen-binding molecules in recombinant production, it would be advantageous to introduce modifications in the Fc domain of the bispecific antigen-binding molecule that promote the desired polypeptide association.

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

There are various methods for modifying the CH3 domain of an Fc domain in order to enhance heterodimerization, which are well described in, for example, WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all of these methods, the CH3 domain of the first unit of the Fc domain and the CH3 domain of the second unit of the Fc domain are engineered in a complementary manner such that each CH3 domain (or a recombinant chain comprising CH3 domains) is no longer able to homodimerize with itself but is forced to heterodimerize with the other complementarily engineered CH3 domain (such that the first CH3 domain and the second CH3 domain heterodimerize and no homodimers are formed between the two first CH3 domains or the two second CH3 domains). These different approaches to improve heavy chain heterodimerization are considered as different options for combining heavy chain-light chain modifications in bispecific antigen binding molecules (e.g., VH and VL exchange/replacement in one binding arm and introduction of substitutions with oppositely charged amino acids in the CH1/CL interface), which reduce heavy chain/light chain mispairing and Bence Jones-type byproducts.

In one embodiment, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-to-hole" modification, which includes a "knob" modification of one of the two subunits of the Fc domain and a "hole" modification of the other of the two subunits of the Fc domain.

The "knob-and-hole" technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide so that the protrusion can be placed in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The protrusion is constructed by replacing smaller amino acid side chains on the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing larger amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a complementary cavity of the same or similar size as the protrusion is formed in the interface of the second polypeptide.

Thus, in a particular embodiment, in the CH3 domain of the first unit of the Fc domain of the bispecific antigen-binding molecule, the amino acid residues are substituted with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first unit, and the protrusion can be placed in the cavity in the CH3 domain of the second unit, and in the CH3 domain of the second unit of the Fc domain, the amino acid residues are substituted with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second unit, and the protrusion in the CH3 domain of the second unit can be placed in the cavity.

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

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

Protrusions and cavities can be produced by altering the nucleic acid encoding the polypeptide, for example by mutagenesis at specific sites or by peptide synthesis.

In one specific embodiment, in the first unit (“knob” unit) of the Fc domain (CH3 domain), the lysine residue at position 366 is substituted by a tryptophan residue (T366W), and in the second unit (“hole” unit) of the Fc domain (CH3 domain), the tyrosine residue at position 407 is substituted by a valine residue (Y407V). In one embodiment, in the second unit of the Fc domain, the lysine residue at position 366 is substituted by a serine residue (T366S), and the leucine residue at position 368 is substituted by an alanine residue (L368A) (numbering according to the Kabat EU index).

In another embodiment, in the first unit of the Fc domain, the serine residue at position 354 is substituted by a cystine residue (S354C) or the glutamine residue at position 356 is substituted by a cystine residue (E356C) (particularly the serine residue at position 354 is substituted by a cystine residue), and in the second unit of the Fc domain, the tyrosine residue at position 349 is substituted by a cystine residue (Y349C) (numbering according to the Kabat EU index). The introduction of these two cysteine residues leads to the formation of a disulfide bond between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

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

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

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

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

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

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

In one embodiment, the heterodimerization method described in WO 2013/157953 can be used instead. In one embodiment, the first CH3 domain comprises an amino acid mutation T366K, and the second CH3 domain comprises an amino acid mutation L351D (numbered according to the Kabat EU index). In another embodiment, the first CH3 domain further comprises an amino acid mutation L351K. In another embodiment, the second CH3 domain further comprises an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numbered according to the Kabat EU index).

In one embodiment, the heterologous dimerization method described in WO 2012/058768 can be used instead. In one embodiment, the first CH3 domain comprises amino acid mutations L351Y, Y407A, and the second CH3 domain comprises amino acid mutations T366A, K409F. In another embodiment, the second CH3 domain further comprises an amino acid mutation at position T411, D399, S400, F405, N390 or K392, wherein the position is selected from, for example: 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 (according to the Kabat EU index numbering). In another embodiment, the first CH3 domain comprises amino acid mutations L351Y, Y407A, and the second CH3 domain comprises amino acid mutations T366V, K409F. In another embodiment, the first CH3 domain comprises amino acid mutations Y407A, and the second CH3 domain comprises amino acid mutations T366A, K409F. In another embodiment, the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (according to the Kabat EU index numbering).

In one embodiment, the heterodimerization method described in WO 2011/143545 can be used instead, for example, by making amino acid modifications at positions selected from 368 and 409 (according to Kabat EU index numbering).

In one embodiment, the heterodimerization method described in WO 2011/090762 can be used instead, which method also uses the "knob-in-hole" technology described above. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W, and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y, and the second CH3 domain comprises the amino acid mutation Y407T (according to the Kabat EU index numbering).

In one embodiment, the bispecific antigen binding molecule or its Fc domain is of the _IgG2 subclass and optionally the heterodimerization method described in WO 2010/129304 is used.

In an alternative embodiment, modifications that promote the association of the first and second Fc domain subunits include modifications that mediate electrostatic switching, such as described in PCT Publication No. WO 2009/089004. Typically, this approach involves replacing one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues, thereby making homodimer formation electrostatically unfavorable, but heterodimerization electrostatically favorable. In one such embodiment, the first CH3 domain comprises an amino acid substitution of K392 and N392 with a negatively charged amino acid (e.g., glutamine (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain comprises an amino acid substitution of D399, E356, D356 or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K and more preferably D399K and E356K). In another embodiment, the first CH3 domain further comprises an amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g., glutamine (E) or aspartic acid (D), more preferably K409D or R409D). In another embodiment, the first CH3 domain further or alternatively comprises an amino acid substitution of K439 and/or K370 with a negatively charged amino acid (e.g., glutamine (E) or aspartic acid (D)) (all numbered according to the Kabat EU index).

In yet another embodiment, the heterologous dimerization method described in WO 2007/147901 may be used instead. In one embodiment, the first CH3 domain comprises amino acid mutations K253E, D282K and K322D, and the second CH3 domain comprises amino acid mutations D239K, E240K and K292D (according to Kabat EU index numbering).

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

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

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

The Fc domain confers favorable pharmacokinetic properties to the bispecific antigen-binding molecules, including a long serum half-life, which facilitates good accumulation in target tissues and a favorable tissue-blood distribution ratio. However, at the same time, it may lead to the undesirable targeting of the bispecific antigen-binding molecules to cells expressing Fc receptors rather than to cells that are preferred to carry the antigen. Furthermore, co-activation of Fc receptor signaling pathways may result in cytokine release, which, combined with the T cell activation properties (e.g., in embodiments of bispecific antigen binding molecules, wherein the second antigen binding moiety binds to an activated T cell antigen) and the long half-life of the bispecific antigen binding molecules, results in overactivation of cytokine receptors and produces severe side effects upon systemic administration. Immune cells other than activated T cells (carrying Fc receptors) may even reduce the efficacy of bispecific antigen binding molecules (particularly wherein the second antigen binding moiety binds to an activated T cell antigen) because NK cells may destroy T cells.

Accordingly, in certain embodiments, the Fc domain of the bispecific antigen binding molecule exhibits reduced affinity for Fc receptors and/or effector function compared to a native IgG ₁ Fc domain. In one such embodiment, the Fc domain (or a bispecific antigen-binding molecule comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity for Fc receptors compared to a native _IgG1 Fc domain (or a bispecific antigen-binding molecule comprising an _IgG1 Fc domain), and/or exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function compared to a native _IgG1 Fc domain (or a bispecific antigen-binding molecule comprising an _IgG1 Fc domain). In one embodiment, the Fc domain (or the bispecific antigen binding molecule comprising the Fc domain) does not substantially bind to an Fc receptor and/or induce an effector function. In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically FcγRIIIa. In one embodiment, the effector function is one or more selected from CDC, ADCC, ADCP and cytokine secretion. In a specific embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the native IgG ₁ Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or a bispecific antigen-binding molecule comprising the Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of the native IgG ₁ Fc domain (or a bispecific antigen-binding molecule comprising the native IgG ₁ Fc domain) to FcRn.

