Antibodies binding to GPRC5D

Abstract

The present invention relates generally to antibodies that bind to GPRC5D, including, for example, bispecific antigen binding molecules for activating T cells. In addition, the invention relates to polynucleotides encoding such antibodies, as well as vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing said antibodies, and to methods of using said antibodies for the treatment of disease.

Description

[0001] This application is a divisional application of the invention application filed on July 28, 2020, with Chinese application number 202080054556.2 and invention title "Antibody that binds to GPRC5D". Technical Field

[0002] This invention generally relates to antibodies that bind to GPRC5D, comprising, for example, bispecific antigen-binding molecules for activating T cells. Furthermore, this invention relates to polynucleotides encoding such antibodies, as well as vectors and host cells containing such polynucleotides. The invention further relates to methods for producing said antibodies, and to methods for using them to treat diseases. Background Technology

[0003] Multiple myeloma (MM) is one of the most common hematologic malignancies, with approximately 75,000 new patients annually in the EU and US, and its medical needs remain unmet. MM is characterized by terminally differentiated plasma cells that secrete non-functional monoclonal immunoglobulins. In the short term, immunomodulatory drugs such as lenalidomide and pomalidomide, and proteasome inhibitors such as carfilzomib or bortezomib, are likely to remain the mainstay of first-line treatment for MM (Moreau, P. and SVRajkumar, 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 the plasma cells in MM. The lack of specific markers for plasma cell surface proteins has hindered the development of antibody or cell therapies for multiple myeloma. To date, there have been only a handful of successful biologics, including daratumumab (anti-CD38) and elotuzumab (anti-CD319), but it's important to note that these molecules are not exclusively expressed by plasma cells. Therefore, RNA sequencing has been used to identify novel targets in multiple myeloma plasma cells, such as G protein-coupled receptor class C5 member D (GPRC5D), which is expressed differently in multiple myeloma plasma cells compared to plasma cells formed from healthy donors. It has been reported that GPRC5D is associated with prognosis and tumor burden in 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 ClinInvest, 2012. 42(9): p. 953-60; and Cohen, Y. et al., GPRC5D is a promising marker for monitoring the tumor load and to target multiple myeloma cells. Hematology, 2013. 18(6): p. 348-51).

[0004] GPRC5D is an orphan receptor with no known ligand, and its biological characteristics, particularly in cancer and in men, are generally unknown. The GPRC5D-encoding gene is located on 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 CG-protein coupled receptor GPRC5D. Biochim Biophys Acta, 2001, 1518(3):p.237-48). The large first exon encodes a seven-transmembrane domain. It has been shown that GPRC5D is 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).

[0005] WO 2018 / 017786 A2 discloses a GPRC5D specific antibody or antigen-binding fragment.

[0006] Given that all standard care treatments fail to cure patients with multiple myeloma, there is a clear need to develop new, effective, and specific therapies. One approach involves antibodies that bind to GPRC5D, particularly bispecific antibodies that bind to both GPRC5D on target cells and activating T-cell antigens (such as CD3 on T cells). When such antibodies bind to both of their targets simultaneously, a T-cell synapse is formed, leading to the activation of (cytotoxic) T cells and subsequent lysis of the target cells.

[0007] This invention provides novel antibodies comprising a bispecific antibody that specifically binds to human GPRC5D. In particular, the T-cell bispecific antibody targeting GPRC5D according to the invention has therapeutic efficacy for multiple myeloma. Summary of the Invention

[0008] The inventors in this case developed a bispecific antigen-binding molecule that combines a novel GPRC5D antibody with GPRC5D and activated T-cell antigens.

[0009] A first aspect of the present invention provides a bispecific antigen-binding molecule comprising:

[0010] (a) A first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding moiety comprises (i) a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:84, and HCDR 3 of SEQ ID NO:86; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; (ii) a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:85, and HCDR 3 of SEQ ID NO:86; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89. (iii) a heavy chain variable region (VH) comprising the heavy chain complement determinant region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complement determinant region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; (iv) a heavy chain variable region (VH) comprising the heavy chain complement determinant region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complement determinant region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO:97; or (v) a heavy chain variable region (VH) comprising the heavy chain complement determinant region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; or (v) a heavy chain variable region (VH) comprising the heavy chain complement determinant region (HCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO:97; The heavy chain complementarity determination region (HCDR) 1 of SEQ ID NO:90, the HCDR 2 of SEQ ID NO:92 and the HCDR 3 of SEQ ID NO:93; and the light chain variable region (VL) comprising the light chain complementarity determination region (LCDR) 1 of SEQ ID NO:94, the LCDR 2 of SEQ ID NO:95 and the LCDR 3 of SEQ ID NO:97;and b) a second antigen-binding region that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding region comprises (i) a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:29, HCDR 2 of SEQ ID NO:30, and HCDR 3 of SEQ ID NO:31; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:32, LCDR 2 of SEQ ID NO:33, and LCDR 3 of SEQ ID NO:34; (i) a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102, and SEQ ID NO:34. (ii) LCDR 3 of SEQ ID NO:103; or (ii) a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:106, HCDR 2 of SEQ ID NO:107, and HCDR 3 of SEQ ID NO:108; and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO:109, LCDR 2 of SEQ ID NO:110, and LCDR 3 of SEQ ID NO:111.

[0011] In another embodiment, (i) the VH of the first antigen-binding portion contains at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to the sequence of SEQ ID NO:13, and the VL of the first antigen-binding portion contains at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to the amino acid sequence of SEQ ID NO:14; or (ii) the VH of the first antigen-binding portion contains at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to the sequence of SEQ ID NO:15, and the VL of the first antigen-binding portion contains at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to the amino acid sequence of SEQ ID NO:16; or (iii) the VH of the first antigen-binding portion contains at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to the sequence of SEQ ID NO:14; The sequence of NO:48 is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:53; or (iv) wherein the VH of the first antigen-binding portion contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:49; and wherein the VL of the first antigen-binding portion contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52; or (v) the VH of the first antigen-binding portion contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:48. The sequence of NO:57 is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:64, and the VL of the first antigen-binding portion contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:64; or (vi) the VH of the first antigen-binding portion contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:58, and wherein the VL of the first antigen-binding portion contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:63.In another embodiment, the VH of the second antigen-binding portion comprises (i) 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:35, and the VL of the second antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:36; (ii) 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:104, and the VL of the second antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:105; or (iii) 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:35; The amino acid sequence of NO:112 is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:113, and the VL of the second antigen-binding portion contains 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:113.

[0012] In one embodiment, the first antigen-binding portion and / or the second antigen-binding portion is a Fab molecule. This means that the first antigen-binding portion can be a Fab molecule, or the second antigen-binding portion can be a Fab molecule, or both the first and second antigen-binding portions can be Fab molecules. 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 Fab heavy chain, particularly the variable domains VL and VH, are substituted for each other. In another embodiment, the first antigen-binding moiety 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 glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing). In another embodiment, the first antigen-binding moiety and the second antigen-binding moiety are fused together, optionally via a peptide linker. In another embodiment, the first antigen-binding portion and the second antigen-binding portion are each Fab molecules, and wherein (i) the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion, or (ii) the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion. In another embodiment, the bispecific antigen-binding molecule includes a third antigen-binding portion. In another embodiment, the third antigen portion is identical to the first antigen-binding portion. In another embodiment, the bispecific antigen-binding molecule includes an Fc domain containing a first subunit and a second subunit. In another embodiment, the first antigen-binding portion, the second antigen-binding portion, and the third antigen-binding portion (in the presence) are each Fab molecules; and wherein (i) the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion, and the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion, and the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; and wherein the third antigen-binding portion (in the presence) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. In another embodiment, the Fc domain is an IgG Fc domain.In another embodiment, the Fc domain is an IgG1 Fc domain. In yet another embodiment, the Fc domain is a human Fc domain. In yet another embodiment, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced by an amino acid residue with a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced by an amino acid residue with a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit, and the protrusion within the CH3 domain of the first subunit can be positioned within the cavity. In another embodiment, the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor and / or effector function.

[0013] Another aspect of the invention provides one or more isolated polynucleotides encoding a bispecific antigen-binding molecule as described herein. Yet another aspect of the invention provides one or more vectors, particularly expression vectors, comprising polynucleotides as described herein. Another aspect of the invention provides a host cell comprising polynucleotides or vectors as described herein.

[0014] Another aspect of the present invention provides a method for producing a bispecific antigen-binding molecule that binds to GPRC5D, the method comprising the steps of: a) culturing host cells as described herein under conditions suitable for expressing the bispecific antigen-binding molecule; and b) optionally, recovering the bispecific antigen-binding molecule.

[0015] In another aspect, the present invention provides a bispecific antigen-binding molecule that binds to GPRC5D, which is produced by the method according to claim 21.

[0016] In another aspect, the present invention provides a pharmaceutical composition comprising a bispecific antigen-binding molecule as described herein and a pharmaceutical carrier.

[0017] In another aspect, the present invention provides bispecific antigen-binding molecules as described herein or pharmaceutical compositions as described herein, which are used as medicines.

[0018] In another aspect, the present invention provides bispecific antigen-binding molecules as described herein or pharmaceutical compositions as described herein for the treatment of diseases.

[0019] In another aspect, the present invention provides bispecific antigen-binding molecules or pharmaceutical compositions as described herein, wherein the disease is cancer or an autoimmune disease.

[0020] In another aspect, the present invention provides a bispecific antigen-binding molecule as described herein or a pharmaceutical composition as described herein, wherein the disease is multiple myeloma.

[0021] In a further aspect, the present invention provides the use of a bispecific antigen-binding molecule as disclosed herein for the manufacture of a medicament for treating diseases.

[0022] On the other hand, the present invention relates to a method for treating a disease, particularly cancer, and more particularly multiple myeloma, in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition comprising a bispecific antigen-binding molecule in pharmaceutical form as described herein. Alternatively, the disease is an autoimmune disease, such as systemic lupus erythematosus and / or rheumatoid arthritis. In any of the above embodiments, the individual is preferably a mammal, particularly a human. Attached Figure Description

[0023] Figure 1 Figures A to Z show exemplary configurations of the bispecific antigen-binding molecule of the present invention. Figure 1 Small picture A Figure 1 D) A diagram of the "1+1CrossMab" molecule. Figure 1 Small picture B Figure 1 The small figure E) is an illustration of the "2+1IgG Crossfab" molecule, showing the sequential substitution ("inverted") of the Crossfab and Fab components. Figure 1 small picture C, Figure 1 (Figure F) Illustration of the “2+1IgG Crossfab” molecule. Figure 1 small image G, Figure 1 The small diagram (K) illustrates the “1+1 IgG Crossfab” molecule, showing the sequential substitution (“inverted”) of the Crossfab and Fab components. Figure 1 small image H, Figure 1 The small diagram (L) illustrates the "1+1 IgG Crossfab" molecule. Figure 1 Small Image I Figure 1 The small diagram (M) illustrates the "2+1IgG Crossfab" molecule, which has two CrossFabs. Figure 1 small picture J, Figure 1 The small diagram (N) illustrates the “2+1IgG Crossfab” molecule, featuring two CrossFabs and a sequence of substitutions (“inverted”) between Crossfab and Fab. Figure 1 small image O, Figure 1The small diagram (S) illustrates the “Fab-Crossfab” molecule. Figure 1 small image P, Figure 1 The small diagram (T) illustrates the "Crossfab-Fab" molecule. Figure 1 small Q, Figure 1 The small diagram shows an illustration of the "(Fab)2-Crossfab" molecule. Figure 1 small image R, Figure 1 The small diagram (V) illustrates the "Crossfab-(Fab)2" molecule. Figure 1 small image W, Figure 1 The small diagram shows an illustration of the "Fab-(Crossfab)2" molecule. Figure 1 small Figure X , Figure 1 The small figure (Z) is an illustration of the "(Crossfab)2-Fab" molecule. Black dots: optional modifications in the Fc domain that promote heterodimerization. ++, --: optional introduction of amino acids with opposite charges in the CH1 and CL domains. Crossfab molecules are described as containing the exchange of VH and VL regions, but may (in embodiments where no charge modifications are introduced in the CH1 and CL domains) alternately contain the exchange of CH1 and CL domains.

[0024] Figure 2 We analyzed the gene expression of tumor targets on plasma cells and B cells using RNAseq.

[0025] Figure 3 Exemplary configuration of the 5E11-bispecific antigen-binding molecule of the present invention. Black dot: optional modification in the Fc domain that promotes heterodimerization. ++, --: optional introduction of amino acids with opposite charges in the CH1 and CL domains.

[0026] Figure 4 Small figures A to C. Bispecific antigen-binding molecule 5F11-TCB ( Figure 4 Small Figure A) and 5E11-TCB ( Figure 4 Small image B) and control antibody ET150-5-TCB ( Figure 4 Figure C) shows the binding analysis of multiple myeloma cell lines expressing GPRC5D, namely AMO-1, L636, NCI-H929, RPMI-8226, OPM-2, and control cell WSU-DLCL2.

[0027] Figure 5 Small figures A to E. GPRC5D-TCB-mediated T-cell cytotoxicity against multiple myeloma cell line AMO-1 ( Figure 5 Small image A), NCI-H929 ( Figure 5 Small image B), RPMI-8226 ( Figure 5 small figure C) and L363 ( Figure 5 Analysis of small figure D). The control cell line was WSU-DL CL2 ( Figure 5 (See small figure E). Molecules tested: 5E11-TCB, 5F11-TCB. Control molecules: DP47-TCB (untargeted) and ET150-5-TCB.

[0028] Figure 6 Analysis of T cell engagement activated by GPRC5D-TCB and upregulation of CD25 and CD69 in multiple myeloma cell line NCI-H929 and negative control cell line WSU-DLCL2.

[0029] Figure 7 Small graphs A to J. In the presence of AMO-1 ( Figure 7 Small image A), NCI-H929 ( Figure 7 Small image B), RPMI-8226 ( Figure 7 Small image C), L363 ( Figure 7 Small image D) and WSU-DLCL2 ( Figure 7 In the case of Figure E), T cell activation was determined by upregulation of CD25 on CD8+ T cells when T cells were incubated with gradually increasing concentrations of GPRC5D-TCB or the negative control DP47-TCB; and in the presence of AMO-1 (… Figure 7 Small image F), NCI-H929 ( Figure 7 Small image G), RPMI-8226 ( Figure 7 small image H), L363 ( Figure 7 Small Figure I) and WSU-DLCL2 ( Figure 7 In the case of small figure J), ​​T cell activation was determined by upregulation of CD69 on CD8+ T cells when T cells were incubated with gradually increasing concentrations of GPRC5D-TCB or negative control DP47-TCB.

[0030] Figure 8 Small figures A to B. Antibody localization and internalization are visualized using fluorescence confocal microscopy. Figure 8 (small figure A) and analysis of signal intensity of cell membrane and cytoplasm ( Figure 8 (Small image B).

[0031] Figure 9The binding of different anti-GPRC5D antibodies to human, canine, and mouse GPRC5D was evaluated by ELISA and by using stable transfected CHO clones expressing human GPRC5D (clone 12), cynomolgus monkey GPRC5D (clone 13), mouse GPRC5D (clone 4), or human GPRC5A (clone 30).

[0032] Figure 10 Figures A through G show the lysis of various multiple myeloma (MM) cell lines mediated by different GPRC5D- or BCMA-targeting T-cell bispecific molecules during 20 hours of co-incubation (E:T = 10:1, human pan-T cells). The figures are duplicates plus SD.

[0033] Figure 11 Figures A through F show T cell activation induced by different GPRC5D-targeting or BCMA-targeting bispecific molecules during approximately 20 hours of co-incubation of allogeneic human pan-T cells from healthy donors and untreated bone marrow cells (E:T = 10:1, human pan-T cells). Figure 11 5E11-TCB in small figure A; Figure 11 5F11-TCB in small figure B; Figure 11 10B10-TCB in small figure C; Figure 11 BCMA-TCB in small diagram D; Figure 11 B72-TCB in small diagram E; Figure 11 (DP47-TCB in small figure F). The plot depicts a FACS dot plot from a representative donor, showing the upregulation of CD69, an activation marker that represents the percentage of positive cells in CD8 T cells for each CD4 T cell (top row) or CD8 T cells (bottom row).

[0034] Figure 12 Figures A through B. T cell activation induced by different GPRC5D-targeting or BCMA-targeting bispecific molecules during approximately 20 hours of co-incubation of allogeneic human pan-T cells from healthy donors and untreated bone marrow cells (E:T = 10:1, human pan-T cells). The figures depict a summary of all four evaluated donors, showing T cell activation induced by different GPRC5D-targeting or BCMA-targeting molecules during the selection of a fixed dose of 50 nM TCB (…). Figure 12 Small Figure A) or 5nM TCB ( Figure 12 At point B in the small image, CD69, an activation marker on CD8 T cells, is upregulated.

[0035] Figure 13 Small figures A to D. Targeting T cells with different GPRC5D bispecific molecules ( Figure 13 5F11-TCB in small figure A; Figure 13 BCMA-TCB in small figure B; Figure 13 B72-TCB in small image C; Figure 13 The in vivo therapeutic effect induced by the carrier (in small figure D) in a humanized NSG mouse model (implanted with NCI-H929 tumor cells) was depicted by changes in tumor growth kinetics over time. The plot is a spider diagram, with each line representing a single mouse.

[0036] Figure 14 Small figures A to D. Targeting T cells with different GPRC5D bispecific molecules ( Figure 14 5F11-TCB in small figure A; Figure 14 5E11-TCB in small figure B; Figure 14 B72-TCB in small image C; Figure 14 The in vivo therapeutic effect induced by the carrier (in small figure D) in a humanized NSG mouse model (implanted with OPM-2 tumor cells) was depicted by changes in tumor growth kinetics over time. The plot is a spider diagram, with each line representing a single mouse.

