RU2797268C2 - Antibodies binding to gprc5d - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an antibody or its antigen-binding fragment that binds to GPRC5D, compositions containing it and a method for obtaining it, as well as to an isolated polynucleotide encoding it, a vector and a cell containing the above polynucleotide. Also a bispecific antigen-binding molecule that specifically binds to GPRC5D and CD3, a composition containing it and a method of obtaining it, as well as an isolated polynucleotide encoding it, a vector and a cell containing the above polynucleotide are disclosed.

EFFECT: invention is effective for the treatment of multiple myeloma.

37 cl, 15 dwg, 13 tbl, 12 ex

Description

Technical field

The present invention generally relates to antibodies that bind to GPRC5D, including bispecific antigen-binding molecules, for example, to activate T cells. In addition, the present invention relates to polynucleotides encoding such antibodies, as well as to vectors and host cells containing such polynucleotides. The present invention also relates to methods for producing antibodies and methods for their use in the treatment of diseases.

Prior Art

Multiple myeloma (MM), affecting about 75,000 new patients each year in the EU and US, is one of the most common hematological malignancies for which there is no effective treatment. MM is characterized by terminally differentiated plasma cells that secrete nonfunctional monoclonal immunoglobulins. In the short term, immunomodulatory drugs such as lenalidomide and pomalidomide, as well as proteasome inhibitors such as carfilzomib or bortezomib, may remain the mainstay of first-line therapy in MM (Moreau P., Rajkumar S.V., Lancet, 388(10040), 2016, 111- 113). However, these drugs do not directly target diseased tumor cells, such as diseased plasma cells (PCs). Attempts have been made to selectively deplete plasma cells in MM. The absence of surface proteins, specific markers of PK, hinders the development of antibodies or cell therapy in MM. So far, there are several examples of successful biologics, such as daratumumab (anti-CD38) and elotuzumab (anti-CD319), with the caveat that these two molecules are not only expressed by plasma cells. Therefore, new plasma cell targets in multiple myeloma have been identified using RNA sequencing, for example, G protein-coupled receptor class C, member of group 5 D (GPRC5D). GPRC5D is a specific surface protein expressed by plasma cells in multiple myeloma. GPRC5D has been reported to be associated with prognosis and tumor burden in patients with multiple myeloma (Atamaniuk, J. et al. Eur J Clin Invest, 42(9), 2012, 953-960; Cohen Y. et al., Hematology, 18( 6), 2013, 348-351).

GPRC5D is an orphan receptor whose ligand or function is not known in healthy individuals or in cancer cases. The gene encoding GPRC5D is located on chromosome 12p13.3, it contains three exons and is approximately 9.6 kb long. (Brauner-Osborae H. et al., Biochim. Biophys Acta, 1518(3), 2001, 237-248). The large first exon encodes a transmembrane domain spanning the membrane seven times. However, the biology of GPRC5D is largely unknown. GPRC5D has been shown to be involved in keratin formation in animal hair follicles (Gao Y. et al., PLoS One, 11(3), 2016, e0151118; Inoue S. et al., J Invest Dermatol, 122(3), 2004 , 565-573).

WO 2018/017786 A2 describes GPRC5D-specific antibodies or antigen-binding fragments. There is a need to develop additional drugs for the treatment of cancer, in particular multiple myeloma. Particularly useful drugs for this purpose include antibodies that bind GPRC5D, in particular bispecific antibodies that bind GPRC5D on target cells and an activating T cell antigen, eg CD3 on T cells. Simultaneous binding of such an antibody to both of these targets will cause a transient interaction between the target cell and the T cell, causing the activation of any cytotoxic T cell and subsequent lysis of the target cell.

The present invention provides novel antibodies, including bispecific antibodies, that specifically bind human GPRC5D. In particular, the T cell bispecific antibodies of the present invention targeting GPRC5D are capable of treating multiple myeloma.

Brief description of the invention

The present invention provides a new antibody with unexpected improved properties that binds to GPRC5D. In addition, bispecific antigen-binding molecules have been developed that bind to GPRC5D and an activating T-cell antigen, including a novel anti-GPRC5D antibody.