In certain embodiments, the engineered Fc domain has reduced binding affinity and/or reduced effector function for an Fc receptor 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 mutations that reduce the binding affinity and/or effector function of the Fc domain for an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of an amino acid to an Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least 10 times, at least 20 times, or even at least 50 times. In one embodiment, the binding affinity of the bispecific antigen-binding molecule comprising the engineered Fc domain to the Fc receptor is reduced to less than 20%, particularly less than 10%, and more particularly less than 5% compared to a bispecific antigen-binding molecule comprising a non-engineered Fc domain. In a specific embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity to the complementary component, i.e., the specific binding affinity to C1q, is also reduced. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn is achieved when the Fc domain (or a bispecific antigen-binding molecule comprising the Fc domain) exhibits greater than about 70% of the binding affinity of the non-engineered form of the Fc domain (or a bispecific antigen-binding molecule comprising the non-engineered form of the Fc domain) to FcRn, i.e., the binding affinity of the Fc domain to the receptor is maintained. The Fc domain or a bispecific antigen-binding molecule of the invention comprising the 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 compared to an unengineered Fc domain. Reduced effector function may 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-induced apoptosis, reduced cross-linking of target-binding antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is selected from one or more of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a specific embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a bispecific antigen-binding molecule comprising a non-engineered Fc domain).

In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor and/or the effector function is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from E233, L234, L235, N297, P331 and P329 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from L234, L235 and P329 (numbered according to the Kabat EU index). In some embodiments, the Fc domain comprises an amino acid substitution of L234A and L235A (numbered according to the Kabat EU index). In one of these embodiments, the Fc domain is an IgG ₁ Fc domain, particularly a human IgG ₁ 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 (numbered according to the Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329, and another amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbered according to the Kabat EU index). In a more specific embodiment, the another amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a specific embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). Specifically, in a particular embodiment, each subunit of the Fc domain comprises amino acid substitutions L234A, L235A and P329G (according to the Kabat EU index numbering), i.e., in each of the first subunit and the second subunit of the Fc domain, the leucine residue at position 234 is substituted by an alanine residue (L234A), the leucine residue at position 235 is substituted by an alanine residue (L235A), and the proline residue at position 329 is substituted by a glycine residue (P329G) (according to the Kabat EU index numbering).

In one such embodiment, the Fc domain is an IgG ₁ Fc domain, in particular a human IgG ₁ Fc domain. The amino acid substitution "P329G LALA" combination almost completely abolishes Fcγ receptor (and complement) binding of human IgG ₁ 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 for preparing such mutant Fc domains and methods for determining their properties (e.g., Fc receptor binding or effector function).

_IgG4 antibodies show reduced binding affinity to Fc receptors and reduced effector functions compared to _IgG1 antibodies. Therefore, in some embodiments, the Fc domain of the bispecific antigen-binding molecule is an _IgG4 Fc domain, in particular a human _IgG4 Fc domain. In one embodiment, the _IgG4 Fc domain comprises an amino acid substitution at position S228, specifically comprising the amino acid substitution S228P (numbered according to the Kabat EU index). To further reduce its binding affinity to Fc receptors and/or its effector functions, in one embodiment, the _IgG4 Fc domain comprises an amino acid substitution at position L235, in particular the amino acid substitution L235E (numbered according to the Kabat EU index). In another embodiment, the _IgG4 Fc domain comprises an amino acid substitution at position P329, specifically comprising the amino acid substitution P329G (numbered according to the Kabat EU index). In a specific embodiment, the _IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically comprising the amino acid substitutions S228P, L235E and P329G (numbered according to the Kabat EU index). Such _IgG4 Fc domain variants and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012/130831, which is incorporated herein by reference in its entirety.

In a specific embodiment, the Fc domain that exhibits reduced binding affinity for an Fc receptor and/or reduced effector function compared to a native _IgG1 Fc domain is a human _IgG1 Fc domain comprising amino acid substitutions L234A, L235A and optionally P329G or a human _IgG4 Fc domain comprising amino acid substitutions S228P, L235E and optionally P329G (numbered according to the Kabat EU index).

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

In addition to the Fc domains described above and in PCT Publication No. WO 2012/130831, Fc domains with reduced Fc receptor binding and/or effector function also include those with substitutions at one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbered according to the 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, in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

Variant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods known in the art. Genetic methods may include site-specific mutagenesis of the coding DNA sequence, PCR, gene synthesis, etc. The correctness of the nucleotide changes can be verified, for example, by sequencing.

Binding to Fc receptors can be readily determined by ELISA, or by surface plasmon resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors can be obtained, for example, by recombinant expression. Alternatively, the binding affinity of an Fc domain or a bispecific antigen binding molecule comprising an Fc domain to an Fc receptor can be assessed using a cell line known to express a specific Fc receptor, such as human NK cells expressing the FcγIIIa receptor.

The effector function of an Fc domain or a bispecific antigen-binding molecule comprising an Fc domain can be measured by methods known in the art. Examples of in vitro assays for evaluating ADCC activity of target molecules are described, for example, in U.S. Pat. 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. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays can be employed (see, e.g., 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 a target molecule can be assessed in vivo in an animal model such as that disclosed by Clynes et al. in Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, the binding of the Fc domain to complement components is reduced, specifically to C1q. Thus, in some embodiments, wherein the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. A C1q binding assay can be performed to determine whether the Fc domain or a bispecific antigen-binding molecule comprising the Fc domain can bind to C1q and therefore has CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

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

Composition, formulation and route of administration

Another aspect of the present invention provides a pharmaceutical composition comprising any antibody or bispecific antigen-binding molecule provided herein, for example, for use in any of the following treatment methods. In one embodiment, the pharmaceutical composition comprises any antibody or bispecific antigen-binding molecule provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any antibody or bispecific antigen-binding molecule provided herein and at least one additional therapeutic agent (as described below).

Also provided is a method for producing the antibody or bispecific antigen-binding molecule of the present invention in a form suitable for in vivo administration, the method comprising (a) obtaining the antibody or bispecific antigen-binding molecule according to the present invention, and (b) formulating the antibody or bispecific antigen-binding molecule together with a carrier received on at least one drug, thereby formulating a formulation of the antibody or bispecific antigen-binding molecule for in vivo administration.

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of an antibody or bispecific antigen-binding molecule dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to receptors at the dosages and concentrations employed, i.e., do not produce adverse, allergic or other untoward reactions (where appropriate) when administered to animals (e.g., humans). Based on the present disclosure, a person of ordinary skill in the art will recognize methods for preparing pharmaceutical compositions comprising antibodies or bispecific antigen-binding molecules and, if appropriate, additional active ingredients, such as those described in Remington's Pharmaceutical Sciences 18th edition (Mack Printing Company, 1990), which is incorporated herein by reference. Moreover, for animal (e.g., human) administration, it should be understood that the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biologics Standards or other relevant authorities in other countries/regions. Preferred compositions are lyophilized preparations or aqueous solutions. As used herein, "pharmaceutically acceptable carriers" include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and any combination thereof known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical compositions is contemplated.

The immune complexes of the present invention (and any other therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and if local treatment is desired, intralesional administration can be used. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.

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 present invention may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks solution, Ringer solution, or saline buffer. The solution may contain a formulation agent, such as a suspending agent, a stabilizer, and/or a dispersant. Alternatively, the antibody or bispecific antigen-binding molecule may be in powder form for formulation with a suitable carrier, such as sterile, pyrogen-free water, prior to use. Sterile injectable solutions are prepared by mixing the desired amount of the antibody or bispecific antigen-binding molecule of the present invention with an appropriate solvent and, as required, a variety of other ingredients listed below. Sterility can be easily achieved, for example, by filtration through a sterile filter membrane. Dispersions are usually prepared by incorporating the various sterilized active ingredients into a sterile carrier containing a basic dispersion medium and/or other ingredients. For sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred method of preparation is vacuum drying or freeze drying techniques, which can obtain a powder of the active ingredient and any other desired ingredients from a previously filtered sterile liquid medium. If necessary, the liquid medium should be appropriately buffered and the liquid diluent should be made isotonic before injecting sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage and must be resistant to the contaminating action of microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be minimized to safe concentrations, for example, less than 0.5 ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzylammonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; o-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, aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes such as zinc protein complexes; and/or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, etc. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to prepare a high concentration solution. In addition, the suspension of the active compound can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes.