[0037] Figure 15 The images A through B show incubation of PGLALA-CAR-J for approximately 16 hours after activation, determined by luminescence. The latter is based on GPRC5D IgG ( Figure 15 5F11-IgG in small image A; Figure 15 The small figure B shows the induced binding of 5E11-IgG to the L-363 multiple myeloma cell line expressing GPRC5D and the Jurkat-NFAT reporter cell, which is a genetically engineered TCR expressing a TCR targeting the PGLALA mutation in the Fc region of these IgG molecules. The plot shows the number of repeats plus SD.

[0038] Figure 16 Figures A to D. Humanized TCB molecules bind to parental TCB on human GPRC5D on NCI-H929 cells. Figure 16 (small figures A and 16B) and human CD3 on Jurkat cells ( Figure 16 The cells expressed in small figures C and 16D.

[0039] Figure 17 Figures A to G. Jurkat-NFAT activation assays in the presence of different GPRC5DxCD3 bispecific TCB molecules ( Figure 17 The small figures (A to 17G) are compared with the non-targeted control TCB, as shown.

[0040] Figure 18Figures A through D. Tumor cell lysis assays compare the GPRC5D-TCB molecule presented in this paper with known GPRC5D or BCMA-targeting molecules and non-targeting reference TCB molecules.

[0041] Figure 19 Activation of autologous T cells during incubation of primary MM samples with different CD3-mediated bispecific molecules. The GPRC5D-TCB presented herein is compared with known molecules targeting GPRC5D or BCMA in this art, and with a non-targeting reference TCB molecule.

[0042] Figure 20 Figures A through D show the depletion of B cells during incubation of healthy donor PBMCs with various CD3-involved bispecific molecules. The GPRC5D-TCB presented herein is compared with known molecules targeting GPRC5D or BCMA in this art, and with a non-targeted reference TCB molecule. A concentration of 50 nM was used. Figure 20 Small image A), 5nM ( Figure 20 (small image B), 0.5nM ( Figure 20 The small figure C) and 0.05nM ( Figure 20 The antibody in the small figure (D).

[0043] Figure 21 Figures A through B. T cell activation during incubation of bone marrow samples from healthy donors with different CD3-involved bispecific molecules. GPRC5D-TCB, as presented herein, is compared with molecules known in the art. Activation is achieved by detecting CD69. + CD8 + T cells ( Figure 21 Small image A) and CD69 + CD4 + T cells ( Figure 21 Small image B) in all CD8 + Each CD4 + The percentage of T cells used to determine activation.

[0044] Figure 22 Small figures A to B. Release of cytokines in whole blood of healthy donors ( Figure 22 TNFa readings in small figure A; Figure 22 (IL6 readings in small figure B). Compare GPRC5D-TCB as described in this paper with positive (Gazyva, Lemtrada) and negative (Erbitux) reference molecules.

[0045] Figure 23Inset figures A through G. In vivo efficacy of different GPRC5DxCD3 bispecific TCB molecules in NCI-H929 (hNSG mice), including mean tumor volume during treatment in each treatment group. Figure 23 (small figure A), tumor volume on day 37 () Figure 23 (Small figure B) and tumor growth, targeting molecules, each row represents a single mouse (loador: Figure 23 Small figure C; 6623: Figure 23D; 6624: Figure 23 Small figure E; 6625: Figure 23F; 6626: Figure 23 (Small image G).

[0046] Figure 24 Data on the clearance of SDPK and indicated TCB molecules in vivo in .hFcRn Tg and KO mice.

[0047] Figure 25 Figures A to I. Bispecific antigen-binding molecule 5E11(6625)-TCB and multiple myeloma cell line OPM-2 expressing human GPRC5D ( Figure 25 Small picture A Figure 25 Small picture D, Figure 25 Small image G), NCI-H929 ( Figure 25 Small picture B Figure 25 small image E, Figure 25 Small figure H) and RPMI-8226 ( Figure 25 small picture C, Figure 25 small image F, Figure 25 The binding analysis in small figure I) represents a representative example. The number of GPRC5D antibody binding sites (ABS) for each cell line is given in parentheses and was previously determined by QSC (Quantum Simply Cellular, BangsLabs). The values ​​depicted are relative median fluorescence values ​​(MFI) from triple-replica results plus SD. The EC50 values ​​for binding were calculated using GraphPadPrism and are summarized in Table 14.2. Detailed Implementation

[0048] definition

[0049] Unless otherwise defined below, the terms used herein are those generally used in the art.

[0050] 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 include immunoglobulins and their derivatives (e.g., fragments).

[0051] The term "bispecific" refers to an antigen-binding molecule that can specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each of which is specific to a different antigenic determinant. In some embodiments, the bispecific antigen-binding molecule can bind to two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cell types.

[0052] As used herein, the term "valent" indicates the presence of a specified number of antigen-binding sites in an antigen-binding molecule. Therefore, the term "monovalent binding to an antigen" indicates the presence of one (and no more than one) antigen-binding site in an antigen-binding molecule that is specific to the antigen.

[0053] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding moiety is capable of directing its attached entity (e.g., a second antigen-binding moiety) to a target site, such as to a specific type of tumor cell carrying the antigenic determinant. In another embodiment, the antigen-binding moiety is capable of activating signal transduction via its target antigen (e.g., a T-cell receptor complex antigen). The antigen-binding moiety includes antibodies and fragments thereof as further defined herein. A particular antigen-binding moiety includes an antigen-binding domain of an antibody that comprises a variable region of the antibody heavy chain and a variable region of the antibody light chain. In some embodiments, the antigen-binding moiety may include antibody constant regions 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.

[0054] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule to which the antigen-binding moiety binds, forming an antigen-binding moiety-antigen complex (e.g., a sequential extension of amino acids or a conformational configuration consisting of distinct regions of discontinuous 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 monkeys), and rodents (e.g., mice and rats)) unless otherwise stated. In a particular embodiment, the antigen is a human protein. Where a particular protein is mentioned herein, the term covers "full-length," untreated proteins, and any protein form produced by treatment in cells. This term also covers naturally occurring protein variants, such as splice variants or allelic variants. Exemplary human proteins suitable as antigens are CD3, particularly the ε subunit of CD3 (see UniProt No. P07766 (185th edition), NCBI RefSeq No. NP_000724.1, SEQ ID NO:40 for human sequences; or UniProt No. Q95LI5 (69th edition), NCBI GenBank No. BAB71849.1, SEQ ID NO:41 for cynomolgus monkey sequences) or GPRC5D (see UniProt No. Q9NZD1 (115th edition); NCBI RefSeq No. NP_061124.1, SEQ ID NO:45 for human sequences). In some embodiments, the antibodies or bispecific antigen-binding molecules of the present invention bind to conserved CD3 or GPRC5D epitopes in CD3 or GPRC5D antigens from different species. In a particular embodiment, the antibody or bispecific antigen-binding molecule of the present invention binds to human GPRC5D.

[0055] "Specific binding" means that the binding is selective for the antigen and can distinguish between unwanted or non-specific interactions. The ability of an antigen-binding moiety to bind a specific epitope 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) techniques (e.g., analysis on a BIAcore instrument) (Liljeblad et al., Glyco J17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent to which the antigen-binding moiety binds unrelated proteins is less than about 10% of the antigen bound by the antigen-binding moiety, for example, by SPR determination. In some embodiments, the antigen-binding moiety binding the antigen or the antigen-binding molecule containing the antigen-binding moiety has a molecular weight of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M or smaller, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K) of M) D ).

[0056] "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 stated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., the antigen-binding moiety and the antigen or receptor and its ligand). The affinity of molecule X for its partner Y can generally be expressed as a dissociation constant (K0). D ) indicates that its dissociation rate constant and binding rate constant (k, respectively) represent the ratios of the dissociation rate constant and the binding rate constant. off and k on The ratio of the rate constants is given by the ratio of the rate constants to the rate constants. Therefore, equivalent affinity can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be determined by established methods known in this art, including those described herein. A specific method used to determine affinity is surface plasmon resonance (SPR).

[0057] "Reduced binding," such as reduced binding to Fc receptors, refers to a decrease in the affinity of the respective interactions, as measured by SPR, for example. For clarity, the term also includes reducing the affinity to zero (or below the detection limit of the analytical method), i.e., the interaction is completely eliminated. Conversely, "increased binding" refers to an increase in the binding affinity of the respective interactions.

[0058] As used herein, “activating T cell antigen” refers to an antigenic determinant expressed on the surface of T lymphocytes (particularly cytotoxic T lymphocytes) that can induce T cell activation upon interaction with antigen-binding molecules. Specifically, the interaction between antigen-binding molecules and activating T cell antigen can induce T cell activation by triggering a signaling cascade of the T cell receptor complex. In a particular embodiment, the activating T cell antigen is CD3, particularly the ε subunit of CD3 (see UniProt No. P07766 (144th edition), NCBI RefSeq No. NP_000724.1, SEQ ID NO:40 for human sequences; or UniProt No. Q95LI5 (49th edition), NCBI GenBank No. BAB71849.1, SEQ ID NO:41 for cynomolgus monkey sequences).

[0059] As used herein, “T cell activation” refers to one or more cellular responses of T lymphocytes (particularly cytotoxic T lymphocytes) selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Appropriate assays for measuring T cell activation are known in this art and are described herein.

[0060] As used herein, “target cell antigen” refers to an antigenic determinant present on the surface of a target cell (e.g., cells in a tumor, such as cancer cells or cells in the tumor stroma). In a particular embodiment, the target cell antigen is GPRC5D, specifically according to human GPRC5D of SEQ ID NO:45.

[0061] The terms “first,” “second,” or “third,” used in this article to refer to Fab molecules, etc., are for convenience in distinguishing when more than one of each type of part is present. Unless explicitly stated otherwise, the use of such terms is not intended to assign a specific order or direction to bispecific antigen-binding molecules.

[0062] "Fusion" means that components (such as Fab molecules and Fc domain subunits) are linked directly by peptide bonds or through one or more peptide linkers.

[0063] A “Fab molecule” is 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.

[0064] A "crossover" Fab molecule (also known as a "Crossfab") refers to a Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., replaced by each other). Specifically, a crossover Fab molecule comprises a peptide chain consisting of the light chain variable domain VL and the heavy chain constant domain 1CH1 (VL-CH1, in the N-terminal to C-terminal direction) and a peptide chain consisting of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in the N-terminal to C-terminal direction). For clarity, in a crossover Fab molecule where the variable domains of the Fab light and Fab heavy chains are exchanged, the peptide chain containing the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule where the constant domains of the Fab light and Fab heavy chains are exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0065] In contrast, “conventional” Fab molecules refer to Fab molecules in their natural form (i.e., Fab molecules containing heavy chains (VH-CH1, in the N-terminal to C-terminal direction) consisting of heavy chain variable and constant structural domains and light chains (VL-CL, in the N-terminal to C-terminal direction) consisting of light chain variable and constant structural domains).

[0066] The term "immunoglobulin molecule" refers to a protein with a structure that resembles naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has a variable domain (VH), also called the heavy chain variable domain or heavy chain variable region, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant region. Similarly, each light chain has a variable domain (VL), also called the light chain variable domain or light chain variable region, from the N-terminus to the C-terminus, followed by the light chain constant (CL) domain, also called the 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 subdivided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Based on the amino acid sequence of their constant domains, the light chains of immunoglobulins can be classified into one of two types, called kappa (κ) and lamuda (λ). Immunoglobulins are essentially composed of two Fab molecules linked by an immunoglobulin hinge region and an Fc domain.

[0067] The term "antibody" as used in the broadest sense and encompasses a wide range of 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.

[0068] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homologous population of antibodies, meaning that the individual antibodies in the population are identical and / or bind to the same epitope, but does not include, for example, antibodies containing naturally occurring mutations or possible variants generated during the production of a monoclonal antibody formulation, such variants typically present in small quantities. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies intended for use according to the invention can be manufactured using a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0069] "Isolated" antibodies are antibodies separated from their components in their natural environment, i.e., antibodies isolated outside their natural environment. Specific purification levels are not required. For example, isolated antibodies can be removed from their natural or natural environment. For the purposes of this invention, recombinant antibodies expressed in host cells are considered isolated, meaning they are natural or recombinant antibodies that have been isolated, fractionated, or partially or substantially purified by any suitable technique. Therefore, the antibodies and bispecific antigen-binding molecules of this invention are isolated. In some embodiments, antibodies are purified to a purity greater than 95% or 99%, 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, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0070] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably in this document and refer to antibodies with a structure substantially similar to that of natural antibodies.

[0071] "Antibody fragment" refers to a molecule other than the intact antibody, which contains a portion of the intact antibody that binds to the antigen bound by the intact antibody. 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 some antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046. A bifunctional antibody is an antibody fragment having two antigen-binding sites (which may be bivalent or bispecific). See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9,129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90,6444-6448 (1993). Trifunctional and tetrafunctional antibodies are also described in Hudson et al., Nat Med 9,129-134 (2003). A single-domain antibody is an antibody fragment containing all or part of the variable domain of the antibody's heavy chain or all or part of the variable domain of the antibody's light chain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516B1). Antibody fragments can be manufactured using a variety of techniques, including but not limited to the proteolytic digestion of intact antibodies as described herein and the generation of recombinant host cells (e.g., E. coli or bacteriophages).

[0072] The term "antigen-binding domain" refers to a portion of an antibody that contains part or all of a region that specifically binds to and is complementary to an antigen. Antigen-binding domains can be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Specifically, antigen-binding domains include antibody light chain variable domains (VL) and antibody heavy chain variable domains (VH).

[0073] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WHFreeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The "Kabat numbering" used in this paper, referring to the 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).

[0074] The amino acid positions of all constant regions and domains of the heavy and light chains used herein are based on the Kabat numbering system (referred to herein as “according to Kabat numbering” or “Kabat numbering”) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). Specifically, the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647–660) is used for the constant domain CL of the light chain of the κ and λ isoforms, and the Kabat EU index numbering system (see pp. 661–723) is used for the constant domains (CH1, hinge, CH2, and CH3) of the heavy chain, which is further clarified herein by reference to “according to Kabat EU indexing”.

[0075] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in the sequence ("complementarity-determining region" or "CDR"; CDRs of heavy chain variable regions / domains are abbreviated as, for example, HCDR1, HCDR2, and HCDR3; CDRs of light chain variable regions / domains are abbreviated as, for example, LCDR1, LCDR2, and LCDR3) and / or forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact"). Generally, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In this document, exemplary HVRs include:

[0076] (a) The hypervariable ring is present 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));

[0077] (b) CDRs are present 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 edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0078] (c) The 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

[0079] (d) Combinations of (a), (b) and / or (c), including HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3) and 94-102(H3).

[0080] Unless otherwise stated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered in this paper according to Kabat et al. (as stated above).

[0081] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0082] A “humanized” antibody is a chimeric antibody comprising amino acid residues from a nonhuman HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody will include substantially all at least one (and typically two) variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to those of the nonhuman antibody, and all or substantially all FRs correspond to those of the human antibody. Such 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 nonhuman antibody (e.g., an antibody derived from HVR residues) to, for example, restore or improve antibody specificity or affinity. A “humanized form” of an antibody (e.g., a nonhuman antibody) refers to an antibody that has undergone humanization. Other forms of "humanized antibody" covered by this invention are in which the constant region has been further modified or altered from the original antibody form to produce the properties according to the invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0083] "Human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source utilizing an antibody repertoire or other human antibody encoding sequence. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. In some embodiments, human antibodies are derived from non-human transgenic mammals, such as mice, rats, or rabbits. In some embodiments, human antibodies are derived from hybridoma cell lines. Antibodies or antibody fragments isolated from a human antibody library are also considered human antibodies or human antibody fragments herein.

[0084] The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0085] The term "Fc domain" or "Fc region" used herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of the IgG heavy chain may vary slightly, the Fc region of the human IgG heavy chain is generally defined as extending from Cys226 or Pro230 to the C-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. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain may include the full-length heavy chain, or may include cleaved variants of the full-length heavy chain (also referred to herein as "cleaved variant heavy chains"). The last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index number). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. Unless otherwise stated, the amino acid sequence of the heavy chain comprising the Fc domain (or a subunit of the Fc domain as defined herein) represents a C-terminal glycine-lysine dipeptide. In one embodiment of the invention, the heavy chain (comprising a subunit of the Fc domain as specified herein) contained in the antibody or bispecific antigen-binding molecule according to the invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, according to Kabat EU index numbers). In one embodiment of the invention, the heavy chain (comprising a subunit of the Fc domain as specified herein) contained in the antibody or bispecific antigen-binding molecule according to the invention comprises an additional C-terminal glycine residue (G446, according to Kabat EU index number). The compositions of the invention, pharmaceutical compositions as described herein, comprise a population of antibody or bispecific antigen-binding molecules of the invention. The population of antibody or bispecific antigen-binding molecules may comprise molecules having a full-length heavy chain and molecules having cleaved variable heavy chains. A population of antibodies or bispecific antigen-binding molecules may consist of a mixture of molecules having a full-length heavy chain and molecules having a cleaved variable heavy chain, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or bispecific antigen-binding molecules have a cleaved variable heavy chain. In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antigen-binding molecules of the invention comprises an antibody or bispecific antigen-binding molecule having a heavy chain having a subunit of the Fc domain as specified herein and additional C-terminal glycine-lysine dipeptides (G446 and K447, according to Kabat EU index numbers).In one embodiment of the invention, a composition comprising a population of antibodies or bispecific antigen-binding molecules of the invention comprises an antibody or bispecific antigen-binding molecule comprising a heavy chain having a subunit of the Fc domain as specified herein and an additional C-terminal glycine residue (G446, according to Kabat EU index number). In one embodiment of the invention, such compositions comprise a population of antibodies or bispecific antigen-binding molecules consisting of: molecules comprising a heavy chain containing a subunit of the Fc domain as specified herein; molecules comprising a heavy chain containing a subunit of the Fc domain as specified herein and an additional C-terminal glycine residue (G446, according to Kabat EU index number); and molecules comprising a heavy chain containing a subunit of the Fc domain as specified herein and an additional C-terminal glycine-lysine dipeptide (G446 and K447, according to Kabat EU index number). Unless otherwise stated herein, amino acid residues in the Fc region or constant region are numbered 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 above). As used herein, a “subunit” of the Fc domain refers to one of the two polypeptides that form the dimer Fc domain, i.e., a polypeptide containing the C-terminal constant region of a self-associating immunoglobulin heavy chain. For example, the subunit of the IgG Fc domain contains the IgG CH2 and IgG CH3 constant domains.