In a first aspect of the present invention, an antibody is provided that binds to GPRC5D, wherein the antibody comprises (i) 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: 84 and HCDR 3 of SEQ ID NO: 86, and a light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87. LCDR 2 of SEQ ID NO: 88 and LCDR 3 sequences 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 variable light chain 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, (iii) heavy chain variable region (VH) containing 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 a 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; (iv) 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 variable a light chain region (VL) comprising a 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; or (v) and a heavy chain variable region (VH) comprising a 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 a 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. Alternatively, the antibody may include (I) a heavy chain variable region (VH) comprising a 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 a 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; or (II) a heavy chain variable region (VH, including 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 variable a light chain region (VL) comprising a 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. In one embodiment of the present invention (i) VH contains 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 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: 14; or (ii) VH contains 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 VL contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence SEQ ID NO: 16; or (iii) VH 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: 48, and VL contains an amino acid sequence that is at least least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53; or (iv) VH contains 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 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: 52; or (v) VH contains 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 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: 64; or (vi) VH contains 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 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. In another embodiment of the present invention (i) VH contains the amino acid sequence of SEQ ID NO: 13, VL contains the amino acid the sequence of SEQ ID NO: 14; or (ii) VH contains the amino acid sequence of SEQ ID NO: 15, and VL contains the amino acid sequence of SEQ ID NO: 16; or (iii) VH contains the amino acid sequence of SEQ ID NO: 48, and VL contains the amino acid sequence of SEQ ID NO: 53; or (iv) VH contains the amino acid sequence of SEQ ID NO: 49 and VL contains the amino acid sequence of SEQ ID NO: 52; or (v) VH contains the amino acid sequence of SEQ ID NO: 57 and VL contains the amino acid sequence of SEQ ID NO: 64; or (vi) VH contains the amino acid sequence of EQ ID NO: 58, and VL contains the amino acid sequence of SEQ ID NO: 63. In another embodiment of the present invention, the antibody is an IgG, especially an IgG ₁ antibody. In one embodiment of the present invention, the antibody is a full length antibody. In another embodiment of the present invention, the antibody is an antibody fragment selected from the group consisting of Fv molecules, scFv molecules, Fab molecules and F(ab') _{2 molecules} . In one of the embodiments of the present invention, the antibody is a multispecific antibody.