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

In addition to the compositions described above, the bispecific antigen-binding molecules can also be formulated as depot preparations. Such long-acting preparations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the antibody or bispecific antigen-binding molecule can be formulated with a suitable polymeric or hydrophobic substance (e.g., as an emulsion in an acceptable oil) or ion exchange resin, or as a sparingly soluble derivative, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the antibodies or bispecific antigen-binding molecules of the present invention can be prepared using conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing methods. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that facilitate processing of the protein into a pharmaceutically acceptable preparation. Suitable formulations depend on the selected route of administration.

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

Immunomodulatory imide drugs (IMiD)

The term "immunomodulatory imide drugs (IMiDs)" refers to a class of immunomodulatory drugs (drugs that modulate immune responses) that contain an imide group. IMiDs include first-generation IMiDs and cerebellum E3 ligase modulators (CELMoDs; also called next-generation IMiDs), both of which contain a retained glutarimido ring. First- and next-generation IMiDs bind cerebellum ribonuclein (CRBN), a receptor for the Cullin ring 4 ubiquitin ligase (CRL4) complex, and modulate ubiquitin ligase activity. Ligase specificity is redirected toward non-physiological protein targets, also called "neosubstrates," which are subsequently ubiquitinated and/or degraded. The retained glutarimido ring binds to CRBN, while the variable side groups interact with both CRBN and the neosubstrate. The term "first generation IMiD" refers to thalidomide and the thalidomide derivatives lenalidomide and pomalidomide. The term "CELMoD" or "next generation IMiD" refers to thalidomide derivatives with expanded side groups that improve interaction with CRBN and/or new substrates, and include but are not limited to ibedomide (also known as CC-220), avadomide (also known as CC-122), mezigedolamide (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 of lenalidomide is C ₁₃ H ₁₃ N ₃ O ₃ , CAS registration number 191732-72-6, and the molecular weight is 259.3. Lenalidomide is a thalidomide analog sold under the trade name REVLIMID ^® .

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

The empirical formula of pomalidomide is C ₁₃ H ₁₁ N ₃ O ₄ , CAS registration number 19171-19-8, and the gram molecular weight is 273.24. Pomalidomide is a thalidomide analog, sold under the trade name Imnovid in the EU and Pomalyst in the United States.

The term "ibedomide" refers to a compound having the following chemical structure:

The empirical formula of ibedomide is C ₂₅ H ₂₇ N ₃ O ₅ , CAS registration number 1323403-33-3, and the gram molecular weight is 449.5.

The term "mezilagidine" refers to compounds with the following chemical structure:

The empirical formula of mezilig polyamine is C ₃₂ H ₃₀ FN ₅ O ₄ , CAS registration number 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 and 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 consisting of thalidomide, lenalidomide, and pomalidomide. In one embodiment, the IMiD is a CELMoD and is selected from the group consisting of ibedomide, avadomide, mezigedomide (CC-92480), CC885, CC647, CC-90009, and CC3060. In one embodiment, the IMiD is selected from the group consisting of lenalidomide, pomalidomide, ibedomide and mezitropium.

Glucocorticoids

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.

As used herein, "glycocorticosteroids" or "glycocorticin" refers to a class of corticosteroid compounds that participate in the metabolism of carbohydrates, proteins, and fats and have anti-inflammatory activity. Glucocorticosteroids are mainly used in therapy for their anti-inflammatory and immunosuppressive effects. Glucocorticosteroids include, but are not limited to, dexamethasone, prednisone, penicillin, methyl penicillin, and substitutes.

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

The empirical formula of dexamethasone is C ₂₂ H ₂₉ FO ₅ , CAS registration 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 glycocorticosteroid. In one embodiment, the glycocorticosteroid is dexamethasone.

Treatment methods and compositions

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

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

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

One embodiment of the present invention is a GPRC5D/anti-CD3 bispecific antibody as described herein, used in combination with an IMiD as described herein for treating cancer or tumors. One embodiment of the present invention is a GPRC5D/anti-CD3 bispecific antibody as described herein, used in combination with an IMiD as described herein and a glycocorticosteroid as described herein for treating cancer or tumors.

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

Yet another embodiment is a glycocorticosteroid as described herein, used in combination with an anti-GPRC5D/anti-CD3 bispecific antibody as described herein and an IMiD as described herein for treating cancer or tumor.

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

One embodiment of the present invention is a combination of an anti-GPRC5D/anti-CD3 bispecific antibody as described herein and an IMiD as described herein for use in treating any of the above cancers or tumors. One embodiment of the present invention is a combination of an anti-GPRC5D/anti-CD3 bispecific antibody as described herein and an IMiD and a glycocorticosteroid as described herein for use in treating any of the above cancers or tumors.

One embodiment of the present invention is a combination of an anti-GPRC5D/anti-CD3 bispecific antibody as described herein and an IMiD as described herein for the treatment of multiple myeloma. One embodiment of the present invention is a combination of an anti-GPRC5D/anti-CD3 bispecific antibody as described herein and an IMiD and a glycocorticosteroid as described herein for the treatment of multiple myeloma.

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

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

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

The present invention includes the use of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD according to the present invention for the combination therapy. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above combination therapy and medical use comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29. In another embodiment, the IMiD for the above combination therapy and medical use is selected from the group consisting of lenalidomide, pomalidomide, ibedomide and mezigtamide. In another embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above-mentioned combination therapy and medical use 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 consisting of lenalidomide, pomalidomide, ibedomide and mezigedolamide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above-mentioned combination therapy and medical use comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD is lenalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above-mentioned combined therapy and medical use comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD is pomalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above-mentioned combined therapy and medical use 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 ibedomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above-mentioned combination therapy and medical use 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 mezimidol. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above-mentioned combination therapy and medical use is voritumimab, and the IMiD is lenalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above-mentioned combination therapy and medical use is voritumimab, and the IMiD is pomalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for use in the above combination therapy and medical use is voritumimab, and the IMiD is ibedomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for use in the above combination therapy and medical use is voritumimab, and the IMiD is ibedomide.

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

In a preferred embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above combination therapy and medical use comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29. In another embodiment, the IMiD for the above combination therapy and medical use is selected from the group consisting of lenalidomide, pomalidomide, ibedomide and mezigtamide. In another embodiment, the glycocorticosteroid for the above combination therapy and medical use is dexamethasone. In another embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above-mentioned combination therapy and medical use comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the above-mentioned combination therapy and medical use is selected from the group consisting of lenalidomide, pomalidomide, ibedomide and mezitropium. In another embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above-mentioned combination therapy and medical use comprises the polypeptide sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD is selected from the group consisting of lenalidomide, pomalidomide, ibedomide and mezigtamide, and the glycocorticosteroid is dexamethasone. In another embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above-mentioned combination therapy and medical use comprises the polypeptide sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD is lenalidomide, and the glycocorticosteroid is dexamethasone. In another embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the above combination therapy and medical use is voritumimab, and the IMiD is lenalidomide, and the glycocorticosteroid is dexamethasone.

The present 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 present invention comprises an anti-GPRC5D/anti-CD3 bispecific antibody as described herein in combination with an IMiD as described herein and a glycocorticosteroid 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, such as a pharmaceutical composition, containing an anti-GPRC5D/anti-CD3 bispecific antibody as described herein and an IMiD as described herein, formulated with a pharmaceutically acceptable carrier. In another aspect, the present invention provides a composition, such as a pharmaceutical composition, containing an anti-GPRC5D/anti-CD3 bispecific antibody as described herein and an IMiD as described herein and a glycocorticosteroid as described herein, formulated with a pharmaceutically acceptable carrier.

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

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

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically active substances is known in the art. In addition to water, the carrier may be, for example, an isotonic buffered saline solution.