[0086] "Modifications that promote association of the first and second subunits of the Fc domain" are operations on the peptide backbone or post-translational modifications to the Fc domain subunits that reduce or prevent the association of peptides containing Fc domain subunits with the same peptide to form homodimers. The association-promoting modifications used herein specifically include individual modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) to which association is desired, wherein the modifications are complementary to each other to promote association of the two Fc domain subunits. For example, association-promoting modifications may alter the structure or charge of one or both Fc domain subunits to make them sterically or electrostatically advantageous, respectively. Thus, (hybrid)dimerization occurs between the peptide containing the first Fc domain subunit and the peptide containing the second Fc domain subunit, which may differ in terms of the components further fused to each subunit (e.g., the antigen-binding moiety). In some embodiments, association-promoting modifications include amino acid mutations in the Fc domain, particularly amino acid substitutions. In one particular embodiment, the association-promoting modifications include individual amino acid mutations, particularly amino acid substitutions, in each of the two subunits of the Fc domain.

[0087] The term "effective function" refers to those biological activities attributed to the Fc region of an antibody, which vary across antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent 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.

[0088] As used herein, the term "engineering" is considered to include any manipulation of the peptide backbone or post-translational modifications of naturally occurring or recombinant peptides or fragments thereof. Engineering includes modifications to the amino acid sequence, glycosylation patterns, or side chain groups of individual amino acids, as well as combinations of these methods.

[0089] As used herein, the term "amino acid mutation" refers to amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as reduced binding to the Fc receptor or increased association with another peptide. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of amino acids. Specific amino acid mutations are amino acid substitutions. Non-conserved amino acid substitutions, i.e., replacing one amino acid with another amino acid having a different structure and / or chemical properties, are particularly preferred to alter, for example, the binding characteristics of the Fc region. Amino acid substitutions include substitution with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods involving alteration of the side chain groups of amino acids by means other than genetic engineering, such as chemical modification, may also be useful. Various names may be used herein to refer to the same amino acid mutation. For example, the proline at position 329 of the Fc domain is replaced with glycine, which can be represented as 329G, G329, G 329 P329G or Pro329Gly.

[0090] The "percentage (%) amino acid residue identity" relative to the reference polypeptide sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence. This percentage represents the maximum possible sequence identity achieved after sequence alignment and introducing differences (if necessary), without considering any conserved substitutions as part of the sequence identity. Alignments performed to determine the percentage amino acid sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine suitable parameters for sequence alignment, including any algorithm required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this document, the % amino acid sequence identity value is generated using the ggsearch program of FASTA package version 36.3.8c or later and the BLOSUM50 comparison matrix. The FASTA package was developed by WRPearson and DJ Lipman (1988) (“Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448); WRPearson (1996) (“Effective protein sequence comparison” Meth. Enzymol. 266:227-258); and Pearson et al. (1997) (Genomics 46:24-36), and is publicly available at http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml. Alternatively, sequences can be compared using the ggsearch(global protein:protein) program with default options (BLOSUM50; open:-10; ext:-2; Ktup=2) on a public server accessible via http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi to ensure global alignment rather than local alignment. The percentage of amino acid identity is provided in the output comparison header.

[0091] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA). Polynucleotides can contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, as seen in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid fragments, such as DNA or RNA fragments, present in a polynucleotide.

[0092] "Isolated" nucleic acid molecules or polynucleotides refer to nucleic acid molecules (DNA or RNA) that have been isolated from their natural environment. For example, for the purposes of this invention, recombinant polynucleotides encoding polypeptides contained in vectors are considered to be isolated. Further examples of isolated polynucleotides include recombinant polynucleotides held in heterologous host cells or purified (partial or substantially) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that typically contain polynucleotide molecules, but which are located extrachromosomally or at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this invention, as well as positive-stranded and negative-stranded forms and double-stranded forms. Isolated polynucleotides or nucleic acids according to the invention further include such molecules synthesized. Furthermore, polynucleotides or nucleic acids may be or may include regulatory components such as promoters, ribosome binding sites, or transcription terminators.

[0093] "Separated polynucleotide (or nucleic acid) encoding [e.g., the antibody or dual antibody antigen-binding molecule of the present invention]" refers to one or more polynucleotide molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such polynucleotide molecules in a single vector or a single antibody, and such nucleic acid molecules being present at one or more locations in the host cell.

[0094] The term "expression cassette" refers to a recombinant or synthetically produced polynucleotide having a series of specific nucleic acid components that allow specific nucleic acids to be transcribed in target cells. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, chloroplast DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassette contains a polynucleotide sequence encoding an antibody or bispecific antigen-binding molecule of the present invention, or a fragment thereof.

[0095] The term "vector" or "expression vector" refers to a DNA molecule used to introduce and direct the expression of a specific gene operatively linked to it in a cell. This term includes vectors as self-replicating nucleic acid structures as well as vectors incorporated into the genome of a host cell. The expression vector of the present invention comprises an expression cassette. The expression vector transcribes a large amount of stable mRNA. Once the expression vector enters the cell, the ribonucleic acid molecule or protein encoded by the gene is produced through cellular transcription and / or translation mechanisms. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence encoding an antibody or bispecific antigen-binding molecule of the present invention or a fragment thereof.

[0096] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including progeny cells of such cells. Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny cells derived therefrom, regardless of passage number. The nucleic acid content of progeny cells may not be exactly the same as that of parent cells, but may contain mutations. This document includes mutant progeny that have the same function or biological activity as the original transformed cells, screened or selected. Host cells are any type of cell system that can be used to produce the antibodies or bispecific antigen-binding molecules of the present invention. Host cells include cultured cells, such as cultured mammalian cells, such as HEK cells, CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, etc., and also include cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0097] "Activated Fc receptors" are Fc receptors that, upon the involvement of the Fc domain of an antibody, trigger signal transduction events that stimulate the receptor to carry out cellular effector functions. Human activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0098] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells containing an Fc region of the antibody or a derivative thereof, which typically binds specifically to the Fc region via a protein portion as its N-terminus. As used herein, the term "reduced ADCC" refers to a reduction in the number of target cells lysed within a given time period by the ADCC mechanism as defined above, at a given concentration of antibody in the culture medium surrounding the target cells, 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 within a given time period by the ADCC mechanism. A reduction in ADCC is relative to ADCC mediated by the same antibody (but not engineered) produced from the same type of host cells using the same standard production, purification, formulation, and storage methods (methods known to those skilled in the art). For example, a reduction in ADCC mediated by an antibody containing an amino acid substitution in the Fc domain that reduces ADCC is relative to ADCC mediated by the same antibody that does not contain this amino acid substitution in the Fc domain. Suitable methods for measuring ADCC are well known in the art (see, for example, PCT Publication No. WO 2006 / 082515 or PCT Publication No. WO 2012 / 130831).

[0099] The "effective amount" of a drug refers to the amount required to induce physiological changes in the cells or tissues to which it is applied.

[0100] The "therapeutic effective amount" of a pharmaceutical agent, such as a pharmaceutical composition, refers to the amount that effectively achieves the desired therapeutic or preventative effect within the required dosage and time period. For example, a therapeutically effective amount of a pharmaceutical agent can eliminate, reduce, delay, minimize, or prevent adverse reactions to a disease.

[0101] "Individual" or "subject" refers to 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). In particular, the individual or subject is a human.

[0102] The term "pharmaceutical composition" refers to a formulation in which the bioactivity of the active ingredient contained herein is permitted to be effective and which contains no other components that would have unacceptable toxicity to the subject to whom the composition will be administered.

[0103] "Pharmaceutical carrier" refers to any component in a pharmaceutical composition other than the active ingredient, which is non-toxic to the subject. Pharmaceutical carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0104] As used herein, “treatment” (and its grammatical variations, such as “treatment process” or “under treatment”) refers to a clinical intervention that attempts to alter the natural course of disease in an individual being treated, and may be preventative or performed during clinicopathological proceedings. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some embodiments, the antibodies or bispecific antigen-binding molecules of the present invention are used to delay the development of disease or slow its progression.

[0105] The term "packaging insert" is used to refer to instructions typically included in the commercial packaging of therapeutic products, which contain information about the indications, usage, dosage, route of administration, combination therapy, contraindications, and / or warnings for the use of such therapeutic products. Detailed Implementation

[0107] This invention provides antibodies and bispecific antigen-binding molecules that bind to GPRC5D, particularly human GPRC5D. Furthermore, the molecule possesses other advantageous properties for therapeutic applications, such as efficacy and / or safety, and manufacturability.

[0108] GPRC5D antibody

[0109] A first aspect of the present invention provides an antibody that binds to GPRC5D, the antibody comprising: (i) a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:84, and HCDR 3 of SEQ ID NO:86; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; (ii) the heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:85, and HCDR 3 of SEQ ID NO:86; and the light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89. 3; (iii) a heavy chain variable region (VH) comprising the heavy chain complement determinant region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complement determinant region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; (iv) a heavy chain variable region (VH) comprising the heavy chain complement determinant region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complement determinant region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO:97; or (v) a heavy chain variable region (VH) comprising the heavy chain complement determinant region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and LCDR 3 of SEQ ID NO:97; or (v) a heavy chain variable region (VH) comprising the heavy chain complement determinant region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; The heavy chain complementarity determination region (HCDR) 1 of SEQ ID NO:90, the HCDR 2 of SEQ ID NO:92 and the HCDR 3 of SEQ ID NO:93; and the light chain variable region (VL) comprising the light chain complementarity determination region (LCDR) 1 of SEQ ID NO:94, the LCDR 2 of SEQ ID NO:95 and the LCDR 3 of SEQ ID NO:97.

[0110] In some embodiments, the antibody is a humanized antibody. In one embodiment, VH is a humanized VH and / or VL is a humanized VL. In one embodiment, the antibody comprises a CDR as described in any of the above embodiments, and further comprises a human receptor framework, such as a human immunoglobulin framework or a human common framework.

[0111] In one particular embodiment, (i) VH contains at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to the sequence of SEQ ID NO: 13, and VL contains at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to the amino acid sequence of SEQ ID NO: 14; or (ii) VH contains at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to the sequence of SEQ ID NO: 15, and VL contains at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to the amino acid sequence of SEQ ID NO: 16; or (iii) VH contains at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to the sequence of SEQ ID NO: 48, and VL contains at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to the sequence of SEQ ID NO: 13. The amino acid sequence of NO:53 is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:53; or (iv) VH contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:49, and VL contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52; or (v) VH contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:57, and VL contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:64; or (vi) VH contains at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:64; The sequence of NO:58 is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:63, and VL contains 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:63.

[0112] In one particular embodiment, the antibody comprises: (i) VH, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:13; and VL, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:14; or (ii) VH, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:15; and VL, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:16; or (iii) VH, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:48; and VL, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:13 ... The amino acid sequence of NO:53 is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical; or (iv) VH, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:49; and VL, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:52; or (v) VH, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:57; and VL, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:64; or (vi) VH, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the ...57; and VL, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:64; or (vi) VH, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:64; and VL, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:64; and VL, which is at least about 95%, 9 The sequence of NO:58 is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical; and VL is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:63.

[0113] In another embodiment, the antibody is IgG, particularly IgG1 antibody. In one embodiment, the antibody is a full-length antibody. In another embodiment, the antibody is an antibody fragment selected from Fv molecules, scFv molecules, Fab molecules, and F(ab')2 molecules. In one embodiment, the antibody is a multispecific antibody.

[0114] In some embodiments, the VH or VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the antibody containing the sequence retains its ability to bind to GPRC5D. In some embodiments, SEQ ID NO:13 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:14 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:15 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:16 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:48 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:53 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:49 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:52 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:57 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:16 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:17 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:18 contains 1 to 10 amino acids that are substituted, inserted, and / or deleted; and / or SEQ ID NO:19 ... In NO:64, 1 to 10 amino acids are substituted, inserted, and / or deleted; and / or in SEQ ID NO:58, 1 to 10 amino acids are substituted, inserted, and / or deleted; and / or in SEQ ID NO:63, 1 to 10 amino acids are substituted, inserted, and / or deleted.

[0115] In some embodiments, substitution, insertion, or deletion occurs in regions outside the HVR (i.e., in the FR). Optionally, the antibody comprises the VH sequence of SEQ ID NO:13 and / or the VL sequence of SEQ ID NO:14, including post-translational modifications of the sequences. Optionally, the antibody comprises the VH sequence of SEQ ID NO:15 and / or the VL sequence of SEQ ID NO:16, including post-translational modifications of the sequences. Optionally, the antibody comprises the VH sequence of SEQ ID NO:448 and / or the VL sequence of SEQ ID NO:53, including post-translational modifications of the sequences. Optionally, the antibody comprises the VH sequence of SEQ ID NO:49 and / or the VL sequence of SEQ ID NO:52, including post-translational modifications of the sequences. Optionally, the antibody comprises the VH sequence of SEQ ID NO:57 and / or the VL sequence of SEQ ID NO:64, including post-translational modifications of the sequences. Optionally, the antibody comprises the VH sequence in SEQ ID NO:58 and / or the VL sequence in SEQ ID NO:63, which includes post-translational modifications of the sequence.

[0116] In one embodiment, the antibody comprises: VH, which comprises an amino acid sequence selected from SEQ ID NO:13 and SEQ ID NO:15; and VL, which comprises an amino acid sequence selected from SEQ ID NO:14.

[0117] In one embodiment, the antibody comprises a VH sequence selected from the group consisting of SEQ ID NO:13 and SEQ ID NO:12 and a VL sequence selected from SEQ ID NO:16.

[0118] In one particular embodiment, the antibody comprises: VH, which comprises the amino acid sequence of SEQ ID NO:13; and VL, which comprises the amino acid sequence of SEQ ID NO:14. In another particular embodiment, the antibody comprises the VH sequence of SEQ ID NO:13 and the VL sequence of SEQ ID NO:14.

[0119] In one particular embodiment, the antibody comprises: VH, which comprises the amino acid sequence of SEQ ID NO:15; and VL, which comprises the amino acid sequence of SEQ ID NO:16. In another particular embodiment, the antibody comprises the VH sequence of SEQ ID NO:15 and the VL sequence of SEQ ID NO:16.

[0120] In one particular embodiment, the antibody comprises: VH, which comprises the amino acid sequence of SEQ ID NO:48; and VL, which comprises the amino acid sequence of SEQ ID NO:53. In another particular embodiment, the antibody comprises the VH sequence of SEQ ID NO:48 and the VL sequence of SEQ ID NO:53.

[0121] In one particular embodiment, the antibody comprises: VH, which comprises the amino acid sequence of SEQ ID NO:49; and VL, which comprises the amino acid sequence of SEQ ID NO:52. In another particular embodiment, the antibody comprises the VH sequence of SEQ ID NO:49 and the VL sequence of SEQ ID NO:52.

[0122] In one particular embodiment, the antibody comprises: VH, which comprises the amino acid sequence of SEQ ID NO:57; and VL, which comprises the amino acid sequence of SEQ ID NO:64. In another particular embodiment, the antibody comprises the VH sequence of SEQ ID NO:57 and the VL sequence of SEQ ID NO:64.

[0123] In one particular embodiment, the antibody comprises: VH, which comprises the amino acid sequence of SEQ ID NO:58; and VL, which comprises the amino acid sequence of SEQ ID NO:63. In another particular embodiment, the antibody comprises the VH sequence of SEQ ID NO:58 and the VL sequence of SEQ ID NO:63.

[0124] In one embodiment, the antibody comprises a human constant region. In one embodiment, the antibody is an immunoglobulin molecule comprising a human constant region, particularly an IgG-type immunoglobulin molecule comprising human CH1, CH2, CH3, and / or CL domains. Exemplary sequences of the human constant domains are given in SEQ ID NO 37 and 38 (human κ and λCL domains, respectively) and SEQ ID NO:39 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In some embodiments, the antibody comprises a light chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:39, particularly the amino acid sequence of SEQ ID NO:38. In some embodiments, the antibody 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 amino acid sequence of SEQ ID NO:39. In particular, as described in this paper, the heavy chain constant region can contain amino acid mutations in the Fc domain.

[0125] In one embodiment, the antibody is a monoclonal antibody.

[0126] In one embodiment, the antibody is IgG, particularly IgG1 antibody. In another embodiment, the antibody is a full-length antibody.

[0127] In one embodiment, the antibody comprises an Fc domain, particularly an IgG Fc domain, and more particularly an IgG1 Fc domain. In another embodiment, the Fc domain is a human Fc domain. The Fc domain of the antibody may, alone or in combination, incorporate any of the features described herein with respect to the Fc domain of the bispecific antigen-binding molecule of the present invention.