Another aspect of the present invention relates to a bispecific antigen-binding molecule comprising (a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment comprises (i) a heavy chain variable region (VH) comprising a heavy chain region, complementarity determining (HCDR) 1 sequences of SEQ ID NO: 83, HCDR 2 sequences of SEQ ID NO: 84, and HCDR 3 sequences of SEQ ID NO: 86, and a light chain variable region (VL) including a light chain complementarity determining region (LCDR ) 1 sequences of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences 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 variable a light chain 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 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 variable a light chain region (VL) comprising a 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; (iv) 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 variable a light chain region (VL) comprising a 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; or (v) a heavy chain variable region (VH) comprising a 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 a 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. In another embodiment, the first antigen-binding fragment may include (I) a heavy chain variable region (VH) comprising a 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 a 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; or (II) a heavy chain variable region (VH) comprising a 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 a 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 fragment that specifically binds to the second antigen. In one embodiment of the present invention, (i) the VH of the first antigen-binding fragment contains 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 The VL of the first antigen-binding fragment 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: 14; or (ii) the VH of the first antigen-binding fragment contains 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 the VL of the first antigen-binding fragment contains an amino acid a 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) the VH of the first antigen-binding fragment contains 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 the VL of the first antigen-binding fragment contains an amino acid a 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) the VH of the first antigen-binding fragment contains 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 the VL of the first antigen-binding fragment contains an amino acid a 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) the VH of the first antigen-binding fragment contains 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 the VL of the first antigen-binding fragment contains an amino acid a 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) the VH of the first antigen-binding fragment contains 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 the VL of the first antigen-binding fragment contains an amino acid a sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 63. the sequence of SEQ ID NO: 13, and the VL of the first antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 14; or (ii) the VH of the first antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 15, and the VL of the first antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 16; or (iii) the VH of the first antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 48, and the VL of the first antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 53; or (iv) the VH of the first antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 49, and the VL of the first antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 52; or (v) the VH of the first antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 57, and the VL of the first antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 64; or (vi) the VH of the first antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 58, and the VL of the first antigen-binding fragment contains the amino acid sequence of SEQ ID NO: 63. In another embodiment of the present invention, the second antigen is CD3, especially CD3e. In one embodiment, the second antigen-binding fragment comprises a VH comprising HCDR 1 of SEQ ID NO: 29, HCDR 2 of SEQ ID NO: 30, and HCDR 3 of SEQ ID NO: 31, and a VL comprising LCDR 1 of SEQ ID NO: 32, LCDR 2 of SEQ ID NO: 33, and LCDR 3 of SEQ ID NO: 34. In another embodiment of the present invention, the VH of the second antigen binding fragment 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: 35, and the VL of the second antigen-binding fragment 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: 36. In another embodiment of the present invention, the VH of the second antigen-binding fragment contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identical. SEQ ID NO: 35 and the VL of the second antigen binding fragment comprises the amino acid sequence of SEQ ID NO: 36. In one embodiment of the present invention, the first and/or second antigen binding fragment is a Fab molecule. This means that either the first antigen-binding fragment may be a Fab molecule, or the second antigen-binding fragment may be a Fab molecule, or both the first antigen-binding fragment and the second antigen-binding fragments may be Fab molecules. In another embodiment of the present invention, the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL and CH1 constant domains, in particular the VL and VH variable domains, of the Fab light chain and the Fab heavy chain are exchanged for each other. In another embodiment of the present invention, the first antigen-binding fragment is a Fab molecule in which the constant domain at amino acid position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (Kabat numbering) and the amino acid at position 123 is independently replaced by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the CH1 constant domain the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (EC numbering according to Kabat), and the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (EC Kabat numbering). In another embodiment of the present invention, the first and second antigen binding fragments are fused to each other, optionally via a peptide linker. In another embodiment of the present invention, the first and second antigen-binding fragments are both in a Fab molecule, wherein either (i) the second antigen-binding fragment 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 fragment, or (ii) the first antigen-binding fragment the fragment 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 fragment. In another embodiment of the present invention, the bispecific antigen-binding molecule comprises a third antigen-binding moiety. In another embodiment of the present invention, the third antigennegative fragment is identical to the first antigennegative fragment. In another embodiment of the present invention, the bispecific antigen-binding molecule comprises an Fc domain consisting of first and second subunits. In another embodiment of the present invention, each of the first, second and, if present, third antigen-binding fragments is a Fab molecule; and either (i) the second antigen-binding fragment 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 fragment, and the first antigen-binding fragment is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain towards the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding fragment 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 fragment, and the second antigen-binding fragment is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the domain Fc; and the third antigen-binding fragment, if present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. In another embodiment of the present invention, the Fc domain is an IgG domain, in particular an IgG ₁ Fc domain. In yet another embodiment of the present invention, the Fc domain is a human Fc domain. In another embodiment of the present invention, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain, thereby creating an overhang in the CH3 domain of the first subunit that can be located in the recess of the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit The CH3 of the second subunit of the Fc domain is replaced by an amino acid residue having a smaller side chain, thereby forming a trough in the CH3 domain of the second subunit, within which the overhang of the CH3 domain of the first subunit can be located. In another embodiment of the present invention, the Fc domain contains one or more amino acid substitutions that reduce Fc receptor binding and/or effector function. This means that Fc receptor binding may be reduced, or effector function may be reduced, or Fc receptor binding and effector function may be reduced.

In another aspect of the present invention, one or more isolated polynucleotides are provided that encode an antibody or a bispecific antigen-binding molecule as described herein. In another aspect of the present invention, one or more vectors are provided, in particular an expression vector containing the polynucleotide(s) of the present invention. In another aspect of the present invention, a host cell is provided comprising the polynucleotide(s) of the present invention or the vector(s) of the present invention. In some embodiments of the present invention, the host cell is a eukaryotic cell, especially a mammalian cell.

In another aspect of the present invention, a method for producing an antibody that binds to GPRC5D is provided, comprising the steps of: a) culturing a host cell as described herein under conditions suitable for antibody expression, and b) optionally isolating the antibody. In another aspect of the present invention, an antibody is provided that binds to GPRC5D obtained by the method described in the present invention. Another object of the present invention relates to a pharmaceutical composition comprising an antibody or bespecifically antigen-binding molecule described in the present invention, and a pharmaceutically acceptable carrier. Another object of the present invention relates to an antibody, or a bispecific antigen-binding molecule described in the present invention, or a pharmaceutical composition described in the present invention, for use as a medicine. Another object of the present invention relates to an antibody, or a bispecific antigen-binding molecule described in the present invention, or a pharmaceutical composition described in the present invention, for use in the treatment of a disease. In one of the embodiments of the present invention, the disease is cancer, especially multiple myeloma. In another embodiment of the present invention, the disease is an autoimmune disease, such as systemic lupus erythematosus and/or rheumatoid arthritis.