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

The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient (effective amount) that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration that is non-toxic to the patient. This dosage may include a step-up dosing cycle of the active ingredient. The term "dosage" refers to the amount (i.e., dosage) and frequency of administration of the active ingredient. The term "step-up dosing cycle" refers to a treatment period in which the amount of the active ingredient is gradually increased during the treatment period. This can be achieved by increasing the dose of the active ingredient and/or increasing the frequency of administration. Thus, in one embodiment, the dosage of the anti-GPRC5D/anti-CD3 bispecific antibody and IMiD combination as described herein comprises at least one step-up dosing cycle. In one embodiment, the dosage of the anti-GPRC5D/anti-CD3 bispecific antibody and IMiD combination as described herein comprises at least one step-up dosing cycle of the anti-GPRC5D/anti-CD3 bispecific antibody. In one embodiment, the dosage of the anti-GPRC5D/anti-CD3 bispecific antibody and IMiD combination 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 for combination therapy and medical use described herein is administered in an effective amount, wherein the dosage comprises at least one step-up dosing cycle. In one embodiment, voritumimab for combination therapy and medical use described herein is administered in 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 composition of the invention employed, or its esters, salts or amides, the route of administration, the time of administration, the rate of excretion of the particular compound employed, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, disease, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts.

The present 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 present invention comprises an IMiD as described herein in combination with an anti-GPRC5D/anti-CD3 bispecific antibody as described herein and a glycocorticosteroid as described herein for use in the manufacture of a medicament for the treatment of cancer. The present invention comprises a glycocorticosteroid 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 the manufacture of a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for treating cancer according to the present invention is voritumimab. In one embodiment, the IMiD for the manufacture of a medicament for treating cancer according to the present invention is selected from the group consisting of lenalidomide, pomalidomide, ibedomide and mezigtamide. In one embodiment, the glycocorticosteroid for manufacturing a medicament for treating cancer according to the present invention is dexamethasone. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for manufacturing a medicament for treating cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for manufacturing a medicament for treating cancer according to the present invention is selected from the group consisting of lenalidomide, pomalidomide, ibedomide and mezigtamide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for the treatment of cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for the treatment of cancer according to the present invention is lenalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for the treatment of cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for the treatment of cancer according to the present invention is pomalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for the treatment of cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for the treatment of cancer according to the present invention is ibedomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for the treatment of cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for the treatment of cancer according to the present invention is mezigotamine. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for the treatment of cancer according to the present invention is voritumimab, and the IMiD for the manufacture of a medicament for the treatment of cancer according to the present invention is lenalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for the treatment of cancer according to the present invention is voritumimab, and the IMiD for the manufacture of a medicament for the treatment of cancer according to the present invention is pomalidomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for the treatment of cancer according to the present invention is voritumimab, and the IMiD for the manufacture of a medicament for the treatment of cancer according to the present invention is ibedomide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for the treatment of cancer according to the present invention is voritumimab, and the IMiD for the manufacture of a medicament for the treatment of cancer according to the present invention is mezidoramide. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for the treatment of cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for the treatment of cancer according to the present invention is selected from the group consisting of lenalidomide, pomalidomide, ibedomide and mezitropium, and the glycocorticosteroid for the manufacture of a medicament for the treatment of cancer according to the present invention is dexamethasone. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for the treatment of cancer according to the present invention comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the IMiD for the manufacture of a medicament for the treatment of cancer according to the present invention is lenalidomide, and the glycocorticosteroid for the manufacture of a medicament for the treatment of cancer according to the present invention is dexamethasone. In one embodiment, the anti-GPRC5D/anti-CD3 bispecific antibody for the manufacture of a medicament for the treatment of cancer according to the present invention is voritumimab, and the IMiD for the manufacture of a medicament for the treatment of cancer according to the present invention is lenalidomide, and the glycocorticosteroid for the manufacture of a medicament for the treatment of cancer according to the present invention is dexamethasone.

The present invention further provides an anti-GPRC5D/anti-CD3 bispecific antibody according to the present invention as described herein and an IMiD according to the present invention as described herein, which is used for the manufacture of a medicament, preferably with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer. The present invention further provides an anti-GPRC5D/anti-CD3 bispecific antibody according to the present invention as described herein and an IMiD according to the present invention as described herein and a glycocorticosteroid according to the present invention as described herein, which is used for the manufacture of a medicament, preferably with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.

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

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

In addition, the kit may include (a) a first container containing a composition, wherein the composition comprises an anti-GPRC5D/anti-CD3 bispecific antibody as described herein; (b) a second container containing a composition, wherein the composition comprises an IMiD as described herein; (c) a third container containing a composition, wherein the composition comprises a glycocorticosteroid as described herein, and optionally (d) a fourth container containing a composition, wherein the composition comprises another cytotoxic agent or other therapeutic agent. The kit in this embodiment of the invention may further include a drug instruction indicating that the composition can be used to treat a specific disease. 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. From a commercial and user perspective, it may further comprise other materials, including other buffers, diluents, filters, needles and syringes.

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

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

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

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

The term "treatment" or its equivalent, when applied to, for example, cancer, refers to a procedure or course of action intended to reduce or eliminate the number of cancer cells in a patient's body or to alleviate the symptoms of cancer. A method of "treating" cancer or another proliferative disorder does not necessarily mean that cancer cells or other disease will actually be eliminated, that the number of cells or disease will actually be reduced, or that the cancer or other disease will actually be alleviated. Often, a method of treating cancer will be performed even if the probability of success is low, but, in view of the patient's medical history and estimated survival expectancy, it is still believed that the method will induce an overall beneficial course of action.

The term "administered in combination with" or "co-administered", "co-administered", "combination therapy" or "combination treatment" refers to the administration of an anti-GPRC5D/anti-CD3 bispecific antibody as described herein and an IMiD as described herein and, optionally, a glycocorticosteroid as described herein, e.g., as separate formulations/applications (or as a single formulation/application). Co-administration can be performed simultaneously or sequentially in either order, wherein preferably, the active agents exert their biological activity over a period of time or all at the same time. The active agents are co-administered simultaneously or sequentially (e.g., intravenously (i.v.)) by continuous infusion or oral administration. When all therapeutic agents are co-administered sequentially, the doses are administered as two separate doses on the same day, or one drug is administered on day 1 and the second drug is co-administered on day 2 to day 7 (preferably on day 2 to day 4). Thus, in one embodiment, the term "sequentially" means within 7 days after administration of the first component, preferably within 4 days after administration of the first component; and the term "concurrently" means at the same time. The term "co-administered" with respect to a maintenance dose of anti-GPRC5D/anti-CD3 bispecific antibody and/or IMiD and/or glycocorticosteroid (if applicable) means that the maintenance dose can be co-administered at the same time, for example, weekly if the treatment cycle is applicable to all drugs.

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

The co-administration amount and timing of co-administration will depend on the type (species, sex, age, weight, etc.) and condition of the patient being treated and the severity of the disease or disorder being treated. The anti-GPRC5D/anti-CD3 bispecific antibody and/or IMiD and/or glycocorticosteroid (if applicable) are suitably co-administered to the patient at one time or over a series of treatments, e.g., on the same day or the next day or at weekly intervals.

A skilled artisan will readily recognize that in many cases, the combination therapy may not provide a cure, but only a partial benefit. In some embodiments, a physiological change that has some benefit is also considered therapeutically beneficial. Therefore, in some embodiments, the amount of the therapeutic combination that is believed to provide a physiological change is considered an "effective amount" or a "therapeutically effective amount."