[0128] In another embodiment, the antibody is an antibody fragment selected from Fv molecules, scFv molecules, Fab molecules, and F(ab')2 molecules, particularly Fab molecules. In another embodiment, the antibody fragment is a bifunctional antibody, a trifunctional antibody, or a tetrafunctional antibody.

[0129] On the other hand, as described in the following sections, the antibodies according to any of the above embodiments can bind any feature alone or in combination.

[0130] Glycosylation variants

[0131] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites in an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0132] When an antibody contains an Fc region, the oligosaccharide linked to it can be modified. Naturally occurring antibodies produced by mammalian cells typically contain branched, biantennary-type oligosaccharides, which are usually linked to Asn297 of the CH2 domain of the Fc region via an N-bond. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the “stem” of the biantennary-type oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with certain improved properties.

[0133] In one embodiment, an antibody variant is provided having a non-fucosylated oligosaccharide, i.e., an oligosaccharide structure lacking (directly or indirectly) fucose linked to the Fc region. Such non-fucosylated oligosaccharides (also known as “defucosylated” oligosaccharides) are specifically N-linked oligosaccharides lacking the fucose residue linked to the first GlcNAc in the stem of the dual-antenna oligosaccharide structure. In one embodiment, an antibody variant is provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to the natural or parental antibody. For example, the proportion of non-fucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucose-linked oligosaccharides present). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues relative to the sum of all oligosaccharides linked to Asn 297 (e.g., complex, hybrid, and high-mannose structures), determined by MALDI-TOF mass spectrometry, as described, for example, in WO 2006 / 082515. Asn 297 refers to the asparagine residue (EU number of Fc region residues) located near position 297 in the Fc region; however, Asn 297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300 due to minor sequence variations in the antibody. Antibodies with such an increased proportion of non-fucosylated oligosaccharides in the Fc region may exhibit improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, US2003 / 0157108; US2004 / 0093621.

[0134] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 0157108; and WO 2004 / 056312, especially in Example 11); and knockout cell lines, such as CHO cells with the α-1,6-fucosyltransferase gene FUT8 knocked out (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107); or cells with reduced or absent GDP-fucosyl synthesis or transporter activity (see, for example, US2004259150, US2005031613, US2004132140, US2004110282).

[0135] In another embodiment, the antibody variant is provided with bipartite oligosaccharides, for example, wherein a dual-antenna oligosaccharide linked to the Fc region of the antibody is bipartitely divided by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function as described above. Examples of such antibody variants are described, for example: Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO2004 / 065540, WO 2003 / 011878.

[0136] Antibody variants having at least one galactose residue linked to the Fc region on the oligosaccharide are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964 and WO1999 / 22764.

[0137] Cysteine-engineered antibody variants

[0138] In some embodiments, it may be desirable to create cysteine-engineered antibodies, such as "thioMAb," in which one or more residues of the antibody are replaced by cysteine ​​residues. In specific embodiments, the substituted residues appear at accessible sites on the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites on the antibody and can be used to conjugate the antibody to other parts (e.g., pharmaceutical parts or linker-pharmaceutical parts) to form immunoconjugates, as further described herein. Cysteine-engineered antibodies can be produced as described, for example, U.S. Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO2016040856.

[0139] Antibody derivatives

[0140] In some embodiments, the antibodies provided herein may be further modified to include additional non-protein portions known and readily available in the art. Suitable portions for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers linked to the antibody may vary, and if more than one polymer is linked, they may be the same or different molecules. Typically, the quantity and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under specified conditions, and so on.

[0141] In another embodiment, a conjugate of the antibody and non-protein fraction is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein fraction is carbon nanotubes (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells but heat the non-protein fraction to near the temperature at which the antibody-non-protein fraction kills cells.

[0142] Immunoconjugates

[0143] The present invention also provides immunoconjugates comprising the anti-GPRC5D antibody as described herein, which are conjugated (chemically bonded) to one or more therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., protein toxins, enzyme-active toxins or fragments thereof derived from bacteria, fungi, plants or animals) or radioisotopes.

[0144] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more of the aforementioned therapeutic agents. Typically, a linker is used to attach the antibody to one or more therapeutic agents. An overview of ADC technology (including examples of therapeutic agents and drugs and linkers) is presented in Pharmacol Review 68:3-19 (2016).

[0145] In another embodiment, the immunoconjugate comprises an antibody conjugated to an enzyme-active toxin or a fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modizoxin A chain, α-Arctica, tung oil protein, carnation toxin, American pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, jatropha toxin, croton toxin, soapwort inhibitor, white tree toxin, mitochondritis, localized aspergillin, phenolmycin, enoxamin, and trichothecene toxins.

[0146] In another embodiment, the immunoconjugate comprises an antibody conjugated to a radioactive atom to form a radioactive conjugate, as described herein. In another embodiment, various radioisotopes can be used to generate the radioactive conjugate. Examples include At... 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi212, P 32 Pb 212 Radioactive isotopes of Lu. When radioactive conjugates are used for detection, they may contain radioactive atoms used for scintillation studies, such as tc99m or I123, or spin-labeled elements used for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0147] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein conjugates, such as N-succinimide-3-(2-pyridyl dithio)propionate (SPDP), succinimide-4-(N-maleimide methyl)cyclohexane-1-carboxylate (SMCC), iminothiones (IT), bifunctional derivatives of imino esters (e.g., dimethyl adipate hydrochloride), active esters (e.g., bissuccinimide octanoic acid), aldehydes (e.g., glutaraldehyde), diazid compounds (e.g., bis(p-azidobenzoyl)hexamethylenediamine), diazide derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and di-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared according to the method described by Vitetta et al. (Science 238:1098 (1987)). An exemplary chelating agent for conjugating radioactive nucleotides to antibodies is carbon-14 labeled 1-isothiocyanate benzoyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA). See WO94 / 11026. The adapter can be a “cleavable adapter” that promotes the release of cytotoxic drugs from cells. For example, acid-labile adapters, peptidase-sensitive adapters, light-labile adapters, dimethyl adapters, or disulfide-containing adapters can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0148] The immunoconjugates or ADCs described herein are explicitly considered, but not limited to, such conjugates prepared with cross-linking agents, including but not limited to commercially available (e.g., from Pierce Biotechnology, Inc. (Rockford, IL., USA)) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfon-EMCS, sulfon-GMBS, sulfon-KMUS, sulfon-MBS, sulfon-SIAB, sulfon-SMCC, and sulfon-SMPB, as well as SVSB (succinimide-(4-vinyl sulfone)benzoate).

[0149] Multispecific antibodies

[0150] In some embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two distinct sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In some embodiments, a multispecific antibody has three or more binding specificities. In some embodiments, one of the binding specificities is specificity to GPRC5D, while the other (two or more) specificities are against any other antigen. In some embodiments, a bispecific antibody may bind to two (or more) distinct epitopes of GPRC5D. Multispecific (e.g., bispecific) antibodies can also be used to target cytotoxic agents or cells to cells expressing GPRC5D. Multispecific antibodies can be formulated as full-length antibodies or antibody fragments.

[0151] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knob-in-hole" engineering (see, for example, U.S. Patent No. 5,731,168, and Atwell et al. J.Mol.Biol.270:26 (1997)). Multispecific antibodies can also be prepared by: engineered electrostatic redirection effects for preparing antibody Fc-heterodimer molecules (see, for example, WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, US Patent No. 4,676,980; and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992); and WO 2011 / 034605); and using common light chain techniques to avoid light chain mismatch problems (see, for example, WO 2009 / 089004). 98 / 50431); preparation of bispecific antibody fragments using “bifunctional antibody” technology (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and preparation of trispecific antibodies using single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and preparation of trispecific antibodies according to, for example, the method described by Tutt et al., J. Immunol. 147:60 (1991).

[0152] This document also includes engineered antibodies having 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 having three or more antigen-binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Bispecific antibodies or their antigen-binding fragments also include “dual-acting FAbs” or “DAFs” that contain antigen-binding sites that bind to GPRC5D and another different antigen or two different epitopes of GPRC5D (see, for example, US 2008 / 0069820 and WO 2015 / 095539).

[0153] Multispecific antibodies can also be provided in asymmetric form, comprising cross-domains in one or more binding arms with the same antigen specificity, i.e., by exchanging VH / VL domains (see, for example, WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, for example, WO 2009 / 080253), or complete Fab arms (see, for example, 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 interfaces to guide correct Fab pairing. See, for example, WO 2016 / 172485.

[0154] Various other molecular forms for multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67 (2015) 95-106).

[0155] This also includes specific types of multispecific antibodies described herein that are bispecific antibodies designed to simultaneously bind to a surface antigen on a target cell (e.g., a tumor cell) and an activation-invariant component of the T-cell receptor (TCR) complex (e.g., Cd3), for redirecting T cells to kill the target cell. Therefore, in some embodiments, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, wherein one binding specificity is against GPRC5D and the other binding specificity is against CD3.

[0156] Bispecific antibody forms 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, for example, WO2004 / 106381, WO2005 / 061547, WO2007 / 042261 and WO2008 / 119567; Nagorsen and Exp Cell Res 317, 1255-1260 (2011)); bifunctional antibodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem bifunctional antibodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (biparental redirection) molecules, which are based on bifunctional antibody forms but have C-terminal disulfide bonds for further stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)); and so-called triomab, which are complete mouse / rat IgG hybrid molecules (see Seimetz et al. review: Cancer Treat Rev 36, 458-467 (2010)). The specific T-cell bispecific antibody forms included in this article are described in: WO 2013 / 026833; WO2013 / 026839; WO 2016 / 020309; and Bacac et al. Oncoimmunology 5(8)(2016)e1203498.

[0157] Bispecific antigen-binding molecules that bind to GPRC5D and the second antigen

[0158] The present invention also provides a bispecific antigen-binding molecule, namely an antigen-binding molecule comprising at least two antigen-binding portions capable of specifically binding to two different antigenic determinants (a first antigen and a second antigen).

[0159] According to a specific embodiment of the present invention, the antigen-binding portion contained in a bispecific antigen-binding molecule is a Fab molecule (i.e., an antigen-binding domain composed of a heavy chain and a light chain, each comprising a variable domain and a constant domain). In one embodiment, the first antigen-binding portion and / or the second antigen-binding portion 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 a human heavy chain and a light chain constant domain.

[0160] Preferably, at least one of the antigen-binding moieties is a cross-Fab molecule. Such modifications reduce mismatches between heavy and light chains from different Fab molecules, thereby improving the yield and purity of the recombinant production of the bispecific antigen-binding molecules of the present invention. In the specific cross-Fab molecules used in the bispecific antigen-binding molecules of the present invention, variable domains of the Fab light chain and Fab heavy chain (VL and VH, respectively) are exchanged. However, even with this domain exchange, the preparation of the bispecific antigen-binding molecules may still involve certain byproducts due to the so-called Bence Jones-type interaction between the mismatched heavy and light chains (see Schaefer et al., PNAS, 108(2011) 11187-11191). To further reduce mismatches between heavy and light chains from different Fab molecules, thereby improving the purity and yield of the desired bispecific antigen-binding molecules, amino acids with opposite charges may be introduced at specific amino acid positions in the CH1 and CL domains of the Fab molecule binding to the first antigen (GPRC5D) or the Fab molecule binding to the second antigen (e.g., an activating T-cell antigen such as CD3), as further described herein. Conventional Fab molecules (e.g., can be included in bispecific antigen-binding molecules) Figure 1 (As shown in AC, GJ) or conventional Fab molecules contained in VH / VL cross-Fab molecules (e.g., AC, GJ) Figure 1 Charge modification is performed in (but not both) the DF and KN (as shown). In a specific embodiment, charge modification is performed in the conventional Fab molecule included in the bispecific antigen-binding molecule (in a specific embodiment, it binds to the first antigen, namely GPRC5D).

[0161] In one specific embodiment of the invention, the bispecific antigen-binding molecule is capable of simultaneously binding to a first antigen (i.e., GPRC5D) and a second antigen (e.g., an activated T-cell antigen, particularly CD3). In one embodiment, the bispecific antigen-binding molecule enables T cells to crosslink with target cells by simultaneously binding to GPRC5D and the activated T-cell antigen. In a more specific embodiment, such simultaneous binding leads to lysis of target cells, particularly tumor cells expressing GPRC5D. In one embodiment, such simultaneous binding leads to T-cell activation. In other embodiments, such simultaneous binding leads to a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one embodiment, binding of the bispecific antigen-binding molecule to the activated T-cell antigen, particularly CD3, rather than simultaneously binding to GPRC5D, does not lead to T-cell activation.

[0162] In one embodiment, a bispecific antigen-binding molecule can redirect the cytotoxic activity of T cells to target cells. In a particular embodiment, the redirection is independent of MHC-mediated peptide antigen presentation of the target cells and / or T cell specificity.

[0163] Specifically, the T cells according to any embodiment of the present invention are cytotoxic T cells. In some embodiments, the T cells are CD4+ cells. + or CD8 + Cells, especially CD8 + T cells.

[0164] First antigen-binding portion

[0165] The bispecific antigen-binding molecule of the present invention comprises at least one antigen-binding moiety (particularly a Fab molecule) that binds to GPRC5D (a first antigen). In some embodiments, the bispecific antigen-binding molecule comprises two antigen-binding moieties, particularly Fab molecules that bind to GPRC5D. In one particular such embodiment, each of these antigen-binding moieties binds to the same antigenic determinant. In a more specific embodiment, all these antigen-binding moieties are identical, i.e., they comprise the same amino acid sequence including the same amino acid substitutions (if any) in the CH1 and CL domains as described herein. In one embodiment, the bispecific antigen-binding molecule comprises no more than two antigen-binding moieties, particularly Fab molecules that bind to GPRC5D.

[0166] In certain embodiments, the antigen-binding portion binding to GPRC5D is a conventional Fab molecule. In such embodiments, the antigen-binding portion binding to a second antigen is a cross-Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains exchange / replace each other.

[0167] In alternative embodiments, the antigen-binding moiety binding to GPRC5D is a cross-Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains exchange / replace each other. In such embodiments, the antigen-binding moiety binding to the second antigen is a conventional Fab molecule.

[0168] The GPRC5D binding moiety can guide bispecific antigen-binding molecules to target sites, such as specific types of tumor cells expressing GPRC5D.

[0169] Unless it is scientifically clearly unreasonable or impossible, the first antigen-binding portion of a bispecific antigen-binding molecule can bind alone or in combination to any of the features described herein with respect to antibodies binding to GPRC5D.

[0170] Therefore, one aspect of the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion comprises a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, the HCDR 2 of SEQ ID NO:84, and the HCDR 3 of SEQ ID NO:86, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, the LCDR 2 of SEQ ID NO:88, and the LCDR 3 of SEQ ID NO:89; and (b) a second antigen-binding portion binding to a second antigen. Another aspect of the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, the HCDR 2 of SEQ ID NO:85, and the HCDR 3 of SEQ ID NO:86, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, the LCDR 2 of SEQ ID NO:88, and the LCDR 3 of SEQ ID NO:89; and (b) a second antigen-binding portion binding to a second antigen. Another aspect of the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion comprises a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, the HCDR 2 of SEQ ID NO:91, and the HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, the LCDR 2 of SEQ ID NO:95, and the LCDR 3 of SEQ ID NO:97; and (b) a second antigen-binding portion binding to a second antigen.Another aspect of the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, the HCDR 2 of SEQ ID NO:91, and the HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, the LCDR 2 of SEQ ID NO:96, and the LCDR 3 of SEQ ID NO:97; and (b) a second antigen-binding portion binding to a second antigen. Another aspect of the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion comprises a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, the HCDR 2 of SEQ ID NO:92, and the HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, the LCDR 2 of SEQ ID NO:95, and the LCDR 3 of SEQ ID NO:97; and (b) a second antigen-binding portion binding to a second antigen. Another aspect of the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:1, HCDR 2 of SEQ ID NO:2, and HCDR 3 of SEQ ID NO:3, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:4, LCDR 2 of SEQ ID NO:5, and LCDR 3 of SEQ ID NO:6; and (b) a second antigen-binding portion binding to a second antigen.Another aspect of the present invention provides a bispecific antigen-binding molecule comprising: (a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding portion comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:7, HCDR 2 of SEQ ID NO:8, and HCDR 3 of SEQ ID NO:9, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:10, LCDR 2 of SEQ ID NO:11, and LCDR 3 of SEQ ID NO:12; and (b) a second antigen-binding portion binding to a second antigen.

[0171] In some embodiments, the first antigen-binding portion is (derived from) a humanized antibody. In one embodiment, VH is a humanized VH and / or VL is a humanized VL. In one embodiment, the first antigen-binding portion comprises a CDR as described in any of the above embodiments, and further comprises a human receptor framework, such as a human immunoglobulin framework or a human common framework.

[0172] 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 selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:57, and SEQ ID NO:58, 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 selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:63, and SEQ ID NO:64.

[0173] 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 selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:57, and SEQ ID NO:58, and a VL sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:63, and SEQ ID NO:64.

[0174] In one embodiment, the first antigen-binding portion comprises: VH, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:57 and SEQ ID NO:58; and VL, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:63 and SEQ ID NO:64.

[0175] In one embodiment, the first antigen-binding portion comprises a VH sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:57 and SEQ ID NO:58, and a VL sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:63 and SEQ ID NO:64.

[0176] In one specific embodiment, the first antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:13; and VL, which contains the amino acid sequence of SEQ ID NO:14. In another specific embodiment, the first antigen-binding portion comprises the VH sequence of SEQ ID NO:13 and the VL sequence of SEQ ID NO:14.