Another object of the present invention relates to a method of treating a disease, in particular cancer, more specifically multiple myeloma, in an individual, comprising administering to said individual a therapeutically effective amount of a composition containing an antibody or a bispecific antigen-binding molecule according to the description of the present invention in a pharmaceutically acceptable form. In another embodiment, the disease is an autoimmune disease, such as systemic lupus erythematosus and/or rheumatoid arthritis. In any of the above embodiments of the present invention, the individual is preferably a mammal, in particular a human.

Brief description of the figures

Fig. 1A-E. Configuration examples of the bispecific antigen-binding molecules of the present invention. (Fig. 1A, Fig. 2D) The structure of the "1+1CrossMab" molecule. (Fig. 1B, Fig. 1D) The structure of the molecule "2+1 IgG Crossfab" with an alternative arrangement of the components of Crossfab and Fab ("inverted"). (Fig. 1B, Fig. 1E) The structure of the "2+1 IgG Crossfab" molecule. (Fig. 1G, Fig. 1L) The structure of the "1 + 1 IgG Crossfab" molecule with an alternative arrangement of the Crossfab and Fab components ("inverted"). (Fig. 1H, Fig. 1M) The structure of the "1+1 IgG Crossfab" molecule. (FIG. 1I, FIG. 1H) Molecule structure of a "2+1 IgG Crossfab" with two CrossFabs. (FIG. 1K, FIG. 10) "2+1 IgG Crossfab" molecular structure with two CrossFabs and an alternate arrangement of the Crossfab and Fab components ("inverted"). (Fig. 1P, Fig. 1U) The structure of the "Fab-Crossfab" molecule. (Fig. 1P, Fig. 1F) The structure of the "Crossfab-Fab" molecule. (Fig. 1C, Fig. 1X) The structure of the "(Fab) ₂ -Crossfab" molecule. (Fig. 1T, Fig. 1C) Molecular structure of "Crossfab-Fab ₂ ". (Fig. 1H, Fig. 1Sh) The structure of the "Fab-(Crossfab) ₂ " molecule. (Fig. 1Sh, Fig. 1E) The structure of the "(Crossfab) ₂ -Fab" molecule. Black dot: optional modification in the Fc domain to promote heterodimerization. ++, --: amino acids of opposite charges, optionally introduced into the CH1 and CL domains. Crossfab molecules are shown to include an exchange of VH and VL regions, but in some embodiments of the present invention, when no charge modifications are introduced into the CH1 and CL domains, in another embodiment, an exchange of CH1 and CL domains may be included.

Fig. 2. Analysis of gene expression of tumor targets on plasma cells and B cells by RNAseq.

Fig. 3. Configuration examples of 5E11 bispecific antigen binding molecules of the present invention. Black dot:

an optional modification in the Fc domain to promote heterodimerization. ++, -: amino acids of opposite charges, optionally introduced into the CH1 and CL domains.

Fig. 4A-B. Binding analysis of the bispecific antigen-binding molecules 5F11-TCB (Fig. 4A) and 5E11-TCB (Fig. 4B) and control antibody ET150-5-TCB (Fig. 4C) to multiple myeloma (MM) cell lines AMO-1, L636, NCI-H929, RPMI-8226, ORM-2 and WSU-DLCL2.

Fig. 5A-D. Analysis of GPRC5D-TCB mediated T cell cytotoxicity in multiple myeloma cell lines AMO-1 (Fig. 5A), NCI-H929 (Fig. 5B), RPMI-8226 (Fig. 5C), and L363 (Fig. 5D). The control cell line is WSU-DL CL2 (FIG. 5E). Molecules under study: 5E11-TCB, 5F11-TCB. Control molecules: DP47-TCB (untargeted) and ET150-5-TCB.

Fig. 6. Analysis of GPRC5D-TCB activated interaction of T cells with multiple myeloma cell lines NCI-H929 and negative control cell line WSU-DLCL2 upregulating CD25 and CD69.

Fig. 7A-K. Analysis of GPRC5D-TCB activated interaction of T-cells with multiple myeloma upregulating CD25 in cell lines AMO-1 (Fig. 7A), NCI-H929 (Fig. 7B), RPMI-8226 (Fig. 7C), L363 (Fig. 7D) and WSU-DLCL2 (Fig. 7E), and upregulating CD69 upregulating AMO-1 (Fig. 7E), NCI-H929 (Fig. 7G), RPMI-8226 (Fig. 73), L363 (Fig. 7I ) and WSU-DLCL2 (FIG. 7K).