Aspects of the invention: 1. A combination of an anti-GPRC5D/anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD) for use as a combination therapy in the treatment of cancer. 2. Use of a combination of an anti-GPRC5D/anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD) in the manufacture of a medicament for the treatment of cancer. 3. A method of treating cancer in an individual, comprising administering to the individual a combination of an anti-GPRC5D/anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD). 4. A kit comprising a first drug comprising an anti-GPRC5D/anti-CD3 bispecific antibody and a second drug comprising an immunomodulatory imide drug (IMiD), and optionally further comprising a package insert comprising instructions for administering the combination of the first drug and the second drug for treating cancer in an individual. 5. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD according to any of the preceding aspects, wherein the anti-GPRC5D/anti-CD3 bispecific antibody comprises (i) a first antigen-binding portion that specifically binds to GPRC5D and comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13, and HCDR 3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16, and LCDR 3 of SEQ ID NO: 17; and (ii) The second antigen-binding portion specifically binds to CD3 and comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23. 6. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD according to any of the preceding aspects, wherein the anti-GPRC5D/anti-CD3 bispecific antibody comprises (i) a first antigen-binding portion that specifically binds to GPRC5D, comprising: a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10; and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11; and (ii) a second antigen-binding portion 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 combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD according to aspect 5 or 6, wherein the first antigen-binding portion and/or the second antigen-binding portion of the anti-GPRC5D/anti-CD3 bispecific antibody is a Fab molecule. 8. A combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibodies and IMiDs according to any one of aspects 5 to 7, wherein the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain, in particular, the variable domains VL and VH are replaced with each other. 9. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD according to any one of aspects 5 to 8, wherein the first antigen-binding portion is a Fab molecule, wherein in the constant domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1, the amino acid at position 147 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering) and the amino acid at position 213 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering). The amino acid at each position is independently substituted with glutamine (E) or aspartic acid (D) (according to the Kabat EU index number). 10. A combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibodies and IMiDs according to any one of aspects 5 to 9, wherein the first antigen-binding portion and the second antigen-binding portion are fused to each other, optionally via a peptide linker. 11. A combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD according to any one of aspects 5 to 10, wherein the first antigen-binding moiety and the second antigen-binding moiety are each Fab molecules, and wherein (i) the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain, or (ii) the first antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen-binding moiety at the C-terminus of the Fab heavy chain. 12. A combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD according to aspects 1 to 11, wherein the anti-GPRC5D/anti-CD3 bispecific antibody comprises a third antigen-binding moiety. 13. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD according to aspect 12, wherein the third antigen portion is the same as the first antigen binding portion. 14. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD according to any of the above aspects, wherein the anti-GPRC5D/anti-CD3 bispecific antibody comprises an Fc domain composed of a first unit and a second unit. 15. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD according to aspects 5 to 14, wherein the first antigen-binding moiety, the second antigen-binding moiety and the third antigen-binding moiety, if present, are each a Fab molecule; and wherein (i) the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain. and wherein the third antigen binding moiety, if present, is fused to the N-terminus of the second unit of the Fc domain at the C-terminus of the Fab recombinant chain. 16. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibodies and IMiDs according to aspects 14 or 15, wherein the Fc domain is an IgG Fc domain. 17. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibodies and IMiDs according to any one of aspects 14 to 16, wherein the Fc domain is an IgG1 Fc domain. 18. The combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD according to any one of aspects 14 to 17, wherein the Fc domain is a human Fc domain. 19. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD according to any one of aspects 14 to 18, wherein the amino acid residues in the CH3 domain of the first unit of the Fc domain are substituted with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first unit, and the protrusion can be placed in the cavity in the CH3 domain of the second unit, and the amino acid residues in the CH3 domain of the second unit of the Fc domain are substituted with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second unit, and the protrusion in the CH3 domain of the first unit can be placed in the cavity. 20. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD according to any one of aspects 14 to 19, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector function. 21. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD according to any one of aspects 1 to 19, wherein the anti-GPRC5D/anti-CD3 bispecific antibody comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29. 22. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD according to any one of aspects 1 to 21, wherein the IMiD is a first generation IMiD or a cerebellar protein E3 ligase modulator (CELMoD). 23. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD according to any one of aspects 1 to 22, wherein the IMiD is selected from the group consisting of lenalidomide, pomalidomide and ibedomide. 24. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD according to any one of aspects 1 to 23, wherein the combination further comprises a glucocorticoid. 25. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD according to aspect 24, wherein the glycocorticosteroid is dexamethasone. Amino acid sequence SEQ ID NO Human kappa CL domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 1 Human λ CL domain QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 2 Human IgG1 heavy chain constant region (CH1-CH2-CH3) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 3 hCD3 MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI 4 cynoCD3 MQSGTRWRVLGLCLLSIGVWGQDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI 5 hIgG1 Fc region DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSP 6 Connector GGGGSGGGGS 7 Connector DGGGGSGGGGS 8 Human GPRC5D MYKDCIESTGDYFLLCDAEGPWGIILESLAILGIVVTILLLLAFLFLMRKIQDCSQWNVLPTQLLFLLSVLGLFGLAFAFIIELNQQTAPVRYFLFGVLFALCFSCLLAHASNLVKLVRGCVSFSWTTILCIAIGCSLLQIIIATEYVTLIMTRGMMFVNMTPCQLNVDFVVLLVYVLFLMALTFFVSKATFCGPCENWKQHGRLIFITV LFSIIIWVVWISMLLRGNPQFQRQPQWDDPVVCIALVTNAWVFLLLYIVPELCILYRSCRQECPLQGNACPVTAYQHSFQVENQELSRARDSDGAEEDVALTSYGTPIQPQTVDPTQECFIPQAKLSPQQDAGGV 9 5E11_VH1c EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSS 10 5E11_VL2b EIVLTQSPGTLSLSPGERATLSCRASQSVSISGINLMNWYQQKPGQQPKLLIYHASILASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTRESPLTFGQGTRLEIK 11 5E11_P1AE5706_Parental-VH-HCDR1 GFTFSKYAMA 12 5E11_P1AE5723_ P1AE5728_VH-HCDR2 SISTGGVNTYYADSVKG 13 5E11_P1AE5706_Parental-VH-HCDR3 HTGDYFDY 14 5E11_P1AE5706_Parent-VL-LCDR1 RASQSVSISGINLMN 15 5E11_P1AE5706_Parent-VL-LCDR2 HASILAS 16 5E11_P1AE5706_Parent-VL-LCDR3 QQTRESPLT 17 CD3-Cl22-VH-HCDR1 SYAMN 18 CD3-Cl22-VH-HCDR2 RIRSKYNNYATYYADSVKG 19 CD3-Cl22-VH-HCDR3 HTTFPSSYVSYYGY 20 CD3-Cl22-VH-LCDR1 GSSTGAVTTSNYAN twenty one CD3-Cl22-VH-LCDR2 GTNKRAP twenty two CD3-Cl22-VH-LCDR3 ALWYSNLWV twenty three CD3-Cl22-VH EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSS twenty four CD3-Cl22-VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL 25 5E11(VH1c+VL2b)-Cl22-TCB-LC1(anti-GPRC5D) (P1AE6625) EIVLTQSPGTLSLSPGERATLSCRASQSVSISGINLMNWYQQKPGQQPKLLIYHASILASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTRESPLTFGQGTRLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 26 5E11(VH1c+VL2b)-Cl22-TCB- LC2(anti-CD3) (P1AE6625) EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC 27 5E11(VH1c+VL2b)-Cl22-TCB- HC1(Fc mortar) (P1AE6625) EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVE PKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 28 5E11(VH1c+VL2b)-Cl22-TCB- HC2(Fc pestle) (P1AE6625) EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYAMAWVRQAPGKGLEWVASISTGGVNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATHTGDYFDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVE PKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTK LTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 29 Examples

The following are examples of methods and compositions of the present invention. It should be understood that various other embodiments may be implemented in view of the general description given above.