[0177] In one specific embodiment, the first antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:15; and VL, which contains the amino acid sequence of SEQ ID NO:16. In another specific embodiment, the first antigen-binding portion comprises the VH sequence of SEQ ID NO:15 and the VL sequence of SEQ ID NO:16.

[0178] In one specific embodiment, the first antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:48; and VL, which contains the amino acid sequence of SEQ ID NO:53. In another specific embodiment, the first antigen-binding portion comprises the VH sequence of SEQ ID NO:48 and the VL sequence of SEQ ID NO:53.

[0179] In one specific embodiment, the first antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:49; and VL, which contains the amino acid sequence of SEQ ID NO:52. In another specific embodiment, the first antigen-binding portion comprises the VH sequence of SEQ ID NO:49 and the VL sequence of SEQ ID NO:52.

[0180] In one specific embodiment, the first antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:57; and VL, which contains the amino acid sequence of SEQ ID NO:64. In another specific embodiment, the first antigen-binding portion comprises the VH sequence of SEQ ID NO:57 and the VL sequence of SEQ ID NO:64.

[0181] In one particular embodiment, the first antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:58; and VL, which contains the amino acid sequence of SEQ ID NO:63. In one particular embodiment, the first antigen-binding portion comprises the VH sequence of SEQ ID NO:58 and the VL sequence of SEQ ID NO:63. In one embodiment, the first antigen-binding portion comprises a human constant region. In one embodiment, the first antigen-binding portion is a Fab molecule containing a human constant region, particularly a human CH1 and / or CL domain. Exemplary sequences of the human constant domain are given in SEQ ID NO 37 and 38 (human κ and λCL domains, respectively) and SEQ ID NO:39 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In some embodiments, the first antigen-binding portion comprises a light chain constant region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to those of SEQ ID NO:37 or SEQ ID NO:38, particularly SEQ ID NO:37. Specifically, the light chain constant region may contain amino acid mutations as described herein under “charge modification,” and / or may include the deletion or substitution of one or more (particularly two) N-terminal amino acids in a cross-Fab molecule. In some embodiments, the first antigen-binding portion comprises a heavy chain constant region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to those of the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO:39. Specifically, the heavy chain constant region (particularly the CH1 domain) may contain amino acid mutations as described herein under “charge modification.”

[0182] Second antigen binding site

[0183] The bispecific antigen-binding molecule of the present invention comprises at least one antigen-binding moiety, particularly a Fab molecule that binds to a second antigen (different from GPRC5D).

[0184] In certain embodiments, the antigen-binding moiety binding to the second antigen is a cross-Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains exchange / replace each other. In such embodiments, the antigen-binding moiety binding 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, particularly a Fab molecule, binding to GPRC5D contained in a bispecific antigen-binding molecule, the antigen-binding moiety binding to the second antigen is preferably a cross-Fab molecule, and the antigen-binding moiety binding to GPRC5D is a conventional Fab molecule.

[0185] In alternative embodiments, the antigen-binding moiety binding to the second antigen is a conventional Fab molecule. In such embodiments, the antigen-binding moiety binding to the first antigen (i.e., GPRC5D) is a cross-Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains exchange / replace each other. In embodiments in which there is more than one antigen-binding moiety, particularly a Fab molecule, binding to the second antigen contained in a bispecific antigen-binding molecule, the antigen-binding moiety binding to GPRC5D is preferably a cross-Fab molecule, and the antigen-binding moiety binding to the second antigen is a conventional Fab molecule.

[0186] In some embodiments, the second antigen is an activated T-cell antigen (also referred to herein as an "activated T-cell antigen-binding portion" or an "activated T-cell antigen-binding Fab molecule"). In one particular embodiment, the bispecific antigen-binding molecule comprises no more than one antigen-binding portion capable of specifically binding to the activated T-cell antigen. In one embodiment, the bispecific antigen-binding molecule provides monovalent binding to the activated T-cell antigen.

[0187] In certain embodiments, the second antigen is CD3, particularly human CD3 (SEQ ID NO:40) or cynomolgus monkey CD3 (SEQ ID NO:41), and most particularly human CD3. In one embodiment, the second antigen binding portion cross-reacts with (i.e. specifically binds to) human and cynomolgus monkey CD3. In some embodiments, the second antigen is the ε subunit of CD3 (CD3ε).

[0188] In one embodiment, the second antigen-binding portion comprises HCDR 1 of SEQ ID NO:29, HCDR 2 of SEQ ID NO:30, HCDR 3 of SEQ ID NO:31, LCDR 1 of SEQ ID NO:32, LCDR 2 of SEQ ID NO:33, and LCDR 3 of SEQ ID NO:34. In one embodiment, the second antigen-binding portion comprises: VH, which comprises HCDR 1 of SEQ ID NO:29, HCDR 2 of SEQ ID NO:30, and HCDR 3 of SEQ ID NO:31; and VL, which comprises LCDR 1 of SEQ ID NO:32, LCDR 2 of SEQ ID NO:33, and LCDR 3 of SEQ ID NO:34. In some embodiments, the second antigen-binding portion is (derived from) a humanized antibody. In one embodiment, VH is a humanized VH and / or VL is a humanized VL. In one embodiment, the second antigen-binding portion comprises a CDR as described in any of the above embodiments, and further comprises a human receptor framework, such as a human immunoglobulin framework or a human common framework. In one embodiment, the second antigen-binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:35. In one embodiment, the second antigen-binding portion comprises a VL sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:36. In one embodiment, the second antigen-binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:35 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:36. In one embodiment, the VH of the second antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:35, and the VL of the second antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:36. In one embodiment, the second antigen-binding portion comprises: a VH comprising the amino acid sequence of SEQ ID NO:35; and a VL comprising the amino acid sequence of SEQ ID NO:36. In one embodiment, the second antigen-binding portion comprises the VH sequence of SEQ ID NO:35 and the VL sequence of SEQ ID NO:36.

[0189] In one embodiment, the second antigen-binding portion comprises HCDR 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, HCDR 3 of SEQ ID NO:100, LCDR 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102, and LCDR 3 of SEQ ID NO:103. In one embodiment, the second antigen-binding portion comprises: VH, which comprises HCDR 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and HCDR 3 of SEQ ID NO:100; and VL, which comprises LCDR 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102, and LCDR 3 of SEQ ID NO:103. In some embodiments, the second antigen-binding portion is (derived from) a humanized antibody. In one embodiment, VH is a humanized VH and / or VL is a humanized VL. In one embodiment, the second antigen-binding portion comprises a CDR as described in any of the above embodiments, and further comprises a human receptor framework, such as a human immunoglobulin framework or a human common framework. In one embodiment, the second antigen-binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:104. In one embodiment, the second antigen-binding portion comprises a VL sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:105. In one embodiment, the second antigen-binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:104 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:105. In one embodiment, the VH of the second antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 104, and the VL of the second antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 105. In one embodiment, the second antigen-binding portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 104; and a VL comprising the amino acid sequence of SEQ ID NO: 105. In one embodiment, the second antigen-binding portion comprises the VH sequence of SEQ ID NO: 104 and the VL sequence of SEQ ID NO: 105.

[0190] In one embodiment, the second antigen-binding portion comprises HCDR 1 of SEQ ID NO:106, HCDR 2 of SEQ ID NO:107, HCDR 3 of SEQ ID NO:108, LCDR 1 of SEQ ID NO:109, LCDR 2 of SEQ ID NO:110, and LCDR 3 of SEQ ID NO:111. In another embodiment, the second antigen-binding portion comprises: VH, which comprises HCDR 1 of SEQ ID NO:106, HCDR 2 of SEQ ID NO:107, and HCDR 3 of SEQ ID NO:108; and VL, which comprises LCDR 1 of SEQ ID NO:109, LCDR 2 of SEQ ID NO:110, and LCDR 3 of SEQ ID NO:111.

[0191] In some embodiments, the second antigen-binding portion is (derived from) a humanized antibody. In one embodiment, VH is a humanized VH and / or VL is a humanized VL. In one embodiment, the second antigen-binding portion comprises a CDR as described in any of the above embodiments, and further comprises a human receptor framework, such as a human immunoglobulin framework or a human common framework. In one embodiment, the second antigen-binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:112. In one embodiment, the second antigen-binding portion comprises a VL sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:113. In one embodiment, the second antigen-binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 112 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: 113. In one embodiment, the VH of the second antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 112, and the VL of the second antigen-binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 113. In one embodiment, the second antigen-binding portion comprises: a VH comprising the amino acid sequence of SEQ ID NO: 112; and a VL comprising the amino acid sequence of SEQ ID NO: 113. In one embodiment, the second antigen-binding portion comprises the VH sequence of SEQ ID NO:112 and the VL sequence of SEQ ID NO:113.

[0192] In one embodiment, the second antigen-binding moiety comprises a human constant region. In another embodiment, the second antigen-binding moiety is a Fab molecule containing a human constant region, particularly the human CH1 and / or CL domains. Exemplary sequences of the human constant domains are given in SEQ ID NO 37 and 38 (human κ and λCL domains, respectively) and SEQ ID NO:39 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In some embodiments, the second antigen-binding moiety comprises a light chain constant region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to those of SEQ ID NO:37 or SEQ ID NO:38, particularly SEQ ID NO:37. In particular, the light chain constant region may contain amino acid mutations as described herein under “charge modification”, and / or may contain the deletion or substitution of one or more (particularly two) N-terminal amino acids in a cross-Fab molecule. In some embodiments, the second antigen-binding portion includes a heavy chain constant region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO:39. In particular, the heavy chain constant region (especially the CH1 domain) may contain amino acid mutations as described herein under “charge modification”.

[0193] In some embodiments, the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light and Fab heavy chains, particularly the variable domains VL and VH, are substituted for each other (i.e., according to such embodiments, the second antigen-binding portion is a cross-Fab molecule in which the variable or constant domains of the Fab light and Fab heavy chains are exchanged). In one such embodiment, the first (and third, if any) antigen-binding portion is a conventional Fab molecule.

[0194] In one embodiment, the bispecific antigen-binding molecule has no more than one antigen-binding moiety that binds to a second antigen (e.g., an activated T-cell antigen, such as CD3) (i.e., the bispecific antigen-binding molecule provides monovalent binding to the second antigen).

[0195] Charge modification

[0196] The bispecific antigen-binding molecule of the present invention may contain amino acid substitutions in the Fab molecule therein, which is particularly effective in reducing mismatches between the light chain and the mismatched heavy chain (Bence-Jones-type byproducts) that may occur in the preparation of Fab-based bispecific / multispecific antigen-binding molecules, wherein VH / VL exchange occurs in one (or more, if the molecule contains more than two antigen-binding Fab molecules) of its binding arm (see also PCT Publication WO 2015 / 150447, particularly the embodiments therein, the entire contents of which are incorporated herein by reference). The ratio of the desired bispecific antigen-binding molecule to the undesirable byproducts, particularly the Bence-Jones-type byproducts occurring in bispecific antigen-binding molecules with 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").

[0197] Therefore, in some embodiments, the first and second antigen-binding portions of the bispecific antigen-binding molecule are both Fab molecules, and in one of the antigen-binding portions (particularly the second antigen-binding portion), the variable domains VL and VH of the Fab light chain and Fab heavy chain substitute for each other.

[0198] i) In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is replaced by a positively charged amino acid (according to Kabat numbering), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is replaced by a negatively charged amino acid (according to Kabat EU indexing); or

[0199] ii) In the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is replaced by a positively charged amino acid (according to the Kabat number), and in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is replaced by a negatively charged amino acid (according to the Kabat EU index number).

[0200] The bispecific antigen-binding molecule does not contain either of the modifications described in i) and ii). The constant domains CL and CH1 of the antigen-binding moiety with VH / VL exchange do not substitute for each other (i.e., they retain the unexchanged state).

[0201] In a more specific embodiment,

[0202] i) In the constant domain CL of the first antigen-binding moiety, 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 moiety, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing); or

[0203] ii) In the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (according to Kabat number), and in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number).

[0204] In one such embodiment, in the constant domain CL of the first antigen-binding moiety, 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 moiety, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing numbering).

[0205] In another embodiment, in the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0206] In one particular embodiment, in the constant domain CL of the first antigen-binding moiety, 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). In the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0207] In a more specific embodiment, in the constant domain CL of the first antigen-binding portion, the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced 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 replaced by glutamic acid (E) (according to Kabat EU indexing), and the amino acid at position 213 is replaced by glutamic acid (E) (according to Kabat EU indexing).

[0208] In one or even more specific embodiments, in the constant domain CL of the first antigen-binding portion, the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced 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 replaced by glutamic acid (E) (according to Kabat EU indexing), and the amino acid at position 213 is replaced by glutamic acid (E) (according to Kabat EU indexing).

[0209] In a specific embodiment, if the amino acid substitution according to the above embodiment occurs in the constant structural domain CL and the constant structural domain CH1 of the first antigen-binding moiety, then the constant structural domain CL of the first antigen-binding moiety is a κ isotype.

[0210] Alternatively, amino acid substitution according to the above embodiments may occur in the constant domains CL and CH1 of the second antigen-binding moiety, rather than in the constant domains CL and CH1 of the first antigen-binding moiety. In certain such embodiments, the constant domain CL of the second antigen-binding moiety is a κ isotype.

[0211] Therefore, in one embodiment, in the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 or the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing numbering).

[0212] In another embodiment, in the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing numbering).

[0213] In another embodiment, in the constant domain CL of the second antigen-binding moiety, 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). In the constant domain CH1 of the second antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0214] In one embodiment, in the constant domain CL of the second antigen-binding portion, the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced 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 replaced by glutamic acid (E) (according to Kabat EU indexing), and the amino acid at position 213 is replaced by glutamic acid (E) (according to Kabat EU indexing).

[0215] In another embodiment, in the constant domain CL of the second antigen-binding portion, the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced by arginine (R) (according to Kabat numbering). In the constant domain CH1 of the second antigen-binding portion, the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat EU indexing), and the amino acid at position 213 is replaced by glutamic acid (E) (according to Kabat EU indexing).

[0216] In one particular embodiment, the bispecific antigen-binding molecule of the present invention comprises

[0217] (a) A first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:84, and HCDR 3 of SEQ ID NO:86, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; and

[0218] (b) A second antigen-binding moiety that binds to a second antigen, wherein the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain substitute for each other.

[0219] In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0220] In one particular embodiment, the bispecific antigen-binding molecule of the present invention comprises

[0221] (a) A first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:85, and HCDR 3 of SEQ ID NO:86, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; and

[0222] (b) A second antigen-binding moiety that binds to a second antigen, wherein the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain substitute for each other.

[0223] In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0224] In one particular embodiment, the bispecific antigen-binding molecule of the present invention comprises

[0225] (a) A first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:85, and HCDR 3 of SEQ ID NO:86, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; and

[0226] (b) A second antigen-binding moiety that binds to a second antigen, wherein the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain substitute for each other.

[0227] In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0228] In one particular embodiment, the bispecific antigen-binding molecule of the present invention comprises

[0229] (a) A first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; and

[0230] (b) A second antigen-binding moiety that binds to a second antigen, wherein the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain substitute for each other.

[0231] In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0232] In one particular embodiment, the bispecific antigen-binding molecule of the present invention comprises

[0233] (a) A first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO:97; and

[0234] (b) A second antigen-binding moiety that binds to a second antigen, wherein the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain substitute for each other.

[0235] In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0236] In one particular embodiment, the bispecific antigen-binding molecule of the present invention comprises

[0237] (a) A first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:92, and HCDR 3 of SEQ ID NO:93, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; and

[0238] (b) A second antigen-binding moiety that binds to a second antigen, wherein the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain substitute for each other.

[0239] In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0240] In one particular embodiment, the bispecific antigen-binding molecule of the present invention comprises

[0241] (a) A first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:1, HCDR 2 of SEQ ID NO:2, and HCDR 3 of SEQ ID NO:3, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:4, LCDR 2 of SEQ ID NO:5, and LCDR 3 of SEQ ID NO:6; and

[0242] (b) A second antigen-binding moiety that binds to a second antigen, wherein the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain substitute for each other.

[0243] In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0244] In one particular embodiment, the bispecific antigen-binding molecule of the present invention comprises

[0245] (a) A first antigen-binding moiety that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:7, HCDR 2 of SEQ ID NO:8, and HCDR 3 of SEQ ID NO:9, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:10, LCDR 2 of SEQ ID NO:11, and LCDR 3 of SEQ ID NO:12; and

[0246] (b) A second antigen-binding moiety that binds to a second antigen, wherein the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain substitute for each other.

[0247] In the constant domain CL of the first antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering) (in one particular embodiment, it is independently substituted with lysine (K) or arginine (R)), and in the constant domain CH1 of the first antigen-binding moiety, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

[0248] Bispecific antigen-binding molecular form

[0249] The components of the bispecific antigen-binding molecule according to the present invention can be fused together in various configurations. Exemplary configurations are as follows: Figure 1 As shown in AZ.

[0250] In a specific embodiment, the antigen-binding portion of the bispecific antigen-binding molecule is a Fab molecule. In such embodiments, the first antigen-binding portion, the second antigen-binding portion, the third antigen-binding portion, etc., may be referred to herein as the first Fab molecule, the second Fab molecule, the third Fab molecule, etc., respectively.

[0251] In one embodiment, the first antigen-binding portion and the second antigen-binding portion of the bispecific antigen-binding molecule are fused together, optionally via a peptide linker. In a particular embodiment, the first antigen-binding portion and the second antigen-binding portion are each Fab molecules. In one such embodiment, the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion. In another such embodiment, the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion. Additionally, in embodiments where (i) the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion or (ii) the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion, the Fab light chain of the first antigen-binding portion may be fused to the Fab light chain of the second antigen-binding portion, optionally via a peptide linker.