Fig. 8A-B. Visualization of antibody localization and internalization by fluorescence confocal microscopy (Fig. 8B).

Fig. 9. Binding of various anti-GPRC5D antibodies to human, cynomolgus, and mouse GPRC5D as determined by ELISA using stably transfected CHO clones expressing human GPRC5D (clone 12), cynomolgus GPRC5D (clone 13), and mouse GPRC5D (clone 4). ) or human GPRC5A (clone 30).

Fig. 10A-G. T cell-mediated lysis of various multiple myeloma (MM) cell lines induced by different GPRC5D- or BCMA-targeted bispecific T cell molecules during 20 h of co-incubation (E:T=10:1, human pan T cells) . Shows the result for two repetitions, taking into account the standard deviation (SD).

Fig. 11A-E. T cell activation induced by different GPRC5D- or BCMA-targeted bispecific T cell molecules (5E11-TCB in Fig. 11A; 5F11-TCB in Fig. 11B; 10B10-TCB in Fig. 11C; BCMA-TCB in Fig. 11D ; BCMA-TCB in Fig. 11E; DP47-TCB in Fig. 11E) for 20 h of co-incubation of allogeneic human pan-T cells and untreated bone marrow cells from healthy donors (E:T=10:1, pan-T human cells). FACS scatter plots from a single representative donor are shown showing upregulation of the CD69 activation marker on CD4 (top row) or CD8 T cells (bottom row) as a percentage of positive cells among all CD4 corresponding CD8 T cells.

Fig. 12A-B. T cell activation induced by various GPRC5D- or BCMA-targeted bispecific T cell molecules during 20 h co-incubation of allogeneic human pan-T cells and untreated bone marrow cells from healthy donors (E:T=10:1, pan- human T cells). Shown is the sum of data from all four donors studied, demonstrating upregulation of the CD69 activation marker on CD8 T cells at selected fixed doses of either 50 nM TCB (FIG. 12A) or 5 nM (FIG. 12B).

Fig. 13A-D. In vivo potency induced by various GPRC5D-targeting T cell bispecific molecules (5F11-TCB in Figure 13A; BCMA-TCB in Figure 13B; B72-TCB in Figure 13C; solvent in Figure 13D), which follows from the kinetics tumor growth over time in a humanized NSG mouse model inoculated with NCI-H929 tumor cells. The graph shows web diagrams, each line of which refers to a single mouse.

Fig. 14A-D. In vivo potency induced by various GPRC5D-targeting T cell bispecific molecules (5F11-TCB in Fig. 14A; 5E11-TCB in Fig. 14B; B72-TCB in Fig. 14C; solvent in Fig. 14D), which follows from the kinetics tumor growth over time in a humanized NSG mouse model inoculated with OPM-2 tumor cells. The graph shows web diagrams, each line of which refers to a single mouse.

Fig. 15A-B. PGLALA-CAR-J activation after about 16 hours of incubation, determined by luminescence. Luminescence is induced by simultaneous binding of GPRC5D IgGs (5F11-IgG in Fig. 15A; 5E11-IgG in Fig. 15B) to a GPRC50-expressing multiple myeloma cell line L-363 and a PGLALA-modified Fc domain to Jurkat-NFAT reporter cells that were genetically engineered to express TCR directed against the PGLALA mutation in the Fc portion of IgG molecules. The result for two repetitions is shown taking into account the standard deviation (SD).

Detailed description of the invention

Definitions

In the present invention, the terms are used in the sense that is generally accepted in this area, unless otherwise indicated below.

In the context of the present invention, the term "antigen-binding molecule" refers in the broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and their derivatives, eg fragments.

The term "bispecific" means that an antigen-binding molecule is capable of specifically binding to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each specific for a different antigenic determinant. In some embodiments, the bispecific antigen-binding molecule is capable of simultaneously binding two antigenic determinants, in particular two antigenic determinants expressed in two different cells.

In the context of the present invention, the term "valent" means the presence of a certain number of antigennegative sites in the antigennegative molecule. Essentially, the term "monovalent antigen binding" means the presence of one (and no more than one) antigen binding site specific for an antigen in an antigen-binding molecule.

"Antigen binding site" refers to a site, i. one or more amino acid residues of the antigen-binding molecule that allow interaction with the antigen. For example, the antigen-binding site of an antibody contains amino acid residues from complementarity determining regions (CDRs). The native immunoglobulin molecule usually has two antigen binding sites, the Fab molecule usually has one antigen binding site.