Materials and methods

All in vivo efficacy and PD experiments were performed in humanized NSG mice bearing subcutaneous multiple myeloma xenograft tumors. Female humanized NSG mice were purchased from Jackson Laboratories and shipped to the animal facility at the Roche Innovation Center Munich 14 to 20 weeks after implantation with human CD34 ⁺ hematopoietic stem cells. After receipt, the animals were housed for one week to acclimate to the new environment and observed. Mice were maintained under specific pathogen-free conditions with a 12 h light/12 h dark day cycle according to agreed guidelines (GV-Solas; Felasa; TierschG). Ongoing health status monitoring was performed regularly. The experimental study protocol has been reviewed and approved by the local authorities (ROB-55.2-2532.Vet_03-16-10 or ROB-55.2-2532.Vet_03-20-170). To evaluate the therapeutic effect on established multiple myeloma tumors, human tumor cell lines were implanted subcutaneously into humanized NSG mice. Tumor cell lines were obtained from different suppliers and stored in the Roche Munich Internal Cell Bank after expansion (Table 1). All tumor cells were cultured at 37°C in a saturated atmosphere of 5% CO ₂ and co-injected with 50 µl of Matrigel into the right flank of anesthetized humanized NSG mice via ventral subcutaneous injection at various cell numbers and > 90% survival (Table 1). When the mean subcutaneous tumor volume reached 200 to 300 mm ³ , humanized mice were randomized into different treatment groups based on tumor volume and body weight. To evaluate the combination of GPRC5D-TCB and meziglutamine, animals were randomly divided into eight different treatment groups when the subcutaneous tumor volume reached a mean volume of 180 mm ³ (Table 3). After randomization, animals were treated with GPRC5D-TCB (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 as disclosed in WO 2021/018859 A1; RO7425781, voritumumab or "forim") as monotherapy or in combination with IMiDs, which are standard of care (SoC) agents for the treatment of multiple myeloma. In addition, the addition of dexamethasone (Dex) to the combination therapy of GPRC5D-TCB and lenalidomide was also studied. The treatment schedule, dose and route of administration for each treatment are summarized in Tables 2 and 3. All treatments were prepared fresh before injection. Animals were monitored daily for clinical control and adverse effects. Animals were terminated based on overt disease (rough fur, hunched back, respiratory problems, motor impairment), weight loss >20%, or tumor size. Tumor growth was monitored twice weekly using caliper measurements. To quantify tumor-infiltrating lymphocytes, GPRC5D-TCB was studied in combination with lenalidomide (Len), pomalidomide, and ibedomide in some experiments. Tumors were harvested from surveilled animals and single-cell suspensions were analyzed by flow cytometry using a FACSFortessa instrument and FlowJo software. To quantify and characterize peripheral immune cells in animals treated with the combination of GPRC5D-TCB and meziglutamine (Mezi), whole blood was collected and processed for flow cytometry, including hemolysis, and flow cytometry was performed using a Cytek Aurora spectrometer and FlowJo software. Cytokines in sera from treated mice were analyzed using the Bio-Plex Multiplex Immunoassay System from BioRad with the Bio-Plex Pro Human Cytokine 27-plex Assay Panel. Statistical analysis was performed using Graph Pad Prism software. To compare the results of immuno-PD, tumor volume, or cytokine levels between the different treatment groups, data were analyzed by one-way ANOVA with correction for multiple comparisons (Tukey test).

Table 1 : Tumor cell lines Cell lines GPRC5D Performance Level original Cell culture medium Cells / animals Survival rate NCI-H929 Culture medium Roche Nutley RPMI 1640 high glucose medium containing 10% FCS, 2 mM L-glutamine, 10 mM HEPES and 1 mM sodium pyruvate 2.5 x10 ⁶ 95% KMS-12BM Ultra Low Chugai (Cello) RPMI 1640; 20% FCS; 2 mM L-glutamine 1.0 x 10 ⁷ 92% OPM-2 high DSMZ RPMI 1640 with 10% FCS, 2 mM L-glutamine 5 x 10 ⁶ 92%

Table 2 : Summary of treatment schedule, dosage, and route of administration. Compound Dosage [mg/kg] Administration route / method Preparation buffer Concentration (mg/mL) GPRC5D-TCB (Voritumizumab) 0.05 (OPM-2); 0.1 (NCI-H929); 1 (RPMI-8226, KMS12-BM) Intravenous, q7d 20 mM histidine, 140 mM NaCl, 0.01% Tween20; pH 6.0 19 mg/ml 0.0005 (NCI-H929; C1D1) 0.002 (NCI-H929; C1D8) 0.04 (NCI-H929; from C1D15); Subcutaneous, q7d Lenalidomide 20 oral 0.5% (w/w) CMC-Na and 1% (w/v) Tween 80 in SWFI 2 mg/ml Pomalidomide 10 Intraperitoneal, 7q7d 5% DMSO, 5% Tween80, 40% PEG300 in physiological saline 1 mg/ml Ibedomide 10 Oral, 7q7d 5% DMSO, 5% Tween80, 40% PEG300 in physiological saline 1 mg/ml Dexamethasone 2 Oral, 7q7d 0.9% NaCl 2 mg/ml Metformin 1 (low); 3 (high) Oral, q7d/3q7d/5q7d 5% DMSO, 5% Tween80, 40% PEG300 in physiological saline 1 mg/ml; 3 mg/ml

Table 3 : Experimental groups used to evaluate the combination of GPRC5D-TCB and meziligamide. Group Number of animals Compound Dosage (mg/kg) Administration route / method 1 A01-A10 Medium - sc,q7d 2 B01-B10 GPRC5D-TCB 0.0005-0.002-0.04 sc,q7d 3 C01-C10 GPRC5D-TCB + Mezigtrol 0.0005-0.002-0.04 + 3 sc,q7d + po,5q7d 4 D01-D10 GPRC5D-TCB + Mezigtrol 0.0005-0.002-0.04 + 3 sc,q7d + po,3q7d 5 E01-E10 GPRC5D-TCB + MetSigmidol 0.0005-0.002-0.04 + 3 sc，q7d + po，q7d 6 F01-F10 GPRC5D-TCB + Mezigtrol 0.0005-0.002-0.04 + 1 sc,q7d + po,5q7d 7 G01-G10 GPRC5D-TCB + Mezigtrol 0.0005-0.002-0.04 + 1 sc,q7d + po,3q7d 8 H01-H10 GPRC5D-TCB + Mezigtrol 0.0005-0.002-0.04 + 1 sc，q7d + po，q7d

result

GPRC5D-TCB and immunomodulatory drugs (IMID) Combination

First-generation IMIDs such as lenalidomide and pomalidomide are approved first-line therapies for patients with multiple myeloma1. When combined with low-dose GPRC5D-TCB therapy against OPM-2 xenografts in humanized mice, lenalidomide was found to exhibit potent synergistic antitumor activity as demonstrated by tumor growth control and statistically significant reductions in tumor burden when compared to monotherapy (Figures ^1A and 1B). In addition, the combination with lenalidomide significantly increased the number of T cells within the tumor when compared to GPRC5D-TCB monotherapy, supporting a synergistic mode of action for the two drugs (Figure 1C). IMIDs are often used in combination with dexamethasone to treat multiple myeloma. Using the poorly responsive multiple myeloma tumor model KMS-12BM, GPRC5D-TCB was combined with lenalidomide and with lenalidomide plus dexamethasone. The combination of lenalidomide and GPRC5D-TCB produced statistically significant tumor growth inhibition in the refractory multiple myeloma xenograft model when compared to the control group (Figures 2A and 2B). Strikingly, when dexamethasone was added to the combination of GPRC5D-TCB and lenalidomide, more robust efficacy was observed, supporting the clinical development of GPRC5D-TCB using this SoC backbone (Figures 2A and 2B). Despite very strong single-agent responses, a high percentage of NCI-H929 xenograft tumors relapsed under GPRC5D-TCB monotherapy (Figures 3A and 5B). When combined with pomalidomide, the frequency of relapse in mice was greatly reduced (Figures 3A and 5C). The improved efficacy was associated with increased cytokine levels detected in mouse serum 48 hours after the first TCB and 24 hours after the first pomalidomide injection, indicating enhanced immune activation with the combination (Figures 3B, 3C, and 3D). Interestingly, the pomalidomide combination did not induce complete tumor eradication, as seen by tumor growth in individual animals when treatment was stopped (Figures 3A and 5C).

Ibedomide belongs to a new class of IMIDs called “cerebellar protein E3 ligase modulators” (CELMoDs) and is currently being evaluated in early clinical development. The study found that ibedomide also prevented relapse of NCI-H929 tumors after GPRC5D-TCB monotherapy (Figure 4A and Figure 5D), and induced a more robust and vigorous T cell response compared to pomalidomide, as evidenced by higher cytokine levels when using the combination (Figure 4B, 4C, and 4D). Ibedomide, but not the pomalidomide combination, induced a complete tumor response in humanized mice, as highlighted by the lack of tumor regrowth after treatment cessation (Figure 5D).