[0252] Bispecific antigen-binding molecules with a single antigen-binding motif (such as Fab molecules) capable of specifically binding to target cell antigens (such as GPRC5D) can be used. Figure 1 (As shown in A, 1D, 1G, 1H, 1K, 1L), especially in cases where the target cell antigen is internalized after binding to the expected high-affinity antigen-binding moiety. In such cases, the presence of more than one antigen-binding moiety targeting a specific target cell antigen can enhance the internalization of the target cell antigen, thereby reducing its availability.

[0253] However, in other cases, bispecific antigen-binding molecules (e.g., such as Fab molecules) containing two or more antigen-binding moieties targeting specific target cell antigens are used. Figure 1 (As shown in B, 1C, 1E, 1F, 1I, 1J, 1M, or 1N) will be advantageous, for example, for optimizing the targeting of the target or enabling the target cell antigen to cross-link.

[0254] Therefore, in a particular embodiment, the bispecific antigen-binding molecule according to the present invention includes a third antigen-binding portion.

[0255] In one embodiment, the third antigen-binding portion binds to the first antigen, GPRC5D. In another embodiment, the third antigen-binding portion is a Fab molecule.

[0256] In one embodiment, the third antigen portion is the same as the first antigen binding portion.

[0257] Unless it is clearly scientifically unreasonable or impossible, the third antigen-binding portion of a bispecific antigen-binding molecule may bind alone or in combination to any of the features described herein in relation to the first antigen-binding portion and / or antibody that binds to GPRC5D.

[0258] In one embodiment, the third antigen-binding portion comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, the HCDR 2 of SEQ ID NO:84, and the HCDR 3 of SEQ ID NO:86; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, the LCDR 2 of SEQ ID NO:88, and the LCDR 3 of SEQ ID NO:89.

[0259] In one embodiment, the third antigen-binding portion comprises: a heavy chain variable region (VH) including the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, the HCDR 2 of SEQ ID NO:85, and the HCDR 3 of SEQ ID NO:86; and a light chain variable region (VL) including the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, the LCDR 2 of SEQ ID NO:88, and the LCDR 3 of SEQ ID NO:89.

[0260] In one embodiment, the third antigen-binding portion comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, the HCDR 2 of SEQ ID NO:91, and the HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, the LCDR 2 of SEQ ID NO:95, and the LCDR 3 of SEQ ID NO:97.

[0261] In one embodiment, the third antigen-binding portion comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, the HCDR 2 of SEQ ID NO:91, and the HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, the LCDR 2 of SEQ ID NO:96, and the LCDR 3 of SEQ ID NO:97.

[0262] In one embodiment, the third antigen-binding portion comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, the HCDR 2 of SEQ ID NO:92, and the HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, the LCDR 2 of SEQ ID NO:95, and the LCDR 3 of SEQ ID NO:97.

[0263] In one embodiment, the third antigen-binding portion comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:1, the HCDR 2 of SEQ ID NO:2, and the HCDR 3 of SEQ ID NO:4; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:5, the LCDR 2 of SEQ ID NO:6, and the LCDR 3 of SEQ ID NO:7.

[0264] In one embodiment, the third antigen-binding portion comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:7, the HCDR 2 of SEQ ID NO:8, and the HCDR 3 of SEQ ID NO:9; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:10, the LCDR 2 of SEQ ID NO:11, and the LCDR 3 of SEQ ID NO:12.

[0265] In some embodiments, the third antigen-binding portion is (derived from) a humanized antibody. In one embodiment, VH is a humanized VH and / or VL is a humanized VL. In one embodiment, the third antigen-binding portion comprises a CDR as described in any of the above embodiments, and further comprises a human receptor framework, such as a human immunoglobulin framework or a human common framework.

[0266] 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 selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:57, and SEQ ID NO:58, 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 selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:63, and SEQ ID NO:64.

[0267] 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 selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:57, and SEQ ID NO:58; and a VL sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:63, and SEQ ID NO:64.

[0268] In one embodiment, the third antigen-binding portion comprises: VH, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:57 and SEQ ID NO:58; and VL, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:63 and SEQ ID NO:64.

[0269] In one embodiment, the third antigen-binding portion comprises a VH sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:57 and SEQ ID NO:58, and a VL sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:63 and SEQ ID NO:64.

[0270] In one specific embodiment, the third antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:13; and VL, which contains the amino acid sequence of SEQ ID NO:14. In one specific embodiment, the third antigen-binding portion comprises the VH sequence of SEQ ID NO:13 and the VL sequence of SEQ ID NO:14.

[0271] In one specific embodiment, the third antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:15; and VL, which contains the amino acid sequence of SEQ ID NO:16. In one specific embodiment, the third antigen-binding portion comprises the VH sequence of SEQ ID NO:15 and the VL sequence of SEQ ID NO:16.

[0272] In one specific embodiment, the third antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:48; and VL, which contains the amino acid sequence of SEQ ID NO:53. In one specific embodiment, the third antigen-binding portion comprises the VH sequence of SEQ ID NO:48 and the VL sequence of SEQ ID NO:53.

[0273] In one specific embodiment, the third antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:49; and VL, which contains the amino acid sequence of SEQ ID NO:52. In one specific embodiment, the third antigen-binding portion comprises the VH sequence of SEQ ID NO:49 and the VL sequence of SEQ ID NO:52.

[0274] In one specific embodiment, the third antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:57; and VL, which contains the amino acid sequence of SEQ ID NO:64. In one specific embodiment, the third antigen-binding portion comprises the VH sequence of SEQ ID NO:57 and the VL sequence of SEQ ID NO:64.

[0275] In one specific embodiment, the third antigen-binding portion comprises: VH, which contains the amino acid sequence of SEQ ID NO:58; and VL, which contains the amino acid sequence of SEQ ID NO:63. In one specific embodiment, the third antigen-binding portion comprises the VH sequence of SEQ ID NO:58 and the VL sequence of SEQ ID NO:63.

[0276] In one embodiment, the third antigen-binding moiety comprises a human constant region. In another embodiment, the third antigen-binding moiety is a Fab molecule containing a human constant region, particularly a human CH1 and / or CL domain. Exemplary sequences of the human constant domains are given in SEQ ID NO 37 and 38 (human κ and λCL domains, respectively) and SEQ ID NO:39 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In some embodiments, the third antigen-binding moiety comprises a light chain constant region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to those of SEQ ID NO:37 or SEQ ID NO:38, particularly SEQ ID NO:37. In particular, the light chain constant region may contain amino acid mutations as described herein under “charge modification”, and / or may contain the deletion or substitution of one or more (particularly two) N-terminal amino acids in a cross-Fab molecule. In some embodiments, the third antigen-binding portion includes a heavy chain constant region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO:39. In particular, the heavy chain constant region (especially the CH1 domain) may contain amino acid mutations as described herein under “charge modification”.

[0277] In certain embodiments, the third and first antigen-binding moieties are each Fab molecules, and the third antigen-binding moieties are identical to the first antigen-binding moieties. Therefore, in these embodiments, the first and third antigen-binding moieties contain the same heavy and light chain amino acid sequences and have the same arrangement of structural domains (i.e., conventional or crossed). Furthermore, in these embodiments, the third antigen-binding moieties contain the same amino acid substitutions (if any) as the first antigen-binding moieties. For example, the “charge-modified” amino acid substitutions described herein will be performed in the constant structural domains CL and CH1 of each of the first and third antigen-binding moieties. Alternatively, the amino acid substitutions may be performed in the constant structural domains CL and CH1 of the second antigen-binding moieties (which, in certain embodiments, are also Fab molecules), but not in the constant structural domains CL and CH1 of the first and third antigen-binding moieties.

[0278] Similar to the first antigen-binding moiety, the third antigen-binding moiety is particularly a conventional Fab molecule. However, embodiments in which the first and third antigen-binding moiety are cross-Fab molecules (and the second antigen-binding moiety is a conventional Fab molecule) are also conceivable. Thus, in a particular embodiment, the first and third antigen-binding moiety are each a conventional Fab molecule, and the second antigen-binding moiety is a cross-Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains exchange / replace each other. In other embodiments, the first and third antigen-binding moiety are each a cross-Fab molecule, and the second antigen-binding moiety is a conventional Fab molecule.

[0279] If a third antigen-binding portion is present, in a particular embodiment, the first and third antigen portions bind to GPRC5D, and the second antigen-binding portion binds to a second antigen, particularly an activated T-cell antigen, more particularly CD3, and most particularly CD3ε.

[0280] In a particular embodiment, the bispecific antigen-binding molecule includes an Fc domain composed of a first subunit and a second subunit. The first and second subunits of the Fc domain are capable of stable association.

[0281] The bispecific antigen-binding molecule according to the invention can have different configurations, i.e., the first antigen-binding moiety, the second antigen-binding moiety (and optionally the third antigen-binding moiety) can be fused to each other and fused to the Fc domain in different ways. These components can be fused directly to each other or preferably fused through one or more suitable peptide linkers. In the case of fusion of the Fab molecule to the N-terminus of a subunit of the Fc domain, it is usually fused through the immunoglobulin hinge region.

[0282] In some embodiments, the first antigen-binding portion and the second antigen-binding portion are each Fab molecules, and the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of either the first or second subunit of the Fc domain. In such embodiments, the first antigen-binding portion may be fused at the C-terminus of the Fab heavy chain to the N-terminus of either the Fab heavy chain or the N-terminus of another subunit of the Fc domain of the second antigen-binding portion. In certain such embodiments, the first antigen-binding portion is a conventional Fab molecule, and the second antigen-binding portion is a cross-Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains exchange / replace each other. In other such embodiments, the first Fab molecule is a cross-Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0283] In one embodiment, the first antigen-binding portion and the second antigen-binding portion are each Fab molecules. The second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of either the first or second subunit of the Fc domain, and the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion. In a specific embodiment, the bispecific antigen-binding molecule is substantially composed of a first Fab molecule and a second Fab molecule, the Fc domain being composed of a first subunit, a second subunit, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of either the first or second subunit of the Fc domain. Figure 1 G and Figure 1 This configuration is schematically described in K (in these examples, the second antigen-binding domain is a VH / VL cross-Fab molecule). Alternatively, the Fab light chains of the first Fab molecule and the Fab light chains of the second Fab molecule may be fused together.

[0284] In another embodiment, the first antigen-binding portion and the second antigen-binding portion are each Fab molecules, and each of the first and second antigen-binding portions is 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 is substantially composed of a first Fab molecule and a second Fab molecule, the Fc domain being composed of a first subunit and a second subunit, and optionally one or more peptide linkers, wherein each of the first and second Fab molecules is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. Figure 1 A and Figure 1This configuration is schematically depicted in D (in these examples, the second antigen-binding domain is a VH / VL cross-linked Fab molecule and the first antigen-binding portion is a conventional Fab molecule). The first and second Fab molecules can be fused to the Fc domain directly or via peptide linkers. In one particular embodiment, the first and second Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In one particular embodiment, the immunoglobulin hinge region is the human IgG1 hinge region, and specifically, the Fc domain is the IgG1 Fc domain.

[0285] In some embodiments, the first antigen-binding portion and the second antigen-binding portion are each Fab molecules, and the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of either the first or second subunit of the Fc domain. In such embodiments, the second antigen-binding portion may be fused at the C-terminus of the Fab heavy chain to the N-terminus of either the Fab heavy chain or the N-terminus of another subunit of the Fc domain (as described above). In certain such embodiments, the first antigen-binding portion is a conventional Fab molecule, and the second antigen-binding portion is a cross-Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains exchange / replace each other. In other such embodiments, the first Fab molecule is a cross-Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0286] In one embodiment, the first antigen-binding portion and the second antigen-binding portion are each Fab molecules. The first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of either the first or second subunit of the Fc domain, and the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion. In a specific embodiment, the bispecific antigen-binding molecule is substantially composed of a first Fab molecule and a second Fab molecule, the Fc domain being composed of a first subunit, a second subunit, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of either the first or second subunit of the Fc domain. Figure 1 H and Figure 1 This configuration is schematically depicted in L (in these examples, the second antigen-binding domain is a VH / VL cross-Fab molecule and the first antigen-binding portion is a conventional Fab molecule). Alternatively, the Fab light chains of the first Fab molecule and the Fab light chains of the second Fab molecule may be fused together.

[0287] In some embodiments, the third antigen-binding portion, particularly the third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In certain such embodiments, the first and third Fab molecules are each conventional Fab molecules, and the second Fab molecule is a cross-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 exchange / replace each other. In other such embodiments, the first and third Fab molecules are each cross-Fab molecules, and the second Fab molecule is a conventional Fab molecule.

[0288] In one particular embodiment of this type, the second and third antigen-binding portions 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 portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion. In one specific embodiment, the bispecific antigen-binding molecule is substantially composed of a first Fab molecule, a second Fab molecule, and a third Fab molecule, the Fc domain being composed of a first subunit and a second subunit, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Figure 1 B and Figure 1 E (in these examples, the second antigen-binding moiety is a VH / VL cross-Fab molecule, and the first and third antigen-binding moieties are conventional Fab molecules) and Figure 1 This configuration is schematically depicted in J and 1N (in these examples, the second antigen-binding portion is a conventional Fab molecule, and the first and third antigen-binding portions are VH / VL cross-Fab molecules). The second and third Fab molecules may be fused to the Fc domain directly or via peptide linkers. In one particular embodiment, the second and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In one particular embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, specifically, the Fc domain is an IgG1 Fc domain. Additionally, optionally, the Fab light chains of the first and second Fab molecules may be fused to each other.

[0289] In another such embodiment, the first antigen-binding portion and the third antigen-binding portion 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 portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion. In one specific embodiment, the bispecific antigen-binding molecule is substantially composed of a first Fab molecule, a second Fab molecule, and a third Fab molecule, the Fc domain being composed of a first subunit and a second subunit, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Figure 1 C and Figure 1 F (in these examples, the second antigen-binding moiety is a VH / VL cross-Fab molecule, and the first and third antigen-binding moieties are conventional Fab molecules) and Figure 1 This configuration is schematically depicted in I and 1M (in these examples, the second antigen-binding portion is a conventional Fab molecule, and the first and third antigen-binding portions are VH / VL cross-Fab molecules). The first and third Fab molecules may be fused to the Fc domain directly or via peptide linkers. In one particular embodiment, the first and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In one particular embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, specifically, the Fc domain is an IgG1 Fc domain. Alternatively, the Fab light chains of the first and second Fab molecules may be fused to each other.

[0290] In a bispecific antigen-binding molecule configuration in which the Fab molecule is fused at the C-terminus of the Fab heavy chain via an immunoglobulin hinge region to the N-terminus of a subunit of the Fc domain, the hinge region and the Fc domain essentially form an immunoglobulin molecule. In one particular embodiment, the immunoglobulin molecule is an IgG class immunoglobulin. In a more specific embodiment, the immunoglobulin is an IgG1 subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG4 subclass immunoglobulin. In yet 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.

[0291] In some bispecific antigen-binding molecules of the present invention, the Fab light chain of the first Fab molecule is fused to the Fab light chain of the second Fab molecule, optionally via a peptide linker. Depending on the configuration of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused to the Fab light chain of the second Fab molecule at its C-terminus, or vice versa. The fusion of the Fab light chains of the first and second Fab molecules further reduces mismatches between the Fab heavy and light chains and also reduces the number of plasmids required to express certain bispecific antigen-binding molecules of the present invention.

[0292] The antigen-binding moieties may fuse to the Fc domain or directly to each other, or fuse via a peptide linker containing one or more amino acids, typically about 2-20 amino acids. Peptide linkers are well known in the art and as described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S). n (SG4) n (G4S) n Or G4 (SG4) n Peptide linker. "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 another embodiment, it is 5 to 100 amino acids long; and in yet another embodiment, it is 10 to 50 amino acids long. In one embodiment, the peptide linker is (GxS). n Or (GxS) n G m Wherein G = glycine, S = serine, and (x = 3, n = 3, 4, 5 or 6, and m = 0, 1, 2 or 3) or (x = 4, n = 2, 3, 4 or 5, and m = 0, 1, 2 or 3), in 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 a first Fab molecule and a second Fab molecule to each other is (G4S)2. An exemplary peptide linker suitable for linking the Fab heavy chains of a first Fab fragment and a second Fab fragment comprises the sequence (D)-(G4S)2 (SEQ ID NO 43 and 44). Another suitable such linker comprises the sequence (G4S)4. Additionally, 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 an Fc domain subunit, fusion can be achieved by including an additional peptide linker or an immunoglobulin hinge region or a portion thereof without an additional peptide linker.

[0293] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises: a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a cross-linked Fab heavy chain, wherein the heavy chain variable region is replaced by the light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fc domain subunit (VL(2)-CH1(2)-CH2-CH3(-CH4)); and a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the Fc domain subunit (VH (1) -CH1 (1) -CH2-CH3(-CH4)). In some embodiments, the bispecific antigen-binding molecule further comprises: a polypeptide, wherein a second Fab molecule (VH (2) -CL (2) The variable region of the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule; and the first Fab molecule (VL) (1) -CL (1) Fab light chain polypeptides. In some embodiments, the polypeptides are covalently linked by, for example, disulfide bonds.

[0294] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises: a polypeptide wherein the variable region of the Fab heavy chain of a second Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (i.e., the second Fab molecule contains a cross-linked Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxyl-terminal peptide bond (VH) with an Fc domain subunit. (2) -CL (2) -CH2-CH3(-CH4)); and polypeptides, wherein the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond (VH) with the Fc domain subunit. (1) -CH1 (1) -CH2-CH3(-CH4)). In some embodiments, the bispecific antigen-binding molecule further comprises: a polypeptide, wherein a second Fab molecule (VL) (2) -CH1 (2) The variable region of the Fab light chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule; and the first Fab molecule (VL) (1) -CL (1) Fab light chain polypeptides. In some embodiments, the polypeptides are covalently linked by, for example, disulfide bonds.