To evaluate the combination of GPRC5D-TCB and meziglutamine in a clinically relevant in vivo setting, humanized mice bearing NCI-H929 tumors were treated weekly (1q7d or q7d) with fixed-duration GPRC5D-TCB using escalating dosing (cycle 1, C1) of 0.0005 mg/kg (escalating dose 1, on day 1; C1D1), 0.002 mg/kg (escalating dose 2, on day 8; C1D8), and 0.04 mg/kg (escalating dose 3, on day 15; C1D15), followed by 5 cycles (C2 to C6) at 0.04 mg/kg. Dosing and treatment-free follow-up for more than 2 weeks were maintained (Figure 6). Metschizophrenia was administered at 3 mg/kg or 1 mg/kg once a week (q7d), three times a week (3q7d), or five times a week (5q7d) 24 h after each GPRC5D-TCB injection. Although GPRC5D-TCB induced transient tumor regression after escalating doses of 1 (C1D1) and 3 (C1D15), mice showed disease progression at the end of cycle 1 (C1), and the progression-free survival (PFS) rate was only 20% at the end of the study (Figure 6B). In contrast, combination with mezigtamine administered at 3q7d and 5q7d resulted in rapid tumor regression during C1, associated with significantly improved PFS rates of 80% (3q7d; Figure 6D) and 100% (5q7d; Figure 6C) at 3 mg/kg and 60% (3q7d; Figure 6G) and 90% (5q7d; Figure 6F) at 1 mg/kg, respectively. Weekly dosing of mexiletamine (1q7d) did not improve the efficacy of GPRC5D-TCB during escalating doses 1 (C1D1) and 2 (C1D8), but achieved a deepening of response after targeted dosing on day 15 of cycle 1, especially at the 3 mg/kg dose (C1D15; Fig. 6H). In association with the lower depth of early response, the 1q7d mexiletamine combination did not improve PFS rates when compared with GPRC5D-TCB monotherapy (Fig. 6E). To investigate the effects of meziglutamine combinations on immune activation, cytokine release was measured in the serum of all mice 48 h after each escalating injection of GPRC5D-TCB at C1D1, C1D8, and C1D15 and 24 h after meziglutamine administration (Figure 7). When meziglutamine was administered at 5q7d or 3q7d, serum levels of IL-10 and IP-10 were comparable to GPRC5D-TCB monotherapy (Figures 7B and 7C), while IL-2 levels slightly increased at all three time points (Figure 7A). Interestingly, a different trend was observed for MIP-1a, as serum levels decreased in relation to the number of meziglutamine administrations (Figure 7D). In contrast to the 5q7d and 3q7d schedules, less frequent mezigtamine dosing (1q7d) induced a robust increase in IL-2, IP-10, and MIP-1a after targeted dosing at C1D15 (Figures 7A, 7B, and 7D). To quantify and phenotype circulating immune cells, we collected blood from all animals at the end of cycle 3 (before C4 dosing) and cycle 5 (before C6 dosing) and performed spectral flow cytometry. When compared to monotherapy, we observed that peripheral CD8a ⁺ (CD8α positive cells) and conventional CD4 ⁺ T cell counts decreased after mezitropium was administered at 3 mg/kg on 3q7d and 1 mg/kg on 5q7d, and increased after 1 mg/kg on 1q7d (peripheral CD8a ⁺ and CD4 ⁺ ) and 1 mg/kg on 3q7d (CD4 ⁺ ) (Figures 8A and 8D). The number of regulatory T cells (Treg) decreased slightly after mezitropium was administered at 3 mg/kg on 3q7d, and increased strongly when 1 mg/kg was administered on 3q7d (Figure 8B). Regardless of the dose level of meziglutamine, the number of peripheral B cells decreased significantly when it was administered at 3q7d and 5q7d (Figure 8C). In contrast, low administration of meziglutamine before C4 administration, but especially before C6 administration, caused a strong increase in not only B cell counts but also NK cell counts (Figures 8C and 8E). We next evaluated whether the combination of GPRC5D-TCB and meziglutamine induces a change in the exhausted state of circulating T lymphocytes. When compared with GPRC5D-TCB monotherapy, we observed a higher frequency of LAG3- and TIGIT-positive CD4 ⁺ and CD8a ⁺ T cells when mice were treated with mezigtamine at 5q7d and 3q7d but not at 1q7d (Figures 9A, 9B, 9C, and 9D), indicating that repeated treatment with mezigtamine induces T cell depletion.

In summary, our data suggest that the combination with mezigtamine significantly improves PFS rates in patients with multiple myeloma. The addition of mezigtamine to GPRC5D-TCB induces profound responses at early time points and overcomes tumor recurrence at later time points. The cytokine data indicate that the combination of escalating dosing of GPRC5D-TCB with mezigtamine does not represent a major risk factor for the development or exacerbation of 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 above invention is described in detail by way of illustrations and examples for the sake of clear understanding, such descriptions and examples should not be interpreted as limiting the scope of the invention. The disclosures of all patents and scientific documents cited herein are expressly incorporated by reference in their entirety.

Figures 1A , 1B , 1C : Results of efficacy/PD experiments evaluating GPRC5D-TCB as a single agent and in combination with lenalidomide. (Figure 1A) Multiple myeloma cell line OPM-2 was injected subcutaneously into stem cell humanized NSG mice to study tumor growth inhibition. GPRC5D-TCB was injected intravenously once a week at a dose of 0.05 mg/kg, and lenalidomide was administered daily by oral gavage at 20 mg/kg, and tumor growth was compared over a period of 18 days. (Figure 1B) Tumor burden was assessed in individual mice at the end of the study (day 18). (Fig. 1C) Five observed tumors in each group were harvested 48 h after the second GPRC5D-TCB injection, and the number of T cells in the tumors 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). Figures 2A , 2B : Results of experiments evaluating the efficacy of GPRC5D-TCB as a single agent and in combination with lenalidomide with or without additional dexamethasone against KMS-12BM multiple myeloma tumors implanted subcutaneously into stem cell humanized NSG mice. (Figure 2A) GPRC5D-TCB was injected intravenously once a week at 1 mg/kg and combined with lenalidomide administered daily by oral gavage at 20 mg/kg with or without additional oral dexamethasone at 2 mg/kg. (Figure 2B) Tumor burden was assessed in individual mice on day 35 (end of study). Statistical analysis, general one-way ANOVA, Tukey test: p = <0.0001 (****); p = 0.0001 to 0.001 (***); p = 0.001 to 0.01 (**); p = 0.01 to 0.05 (*); p = ≥ 0.05 (ns). Figure 3A , 3B , 3C , 3D : Results of experiments evaluating the efficacy of GPRC5D-TCB as a single agent and in combination with pomalidomide against NCI-H929 multiple myeloma tumors implanted subcutaneously into stem cell humanized NSG mice. (Fig. 3A) Growth of NCI-H929 xenograft tumors after weekly intravenous administration of GPRC5D-TCB at 0.1 mg/kg and daily oral gavage administration of pomalidomide at 10 mg/kg. Serum levels of IFN-γ (Fig. 3B), IL-2 (Fig. 3C), and TNF-α (Fig. 3D) were determined in mice 48 h after the first GPRC5D-TCB and 24 h after pomalidomide injection using multiplex techniques. Statistical analysis, ordinary one-way analysis of variance, 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). Figures 4A , 4B , 4C , 4D : Results of experiments evaluating the efficacy of GPRC5D-TCB as a single agent and in combination with ibedomide against NCI-H929 multiple myeloma tumors implanted subcutaneously into stem cell humanized NSG mice. (Figure 4A) Growth of NCI-H929 xenograft tumors following weekly intravenous administration of GPRC5D-TCB at 0.1 mg/kg and daily oral gavage injection of ibedomide at 10 mg/kg. Multiplex techniques were used to determine the levels of IFN-γ (Figure 4B), IL-2 (Figure 4C), and TNF-α (Figure 4D) in the serum of mice 48 hours after the first GPRC5D-TCB and 24 hours after ibedomide injection. 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 (Figure 5A), GPRC5D-TCB monotherapy (Figure 5B), or in combination with pomalidomide (Figure 5C) or ibedomide (Figure 5D), as described in Figures 3 and 4, respectively. Figures 6A , 6B , 6C , 6D , 6E , 6F , 6G , 6H : Results of experiments evaluating the efficacy of GPRC5D-TCB as a single agent and in combination with high or low doses of mezigtamine against NCI-H929 multiple myeloma tumors implanted subcutaneously into stem cell humanized NSG mice. NCI-H929 tumor growth in individual mice following weekly subcutaneous (sc) injections of vehicle (Figure 6A), escalating dosing of GPRC5D-TCB at 0.0005 – 0.002 – 0.04 mg/kg followed by maintenance dosing at 0.04 mg/kg (Figure 6B), and combination of GPRC5D-TCB and meziglutamine at 3 mg/kg for 5 days (5q7d; Figure 6C), 3 days (3q7d; Figure 6D), 1 day (1q7d; Figure 6E) or 1 mg/kg for 5 days (5q7d; Figure 6F), 3 days (3q7d; Figure 6G), 1 day (1q7d; Figure 6H) per week. Figures 7A , 7B , 7C , 7D : Results of cytokine analysis in blood NCI-H929 stem cell-transplanted humanized NSG mice treated with GPRC5D-TCB as a single agent or in combination with mepiquatamide. Cytokines IL-2 (Figure 7A), IP-10 (Figure 7B), IL-10 (Figure 7C), and MIP-1a (Figure 7D) were measured in mouse serum using a multiplex technique 48 hours after GPRC5D-TCB administration on cycle 1 day 1 (C1D1, 0.0005 mg/kg), cycle 1 day 8 (C1D8, 0.002 mg/kg), and cycle 1 day 15 (C1D15, 0.04 mg/kg), and 24 hours after mepiquat administration at 3 mg/kg or 1 mg/kg. Figures 8A , 8B , 8C , 8D , 8E : present the results of quantitative flow cytometric analysis performed in blood NCI-H929 stem cell-transplanted humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with mepiquatamide. CD8a+ T cells (Figure 8A), regulatory T cells (Figure 8B), B cells (Figure 8C), conventional CD4+ cells (Figure 8D), and NK cells (Figure 8E) in the blood of mice were quantified by flow cytometry 164 hours after administration of GPRC5D ^- TCB at 0.04 mg/kg in cycle 3 (= before C4 administration) and cycle 5 (= before C6 administration) and 48h (5q7d), 96h (3q7d), or 144h (1q7d) after administration of mepiquat at 3 mg ^/ kg or 1 mg/kg. Figures 9A , 9B , 9C , 9D : Presents the results of phenotypic flow cytometric analysis of blood NCI-H929 stem cell-transplanted humanized NSG mice treated with GPRC5D-TCB as a single agent and in combination with meziglutamine. Flow cytometric analysis of circulating immune cells in the blood of mice was performed 164 hours after administration of GPRC5D-TCB at 0.04 mg/kg in cycle 3 (= before C4 administration) and cycle 5 (= before C6 administration) and 48h (5q7d), 96h (3q7d) or 144h (1q7d) after administration of meziglutamine at 3 mg/kg or 1 mg/kg. The percentages of CD8a ⁺ T cells expressing TIGIT ( Fig. 9A ) and Lag3 ( Fig. 9B ) and conventional CD4 ⁺ T cells expressing TIGIT ( Fig. 9C ) and Lag3 ( Fig. 9D ) were assessed.

TW202430211A_112138446_SEQL.xml

Claims (26)

A combination of an anti-GPRC5D/anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD) for use as a combination therapy in the treatment of cancer. Use of a combination of an anti-GPRC5D/anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD) in the manufacture of a medicament for the treatment of cancer. A method of treating cancer in an individual comprises administering to the individual a combination of an anti-GPRC5D/anti-CD3 bispecific antibody and an immunomodulatory imide drug (IMiD). A kit comprising a first drug comprising an anti-GPRC5D/anti-CD3 bispecific antibody and a second drug comprising an immunomodulatory imide drug (IMiD), and optionally further comprising a package insert comprising instructions for administering the combination of the first drug and the second drug for treating cancer in an individual. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD as claimed in any of the preceding claims, wherein the anti-GPRC5D/anti-CD3 bispecific antibody comprises (i) a first antigen binding portion that specifically binds to GPRC5D and comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 12, HCDR 2 of SEQ ID NO: 13 and HCDR 3 of SEQ ID NO: 14, and a light chain variable region comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 15, LCDR 2 of SEQ ID NO: 16 and LCDR 3 of SEQ ID NO: 17 (VL); and (ii) a second antigen-binding portion that specifically binds to CD3 and comprises: a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 18, HCDR 2 of SEQ ID NO: 19, and HCDR 3 of SEQ ID NO: 20, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 21, LCDR 2 of SEQ ID NO: 22, and LCDR 3 of SEQ ID NO: 23. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD as claimed in any of the preceding claims, wherein the anti-GPRC5D/anti-CD3 bispecific antibody comprises (i) a first antigen-binding portion that specifically binds to GPRC5D, comprising: a VH that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10; and a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11; and (ii) a second antigen-binding portion that specifically binds to CD3, comprising: a VL that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24. 95%, 96%, 97%, 98%, 99% or 100% identical to a VH; 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. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD of claim 5 or 6, wherein the first antigen-binding portion and/or the second antigen-binding portion of the anti-GPRC5D/anti-CD3 bispecific antibody is a Fab molecule. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD according to any one of claims 5 to 7, wherein the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain, in particular, the variable domains VL and VH are replaced with each other. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD of any one of claims 5 to 8, wherein the first antigen-binding portion is a Fab molecule, wherein in the constant domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1, the amino acid at position 147 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering) and the amino acid at position 213 is independently substituted with glutamine (E) or aspartic acid (D) (according to Kabat EU index numbering). The amino acid at each position was independently substituted by glutamine (E) or aspartic acid (D) (according to the Kabat EU index number). A combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD as claimed in any one of claims 5 to 9, wherein the first antigen binding part and the second antigen binding part are fused to each other via a peptide linker, as the case may be. The combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and IMiD as claimed in any one of claims 5 to 10, wherein the first antigen-binding moiety and the second antigen-binding moiety are each Fab molecules, and wherein (i) the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain, or (ii) the first antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen-binding moiety at the C-terminus of the Fab heavy chain. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD of claim 1 to 11, wherein the anti-GPRC5D/anti-CD3 bispecific antibody comprises a third antigen binding portion. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD of claim 12, wherein the third antigen binding portion is the same as the first antigen binding portion. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD according to claim 1 to 13, wherein the anti-GPRC5D/anti-CD3 bispecific antibody comprises an Fc domain composed of a first unit and a second unit. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD as claimed in claim 5 to 14, wherein the first antigen-binding moiety, the second antigen-binding moiety and the third antigen-binding moiety, if present, are each a Fab molecule; and wherein (i) the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fc domain of the first subunit. domain; and wherein the third antigen binding moiety, if present, is fused to the N-terminus of the second unit of the Fc domain at the C-terminus of the Fab heavy chain. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD of claim 14 or 15, wherein the Fc domain is an IgG Fc domain. A combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD as claimed in any one of claims 14 to 16, wherein the Fc domain is an IgG1 Fc domain. A combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD as claimed in any one of claims 14 to 17, wherein the Fc domain is a human Fc domain. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and IMiD as claimed in any one of claims 14 to 18, wherein the amino acid residues in the CH3 domain of the first unit of the Fc domain are substituted with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first unit, and the protrusion can be placed in the cavity in the CH3 domain of the second unit, and the amino acid residues in the CH3 domain of the second unit of the Fc domain are substituted with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second unit, and the protrusion in the CH3 domain of the first unit can be placed in the cavity. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD as claimed in any one of claims 14 to 19, wherein the Fc domain comprises one or more amino acid substitutions that reduce Fc receptor binding and/or effector function. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD as claimed in any one of claims 1 to 20, wherein the anti-GPRC5D/anti-CD3 bispecific antibody comprises the polypeptide sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD according to any one of claims 1 to 20, wherein the anti-GPRC5D/anti-CD3 bispecific antibody is forimtamig. A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD as claimed in any one of claims 1 to 22, wherein the IMiD is a first generation IMiD or a cereblon E3 ligase modulator (CELMoD). A combination, use, method or kit of an anti-GPRC5D/anti-CD3 bispecific antibody and an IMiD as claimed in any one of claims 1 to 23, wherein the IMiD is selected from the group consisting of lenalidomide, pomalidomide, iberdomide and mezigdomide. A combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD as claimed in any one of claims 1 to 24, wherein the combination further comprises a glycocorticosteroid. The combination, use, method or kit of anti-GPRC5D/anti-CD3 bispecific antibody and IMiD of claim 25, wherein the glycocorticosteroid is dexamethasone.