[0295] 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 C-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a cross-linked Fab heavy chain, wherein the heavy chain variable region is replaced by the light chain variable region), which in turn shares a C-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and subsequently shares a C-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 the first Fab molecule shares a C-terminal peptide bond with the variable region of the Fab light chain of the second Fab molecule, and subsequently shares a C-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule (i.e., the second Fab molecule contains a cross-linked Fab heavy chain, wherein the variable region of the heavy chain is replaced by the variable region of the light chain), and subsequently shares a C-terminal peptide bond (VH) with the Fc domain subunit. (1) -CH1 (1) -VL (2) -CH1 (2) -CH2-CH3(-CH4)).

[0296] In some of these embodiments, the bispecific antigen-binding molecule further comprises: a second Fab molecule (VH (2) -CL (2) A cross-Fab light chain polypeptide, wherein the variable region of the Fab heavy chain of the second Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule; and the first Fab molecule (VL (1) -CL (1) The Fab light chain polypeptide of the second Fab molecule. In other examples of these embodiments, the bispecific antigen-binding molecule further comprises: a polypeptide wherein the variable region of the Fab heavy chain of the second Fab molecule shares a C-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule, and subsequently shares a C-terminal peptide bond (VH) with the Fab light chain polypeptide of the first Fab molecule. (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 variable region of the Fab heavy chain of the second Fab molecule, and subsequently shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (VL (1) -CL (1) -VH (2) -CL (2) (where appropriate).

[0297] 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 the third Fab molecule shares a carboxyl-terminal peptide bond (VH) with the Fc domain subunit. (3) -CH1 (3) -CH2-CH3(-CH4)), and the third Fab molecule (VL) (3) -CL (3) Fab light chain polypeptides. In some embodiments, the polypeptides are covalently linked by, for example, disulfide bonds.

[0298] In some embodiments, the bispecific antigen-binding molecule comprises a polypeptide wherein the variable region of the Fab heavy chain of the second Fab molecule shares a C-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (i.e., the second Fab molecule contains a cross-linked Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), which in turn shares a C-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and subsequently shares a C-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 the first Fab molecule shares a C-terminal peptide bond with the variable region of the Fab heavy chain of the second Fab molecule, and subsequently shares a C-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (i.e., the second Fab molecule contains a cross-linked Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), and subsequently shares a C-terminal peptide bond (VH) with the Fc domain subunit. (1) -CH1 (1) -VH (2) -CL (2) -CH2-CH3(-CH4)).

[0299] In some of these embodiments, the bispecific antigen-binding molecule further comprises: a second Fab molecule (VL (2) -CH1 (2) A cross-Fab light chain polypeptide, wherein the variable region of the Fab light chain of the second Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule; and the first Fab molecule (VL (1) -CL (1) The Fab light chain polypeptide of the second Fab molecule. In other examples, the bispecific antigen-binding molecule further comprises: a polypeptide wherein the variable region of the Fab light chain of the second Fab molecule shares a C-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule, and subsequently shares a C-terminal peptide bond (VL) with the Fab light chain polypeptide of the first Fab molecule.(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 variable region of the Fab heavy chain of the second Fab molecule, and subsequently shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (VL (1) -CL (1) -VL (2) -CH1 (2) (where appropriate).

[0300] 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 the third Fab molecule shares a carboxyl-terminal peptide bond (VH) with the Fc domain subunit. (3) -CH1 (3) -CH2-CH3(-CH4)), and the third Fab molecule (VL) (3) -CL (3) Fab light chain polypeptides. In some embodiments, the polypeptides are covalently linked by, for example, disulfide bonds.

[0301] In some embodiments, the bispecific antigen-binding molecule does not contain an Fc domain. In certain such embodiments, the first Fab molecule and (in the presence) the third Fab molecule are each conventional Fab molecules, and the second Fab molecule is a cross-Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged / substituted for each other. In other such embodiments, the first Fab molecule and (in the presence) the third Fab molecule are each cross-Fab molecules, and the second Fab molecule is a conventional Fab molecule.

[0302] In one such embodiment, the bispecific antigen-binding molecule is substantially composed of a first antigen-binding portion and a second antigen-binding portion, and optionally includes one or more peptide linkers, wherein both the first and second antigen-binding portions are Fab molecules, and the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion. Figure 1 O and Figure 1 This configuration is schematically depicted in S (in these examples, the second antigen-binding domain is a VH / VL cross-Fab molecule and the first antigen-binding part is a conventional Fab molecule).

[0303] In another such embodiment, the bispecific antigen-binding molecule is substantially composed of a first antigen-binding portion and a second antigen-binding portion, and optionally includes one or more peptide linkers, wherein both the first and second antigen-binding portions are Fab molecules, and the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion. Figure 1 P and Figure 1 This configuration is schematically depicted in T (in these examples, the second antigen-binding domain is a VH / VL cross-Fab molecule and the first antigen-binding part is a conventional Fab molecule).

[0304] 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 some such embodiments, the bispecific antigen-binding molecule is substantially composed 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. Figure 1 Q and 1U (in these examples, the second antigen-binding domain is a VH / VL cross-Fab molecule, and the first and third antigen-binding portions are conventional Fab molecules) and Figure 1 This configuration is schematically depicted in X and 1Z (in these examples, the second antigen-binding domain is a conventional Fab molecule, and the first and third antigen-binding portions are each VH / VL cross-Fab molecules).

[0305] 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 some such embodiments, the bispecific antigen-binding molecule is substantially composed 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 its 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 its Fab heavy chain to the C-terminus of the Fab heavy chain of the first Fab molecule. Figure 1R and 1V (in these examples, the second antigen-binding domain is a VH / VL cross-Fab molecule, and the first and third antigen-binding portions are conventional Fab molecules) and Figure 1 This configuration is schematically depicted in W and 1Y (in these examples, the second antigen-binding domain is a conventional Fab molecule, and the first and third antigen-binding portions are each VH / VL cross-Fab molecules).

[0306] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a C-terminal peptide bond with the variable region of the Fab light chain of the second Fab molecule, and subsequently shares a C-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule (i.e., the second Fab molecule comprises a cross-linked Fab heavy chain, wherein the variable region of the heavy chain is replaced by the variable region of the light chain) (VH (1) -CH1 (1) -VL (2) -CH1 (2) In some embodiments, the bispecific antigen-binding molecule further comprises: a polypeptide, wherein a second Fab molecule (VH) (2) -CL (2) The variable region of the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule; and the first Fab molecule (VL) (1) -CL (1) ) Fab light chain polypeptide.

[0307] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide, wherein the Fab light chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a cross-linked Fab heavy chain, wherein the heavy chain variable region is replaced by the light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain 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 a second Fab molecule (VH) (2) -CL (2) The variable region of the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule; and the first Fab molecule (VL) (1) -CL (1) ) Fab light chain polypeptide.

[0308] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide, wherein the variable region of the Fab heavy chain of the second Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (i.e., the second Fab molecule comprises a cross-linked Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxyl-terminal peptide bond (VH) with the Fab heavy chain of the first Fab molecule. (2) -CL (2) -VH (1) -CH1 (1) In some embodiments, the bispecific antigen-binding molecule further comprises: a polypeptide, wherein a second Fab molecule (VL) (2) -CH1 (2) The variable region of the Fab light chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule; and the first Fab molecule (VL) (1) -CL (1) ) Fab light chain polypeptide.

[0309] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide, wherein the Fab light chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a cross-linked Fab heavy chain, wherein the heavy chain variable region is replaced by the light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain 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 a second Fab molecule (VH) (2) -CL (2) The variable region of the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule; and the first Fab molecule (VL) (1) -CL (1) ) Fab light chain polypeptide.

[0310] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide wherein the Fab heavy chain of the third Fab molecule shares a C-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and subsequently shares a C-terminal peptide bond with the variable region of the Fab light chain of the second Fab molecule, and subsequently shares a C-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule (i.e., the second Fab molecule comprises a cross-linked Fab heavy chain, wherein the variable region of the heavy chain is replaced by the variable region of the light chain) (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 a second Fab molecule (VH) (2) -CL (2) The variable region of the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule; and the first Fab molecule (VL) (1) -CL (1) The Fab light chain polypeptide. In some embodiments, the bispecific antigen-binding molecule further comprises a third Fab molecule (VL). (3) -CL (3) ) Fab light chain polypeptide.

[0311] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide, wherein the Fab heavy chain of the third Fab molecule shares a C-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and subsequently shares a C-terminal peptide bond with the variable region of the Fab heavy chain of the second Fab molecule, and subsequently shares a C-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (i.e., the second Fab molecule comprises a cross-linked Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region) (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VH (2) -CL (2) In some embodiments, the bispecific antigen-binding molecule further comprises: a polypeptide, wherein a second Fab molecule (VL) (2) -CH1 (2) The variable region of the Fab light chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule; and the first Fab molecule (VL) (1) -CL (1) The Fab light chain polypeptide. In some embodiments, the bispecific antigen-binding molecule further comprises a third Fab molecule (VL). (3) -CL (3) ) Fab light chain polypeptide.

[0312] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide, wherein the variable region of the Fab light chain of the second Fab molecule shares a C-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule (i.e., the second Fab molecule contains a cross-linked Fab heavy chain, wherein the variable region of the heavy chain is replaced by the variable region of the light chain), which in turn shares a C-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and subsequently shares a C-terminal peptide bond with the Fab heavy chain of the third Fab molecule (VL). (2) -CH1 (2) -VH (1) -CH1(1) -VH (3) -CH1 (3) In some embodiments, the bispecific antigen-binding molecule further comprises: a polypeptide, wherein a second Fab molecule (VH) (2) -CL (2) The variable region of the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule; and the first Fab molecule (VL) (1) -CL (1) The Fab light chain polypeptide. In some embodiments, the bispecific antigen-binding molecule further comprises a third Fab molecule (VL). (3) -CL (3) ) Fab light chain polypeptide.

[0313] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide, wherein the variable region of the Fab heavy chain of the second Fab molecule shares a C-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (i.e., the second Fab molecule contains a cross-linked Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), which in turn shares a C-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and subsequently shares a C-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 a second Fab molecule (VL) (2) -CH1 (2) The variable region of the Fab light chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule; and the first Fab molecule (VL) (1) -CL (1) The Fab light chain polypeptide. In some embodiments, the bispecific antigen-binding molecule further comprises a third Fab molecule (VL). (3) -CL (3) ) Fab light chain polypeptide.

[0314] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide, wherein the Fab heavy chain of the second Fab molecule shares a C-terminal peptide bond with the variable region of the Fab light chain of the first Fab molecule, and subsequently shares a C-terminal peptide bond with the constant region of the Fab heavy chain of the first Fab molecule (i.e., the first Fab molecule contains a cross-linked Fab heavy chain, wherein the variable region of the heavy chain is replaced by the variable region of the light chain), and subsequently shares a C-terminal peptide bond with the variable region of the Fab light chain of the third Fab molecule, and subsequently shares a C-terminal peptide bond with the constant region of the Fab heavy chain of the third Fab molecule (i.e., the third Fab molecule contains a cross-linked Fab heavy chain, wherein the variable region of the heavy chain is replaced by the variable region of the light chain) (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 first Fab molecule (VH) (1) -CL (1) The variable region of the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule; and the variable region of the Fab heavy chain of the second Fab molecule (VL) shares a carboxyl-terminal peptide bond with the variable region of the Fab light chain of the first Fab molecule; (2) -CL (2) The Fab light chain polypeptide. In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide, wherein a third Fab molecule (VH) is a Fab light chain polypeptide. (3) -CL (3) The variable region of the Fab heavy chain of the third Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain.

[0315] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide, wherein the Fab heavy chain of the second Fab molecule shares a C-terminal peptide bond with the variable region of the Fab heavy chain of the first Fab molecule, and subsequently shares a C-terminal peptide bond with the constant region of the Fab light chain of the first Fab molecule (i.e., the first Fab molecule comprises a cross-linked Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), and subsequently shares a C-terminal peptide bond with the variable region of the Fab heavy chain of the third Fab molecule, and subsequently shares a C-terminal peptide bond with the constant region of the Fab light chain of the third Fab molecule (i.e., the third Fab molecule comprises a cross-linked Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region) (VH (2) -CH1 (2) -VH (1) -CL (1) -VH (3) -CL (3) In some embodiments, the bispecific antigen-binding molecule further comprises: a polypeptide, wherein the first Fab molecule (VL) (1) -CH1(1) The variable region of the Fab light chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule; and the variable region of the Fab light chain of the second Fab molecule (VL) shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain of the first Fab molecule. (2) -CL (2) The Fab light chain polypeptide. In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide, wherein a third Fab molecule (VL) is a Fab light chain polypeptide. (3) -CH1 (3) The variable region of the Fab light chain of the third Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain.

[0316] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide wherein the Fab light chain variable region of a third Fab molecule shares a C-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule contains a cross-linked Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a C-terminal peptide bond with the Fab light chain variable region of a first Fab molecule, which in turn shares a C-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule contains a cross-linked Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), and subsequently shares a C-terminal peptide bond with the Fab heavy chain of a 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 first Fab molecule (VH) (1) -CL (1) The variable region of the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule; and the variable region of the Fab heavy chain of the second Fab molecule (VL) shares a carboxyl-terminal peptide bond with the variable region of the Fab light chain of the first Fab molecule; (2) -CL (2) The Fab light chain polypeptide. In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide, wherein a third Fab molecule (VH) is a Fab light chain polypeptide. (3) -CL (3) The variable region of the Fab heavy chain of the third Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain.

[0317] In some embodiments, the bispecific antigen-binding molecule according to the invention comprises a polypeptide wherein the variable region of the Fab heavy chain of a third Fab molecule shares a C-terminal peptide bond with the constant region of the Fab light chain of the third Fab molecule (i.e., the third Fab molecule contains a cross-linked Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), which in turn shares a C-terminal peptide bond with the variable region of the Fab heavy chain of a first Fab molecule, which in turn shares a C-terminal peptide bond with the constant region of the Fab light chain of the first Fab molecule (i.e., the first Fab molecule contains a cross-linked Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), and subsequently shares a C-terminal peptide bond with the Fab heavy chain of a 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 first Fab molecule (VL) (1) -CH1 (1) The variable region of the Fab light chain of the first Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule; and the variable region of the Fab light chain of the second Fab molecule (VL) shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain of the first Fab molecule. (2) -CL (2) The Fab light chain polypeptide. In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide, wherein a third Fab molecule (VL) is a Fab light chain polypeptide. (3) -CH1 (3) The variable region of the Fab light chain of the third Fab molecule shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain.

[0318] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:84, and HCDR 3 of SEQ ID NO:86; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:83, and LCDR 3 of SEQ ID NO:89; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain HCDR 2 of SEQ ID NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102, and LCDR of SEQ ID NO:103. 3; c) a third antigen-binding portion that binds to the first antigen, and the third antigen-binding portion being identical to the first antigen-binding portion; and d) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0319] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:85, and HCDR 3 of SEQ ID NO:86; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, the heavy chain complementarity-determining region (LCDR) 1 of SEQ ID NO:83, the heavy chain complementarity-determining region (LCDR) 2 of SEQ ID NO:87, and LCDR 3 of SEQ ID NO:89; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:87, the HCDR 2 of SEQ ID NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102, and LCDR of SEQ ID NO:103. 3; c) a third antigen-binding portion that binds to the first antigen, and the third antigen-binding portion being identical to the first antigen-binding portion; and d) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0320] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the Fab light chain is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the Fab light chain is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determ HCDR 2 of SEQ ID NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102, and LCDR of SEQ ID NO:103. 3; c) a third antigen-binding portion that binds to the first antigen, and the third antigen-binding portion being identical to the first antigen-binding portion; and d) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0321] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:94, and LCDR 3 of SEQ ID NO:97; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain HCDR 2 of SEQ ID NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102, and LCDR of SEQ ID NO:103. 3; c) a third antigen-binding portion that binds to the first antigen, and the third antigen-binding portion being identical to the first antigen-binding portion; and d) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0322] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:92, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain HCDR 2 of SEQ ID NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102, and LCDR of SEQ ID NO:103. 3; c) a third antigen-binding portion that binds to the first antigen, and the third antigen-binding portion being identical to the first antigen-binding portion; and d) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0323] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:1, HCDR2 of SEQ ID NO:2, and HCDR3 of SEQ ID NO:3; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5, and LCDR3 of SEQ ID NO:6; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:98, ... HCDR 2 of SEQ ID NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102, and LCDR of SEQ ID NO:103. 3; c) a third antigen-binding portion that binds to the first antigen, and the third antigen-binding portion being identical to the first antigen-binding portion; and d) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0324] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8, and HCDR3 of SEQ ID NO:9; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:10, LCDR2 of SEQ ID NO:11, and LCDR3 of SEQ ID NO:12; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:98, the heavy chain complementarity-determining region (LCDR)1 of SEQ ID NO:10, the light chain complementarity-determining region (LCDR)2 of SEQ ID NO:11, and LCDR3 of SEQ ID NO:12; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:98, the heavy chain complementarity-determining region (LCDR HCDR 2 of SEQ ID NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102, and LCDR of SEQ ID NO:103. 3; c) a third antigen-binding portion that binds to the first antigen, and the third antigen-binding portion being identical to the first antigen-binding portion; and d) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0325] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:83, HCDR2 of SEQ ID NO:84, and HCDR3 of SEQ ID NO:86; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:87, LCDR2 of SEQ ID NO:88, and LCDR3 of SEQ ID NO:89; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:98, SEQ ID NO:89, HCDR2 of SEQ ID NO:84, and LCDR3 of SEQ ID NO:89; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:87, LCDR2 of SEQ ID NO:88, and LCDR3 of SEQ ID NO:89; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-de HCDR 2 of NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, an LCDR 2 of SEQ ID NO:102, and an LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding moiety described in a) and the second antigen-binding moiety described in 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 described in c).

[0326] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:83, HCDR2 of SEQ ID NO:85, and HCDR3 of SEQ ID NO:86; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:87, LCDR2 of SEQ ID NO:88, and LCDR3 of SEQ ID NO:89; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:98, the heavy chain complementarity-determining region (LCDR)1 of SEQ ID NO:87, the heavy chain complementarity-determining region (LCDR)2 of SEQ ID NO:88, and LCDR3 of SEQ ID NO:89; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:87, the heavy chain complementarity-determining region ( HCDR 2 of NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, an LCDR 2 of SEQ ID NO:102, and an LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding moiety described in a) and the second antigen-binding moiety described in 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 described in c).

[0327] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:94, and LCDR 3 of SEQ ID NO:97; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complement HCDR 2 of NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, an LCDR 2 of SEQ ID NO:102, and an LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding moiety described in a) and the second antigen-binding moiety described in 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 described in c).

[0328] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:94, and LCDR 3 of SEQ ID NO:97; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complement HCDR 2 of NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, an LCDR 2 of SEQ ID NO:102, and an LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding moiety described in a) and the second antigen-binding moiety described in 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 described in c).

[0329] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:90, HCDR2 of SEQ ID NO:92, and HCDR3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:94, LCDR2 of SEQ ID NO:95, and LCDR3 of SEQ ID NO:97; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:98, HCDR2 of SEQ ID NO:90, HCDR3 of SEQ ID NO:93, and LCDR3 of SEQ ID NO:97; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:94, LCDR2 of SEQ ID NO:95, and LCDR3 of SEQ ID NO:97; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain HCDR 2 of NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, an LCDR 2 of SEQ ID NO:102, and an LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding moiety described in a) and the second antigen-binding moiety described in 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 described in c).

[0330] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:1, HCDR2 of SEQ ID NO:2, and HCDR3 of SEQ ID NO:3; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5, and LCDR3 of SEQ ID NO:6; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:98, ... HCDR 2 of NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, an LCDR 2 of SEQ ID NO:102, and an LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding moiety described in a) and the second antigen-binding moiety described in 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 described in c).

[0331] In another embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8, and HCDR3 of SEQ ID NO:9; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:10, LCDR2 of SEQ ID NO:11, and LCDR3 of SEQ ID NO:12; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:98, SEQ ID NO:10, SEQ ID NO:11, and LCDR3 of SEQ ID NO:12; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:10, SEQ ID NO:11, and LCDR3 of SEQ ID NO:12; b) a second antigen-binding moiety binding 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 is substituted with the variable domains VL and VH or the constant domains CL and CH1 of the Fab heavy chain, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ HCDR 2 of NO:99 and HCDR 3 of SEQ ID NO:100; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, an LCDR 2 of SEQ ID NO:102, and an LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein (i) the first antigen-binding moiety described in a) and the second antigen-binding moiety described in 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 described in c).

[0332] In all the different configurations of the bispecific antigen-binding molecule according to the invention, the amino acid substitutions described herein (in the presence) may be in the CH1 and CL domains of the first antigen-binding moiety and (in the presence) 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 (in the presence) the third antigen-binding moiety / Fab molecule. According to the concept of the invention, if the amino acid substitutions described herein occur in the first antigen-binding moiety (and, in the presence, the third) antigen-binding moiety / Fab molecule, then such amino acid substitutions are not present in the second antigen-binding moiety / Fab molecule. Conversely, if the amino acid substitutions described herein occur in the second antigen-binding moiety / Fab molecule, then such amino acid substitutions are not present in the first (and, in the presence, the third) antigen-binding moiety / Fab molecule. Amino acid substitutions are particularly carried out in bispecific antigen-binding molecules comprising Fab molecules in which the Fab light chain substitutes for the variable domains VL and VH1 of the Fab heavy chain.

[0333] In specific embodiments of the bispecific antigen-binding molecule according to the invention, particularly where amino acid substitutions as described herein occur in the first (and, in the presence, the third) antigen-binding moiety / Fab molecule, the constant domain CL of the first (and, in the presence, the third) Fab molecule is κ isoform. In other embodiments of the bispecific antigen-binding molecule according to the invention, particularly where amino acid substitutions as described herein occur in the second antigen-binding moiety / Fab molecule, the constant domain CL of the second antigen-binding moiety / Fab molecule is κ isoform. In some embodiments, the constant domain CL of the first (and, in the presence, the third) antigen-binding moiety / Fab molecule and the constant domain CL of the second antigen-binding moiety / Fab molecule are κ isoform.

[0334] In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:84, and HCDR 3 of SEQ ID NO:86; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98 and HCDR 3 of SEQ ID NO:99. 2 and HCDR3 of SEQ ID NO:100; and light chain variable region (VL) comprising light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102 and LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described below a), the amino acid at position 124 is replaced by lysine (K) (according to Kabat number), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat number) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described below a), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat EU index number), and the amino acid at position 213 is replaced by glutamic acid (E) (according to Kabat EU index number). (EU index number) is replaced; and wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c).

[0335] In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:85, and HCDR 3 of SEQ ID NO:86; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98 and HCDR 3 of SEQ ID NO:99. 2 and HCDR3 of SEQ ID NO:100; and light chain variable region (VL) comprising light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102 and LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described below a), the amino acid at position 124 is replaced by lysine (K) (according to Kabat number), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat number) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described below a), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat EU index number), and the amino acid at position 213 is replaced by glutamic acid (E) (according to Kabat EU index number). (EU index number) is replaced; and wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c).

[0336] In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:99. 2 and HCDR3 of SEQ ID NO:100; and light chain variable region (VL) comprising light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102 and LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described below a), the amino acid at position 124 is replaced by lysine (K) (according to Kabat number), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat number) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described below a), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat EU index number), and the amino acid at position 213 is replaced by glutamic acid (E) (according to Kabat EU index number). (EU index number) is replaced; and wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c).

[0337] In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:99. 2 and HCDR3 of SEQ ID NO:100; and light chain variable region (VL) comprising light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102 and LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described below a), the amino acid at position 124 is replaced by lysine (K) (according to Kabat number), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat number) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described below a), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat EU index number), and the amino acid at position 213 is replaced by glutamic acid (E) (according to Kabat EU index number). (EU index number) is replaced; and wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c).

[0338] In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:92, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:99. 2 and HCDR3 of SEQ ID NO:100; and light chain variable region (VL) comprising light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102 and LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described below a), the amino acid at position 124 is replaced by lysine (K) (according to Kabat number), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat number) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described below a), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat EU index number), and the amino acid at position 213 is replaced by glutamic acid (E) (according to Kabat EU index number). (EU index number) is replaced; and wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c).

[0339] In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:1, HCDR 2 of SEQ ID NO:2, and HCDR 3 of SEQ ID NO:3; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:4, LCDR 2 of SEQ ID NO:5, and LCDR 3 of SEQ ID NO:6; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the Fab light chain and the variable domains VL and VH of the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and HCDR 3 of SEQ ID NO:6; and a light chain variable region (VL) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:6; HCDR3 of NO:100; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, the LCDR 2 of SEQ ID NO:102, and the LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described below a), the amino acid at position 124 is replaced by lysine (K) (according to Kabat number), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat number) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described below a), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat EU index number), and the amino acid at position 213 is replaced by glutamic acid (E) (according to Kabat EU index number). (EU index number) is replaced; and wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c).

[0340] In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:7, HCDR 2 of SEQ ID NO:8, and HCDR 3 of SEQ ID NO:9; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:10, LCDR 2 of SEQ ID NO:11, and LCDR 3 of SEQ ID NO:12; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:12; and a light chain variable region (VL) comprising the light chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:12; HCDR3 of NO:100; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, the LCDR 2 of SEQ ID NO:102, and the LCDR 3 of SEQ ID NO:103; c) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described below a), the amino acid at position 124 is replaced by lysine (K) (according to Kabat number), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat number) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described below a), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat EU index number), and the amino acid at position 213 is replaced by glutamic acid (E) (according to Kabat EU index number). (EU index number) is replaced; and wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c); or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described in c).

[0341] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:84, and HCDR 3 of SEQ ID NO:86; and a light chain variable region (VL) comprising light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:89. 2 and HCDR 3 of SEQ ID NO:100; and light chain variable region (VL) comprising light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102 and LCDR 3 of SEQ ID NO:103; c) a third antigen-binding moiety that binds to the first antigen, and the third antigen-binding moiety is identical to the first antigen-binding moiety; and d) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat numbering) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat numbering). The amino acid at position 213 was replaced by glutamic acid (E) (according to the Kabat EU index number);And wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d), or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0342] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:83, HCDR 2 of SEQ ID NO:85, and HCDR 3 of SEQ ID NO:86; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:87, LCDR 2 of SEQ ID NO:88, and LCDR 3 of SEQ ID NO:89; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98 and HCDR 3 of SEQ ID NO:99. 2 and HCDR 3 of SEQ ID NO:100; and light chain variable region (VL) comprising light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102 and LCDR 3 of SEQ ID NO:103; c) a third antigen-binding moiety that binds to the first antigen, and the third antigen-binding moiety is identical to the first antigen-binding moiety; and d) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat numbering) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat numbering). The amino acid at position 213 was replaced by glutamic acid (E) (according to the Kabat EU index number);And wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d), or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0343] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:99. 2 and HCDR 3 of SEQ ID NO:100; and light chain variable region (VL) comprising light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102 and LCDR 3 of SEQ ID NO:103; c) a third antigen-binding moiety that binds to the first antigen, and the third antigen-binding moiety is identical to the first antigen-binding moiety; and d) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat numbering) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat numbering). The amino acid at position 213 was replaced by glutamic acid (E) (according to the Kabat EU index number);And wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d), or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0344] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:91, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:96, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:99. 2 and HCDR 3 of SEQ ID NO:100; and light chain variable region (VL) comprising light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102 and LCDR 3 of SEQ ID NO:103; c) a third antigen-binding moiety that binds to the first antigen, and the third antigen-binding moiety is identical to the first antigen-binding moiety; and d) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat numbering) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat numbering). The amino acid at position 213 was replaced by glutamic acid (E) (according to the Kabat EU index number);And wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d), or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0345] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding portion binding to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding portion is a Fab molecule, the Fab molecule comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:90, HCDR 2 of SEQ ID NO:92, and HCDR 3 of SEQ ID NO:93; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:94, LCDR 2 of SEQ ID NO:95, and LCDR 3 of SEQ ID NO:97; b) a second antigen-binding portion binding to a second antigen, wherein the second antigen is CD3 and the second antigen-binding portion is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:99. 2 and HCDR 3 of SEQ ID NO:100; and light chain variable region (VL) comprising light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, LCDR 2 of SEQ ID NO:102 and LCDR 3 of SEQ ID NO:103; c) a third antigen-binding moiety that binds to the first antigen, and the third antigen-binding moiety is identical to the first antigen-binding moiety; and d) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat numbering) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat numbering). The amino acid at position 213 was replaced by glutamic acid (E) (according to the Kabat EU index number);And wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d), or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0346] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:1, HCDR 2 of SEQ ID NO:2, and HCDR 3 of SEQ ID NO:3; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:4, LCDR 2 of SEQ ID NO:5, and LCDR 3 of SEQ ID NO:6; b) a second antigen-binding moiety binding 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 substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and HCDR 3 of SEQ ID NO:6; and a light chain variable region (VL) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:6; HCDR3 of NO:100; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, an LCDR 2 of SEQ ID NO:102, and an LCDR 3 of SEQ ID NO:103; c) a third antigen-binding moiety that binds to the first antigen, and the third antigen-binding moiety being identical to the first antigen-binding moiety; and d) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat numbering) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat numbering). The amino acid at position 213 was replaced by glutamic acid (E) (according to the Kabat EU index number);And wherein (i) the first antigen-binding portion described in a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion described in b), and the second antigen-binding portion described in b) and the third antigen-binding portion described in 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 in d), or (ii) the second antigen-binding portion described in b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding portion described in a), and the first antigen-binding portion described in a) and the third antigen-binding portion described in 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 in d).

[0347] In one specific embodiment, the present invention provides a bispecific antigen-binding molecule comprising: a) a first antigen-binding moiety binding 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 the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:7, HCDR 2 of SEQ ID NO:8, and HCDR 3 of SEQ ID NO:9; and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:10, LCDR 2 of SEQ ID NO:11, and LCDR 3 of SEQ ID NO:12; b) a second antigen-binding moiety binding 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 substituted for each other, and wherein the Fab molecule comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and HCDR 3 of SEQ ID NO:12; and a light chain variable region (VL) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:98, HCDR 2 of SEQ ID NO:99, and LCDR 3 of SEQ ID NO:12; HCDR 3 of ID NO:100; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:101, an LCDR 2 of SEQ ID NO:102, and an LCDR 3 of SEQ ID NO:103; c) a third antigen-binding moiety that binds to the first antigen, and the third antigen-binding moiety being identical to the first antigen-binding moiety; and d) an Fc domain consisting of a first subunit and a second subunit; wherein in the constant domain CL of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced by either lysine (K) or arginine (R) (according to Kabat numbering) (most particularly replaced by arginine (R), and wherein in the constant domain CH1 of the first antigen-binding moiety described in a) and the third antigen-binding moiety described in c), the amino acid at position 147 is replaced by glutamic acid (E) (according t...

Claims

1. 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 comprises: (i) a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 83, HCDR 2 of SEQ ID NO: 84, and HCDR 3 of SEQ ID NO: 86; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 of SEQ ID NO: 88, and LCDR 3 of SEQ ID NO: 89; (ii) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 83, HCDR 2 of SEQ ID NO: 85, and HCDR 3 of SEQ ID NO: 86; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 of SEQ ID NO: 88, and LCDR 3 of SEQ ID NO: 89; (iii) a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 90, HCDR 2 of SEQ ID NO: 91, and HCDR 3 of SEQ ID NO: 93; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 of SEQ ID NO: 95, and LCDR 3 of SEQ ID NO: 97; (iv) a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 90, HCDR 2 of SEQ ID NO: 91, and HCDR 3 of SEQ ID NO: 93; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 of SEQ ID NO: 96, and LCDR 3 of SEQ ID NO: 97; or (v) a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 90, HCDR 2 of SEQ ID NO: 92, and HCDR 3 of SEQ ID NO: 93; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 of SEQ ID NO: 95, and LCDR 3 of SEQ ID NO: 97; and (b) a second antigen binding moiety that binds to a second antigen, wherein the second antigen is CD3, and the second antigen binding moiety comprises: (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 29, HCDR 2 of SEQ ID NO: 30, and HCDR 3 of SEQ ID NO: 31; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 32, LCDR 2 of SEQ ID NO: 33, and LCDR 3 of SEQ ID NO: 34; (i) a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 98, HCDR 2 of SEQ ID NO: 99, and HCDR 3 of SEQ ID NO: 100; and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 101, LCDR 2 of SEQ ID NO: 102, and LCDR 3 of SEQ ID NO: 103; or (ii) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, HCDR 2 of SEQ ID NO: 107, and HCDR 3 of SEQ ID NO: 108; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, LCDR 2 of SEQ ID NO: 110, and LCDR 3 of SEQ ID NO:

111.

2. The bispecific antigen-binding molecule according to claim 1, (i) wherein the VH of the first antigen binding moiety comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 13, and wherein the VL of the first antigen binding moiety comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14; or (ii) wherein the VH of the first antigen binding moiety comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 15, and wherein the VL of the first antigen binding moiety comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16; or (iii) wherein the VH of the first antigen binding moiety comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:48, and wherein the VL of the first antigen binding moiety comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:53; or (iv) wherein the VH of the first antigen binding moiety comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:49, and wherein the VL of the first antigen binding moiety comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:52; or (v) wherein the VH of the first antigen binding moiety comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:57, and wherein the VL of the first antigen binding moiety comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:64; or (vi) wherein 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 sequence of SEQ ID NO:58, and wherein 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:

63.

3. The bispecific antigen binding molecule according to claim 1 or 2, wherein the VH of the second antigen binding moiety comprises (i) 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:35, and the VL of the second antigen binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:36; (ii) 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: 104, and the VL of the second antigen binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 105; or (iii) 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:112, and the VL of the second antigen binding portion comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

113. 4 . The bispecific antigen binding molecule according to claim 1 , wherein the first antigen binding moiety and / or the second antigen binding moiety is a Fab molecule.

5. The bispecific antigen binding molecule according to any one of claims 1 to 4, wherein 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 replaced with each other.

6. A bispecific antigen binding molecule according to any one of claims 1 to 5, wherein the first antigen binding moiety is a Fab molecule, wherein in the constant domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index numbering).

7. The bispecific antigen binding molecule according to any one of claims 1 to 6, wherein the first antigen binding moiety and the second antigen binding moiety are fused to each other, optionally via a peptide linker.

8. The bispecific antigen binding molecule according to any one of claims 1 to 7, wherein the first antigen binding moiety and the second antigen binding moiety are each a Fab molecule, and wherein (i) the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, or (ii) the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety.

9. The bispecific antigen binding molecule according to any one of claims 1 to 8, comprising a third antigen binding moiety.

10. The bispecific antigen binding molecule of claim 9, wherein the third antigen moiety is identical to the first antigen binding moiety.

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