In the context of the present invention, the term "antigen-binding fragment" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment of the present invention, the antigen-binding fragment is capable of directing the entity to which it is attached (eg, the second antigen-binding fragment) to a target site, eg, a particular type of antigenic determinant-bearing tumor cell. In another embodiment of the present invention, the antigen-binding fragment is capable of activating signaling through its target antigen, eg, a T-cell receptor complex antigen. Antigen-binding portions include antibodies and fragments thereof, as discussed below. Specific antigen-binding fragments include an antibody antigen-binding domain comprising an antibody heavy chain variable region and an antibody light chain variable region. In some embodiments, antigen-binding fragments may include antibody constant regions, as is known in the art and described below. Applicable heavy chain constant regions refer to any of the five isotypes: α, δ, ε, γ, or μ.

Applicable light chain constant regions are either of two isotypes: κ and λ.

In the context of the present invention, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site (for example, a continuous stretch of amino acids or a conformational configuration consisting of different sections of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen-binding fragment binds, forming antigen-binding fragment-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, in the free state in the blood serum and/or in the extracellular matrix (extracellular matrix, ECM). The proteins referred to herein as antigens (e.g., GPRC5D, CD3) may be any native form of proteins from any vertebrate animal, including those derived from mammals such as primates (e.g., humans, or primates other than humans). , e.g., cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise noted. In one embodiment of the present invention, the antigen is a human protein. When reference is made in the present invention to a specific protein, the term encompasses the unaltered "full length" protein as well as any form of the protein that is the result of processing in the cell. The term also includes naturally occurring protein variants, such as splicing variants or allelic variants. An example of a human protein used as an antigen is CD3, especially the epsilon subunit of CD3 (see UniProt no. P07766 (version 185), NCBI RefSeq no. NP_000724.1, SEQ ID NO: 40 human sequence; or UniProt no. Q95LI5 (version 69), NCBI GenBank no. BAB71849.1, SEQ ID NO: 41 for the cynomolgus macaque [Macaca fascicularis] sequence), or GPRC5D (see UniProt no. Q9NZD1 (version 115); NCBI RefSeq no. NP_061124.1, SEQ ID NO: 45 for human sequence). In some embodiments, the antibody or bispecific antigen-binding molecule of the present invention binds to a CD3 or GPRC5D epitope that is conserved among CD3 or GPRC5D antigens of various species. In one of the embodiments of the present invention, the antibody or bispecific antigen-binding molecule of the present invention binds to human GPRC5D.

The term "specific binding" means that the binding is selective for the antigen and may be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding moiety to bind to a specific antigenic determinant can be measured either by an enzyme-linked immunosorbent assay (ELISA) or by other methods known to those skilled in the art, such as surface plasmon resonance (SPR) (for example, on the BIAcore instrument). ) (Liljeblad et al., Glyco J 17, 2000, 323-329) and traditional binding methods (Heeley, Endocr Res 28, 2002, 217-229). In one of the embodiments of the present invention, the degree of binding of the antigen-binding fragment to the unrelated protein is less than about 10% of the binding of the antigen-binding fragment to the antigen, for example, as measured by the SPR method. In some embodiments of the present invention, the antigen-binding fragment that binds to the antigen, or the antigen-binding molecule containing this antigen-binding fragment, has a dissociation constant (K _D ) ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM or ≤ 0.001 nM (eg 10 ^-8 M or less, eg 10 ^-8 M to 10 ^-13 M, eg 10 ^-9 M to 10 ^-13 M).

The concept of "affinity (affinity)" refers to the strength of the total amount of non-covalent interactions between one binding site of a molecule (eg, receptor) and its binding partner (eg, ligand). Unless otherwise indicated, in the context of the present invention, the term "binding affinity (binding affinity)" refers to intrinsic binding affinity, which reflects a 1:1 interaction between representing a binding pair (for example, an antigen-binding fragment and an antigen or receptor and its ligand). The affinity of a molecule X for its partner Y can usually be expressed by the dissociation constant (K _D ), which is the ratio of the dissociation and association rate constants (k _off and k _on , respectively). Thus, equivalent affinities may include different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by well established methods known in the art, including those described in the present invention. One method for measuring affinity is surface plasmon resonance (SPR).

The term "reduced binding", for example, reduced binding to the Fc receptor, means a decrease in affinity for the corresponding interaction, measured, for example, using SPR. It is worth clarifying that this concept also includes a decrease in affinity to zero (or below the detection limit of the analytical method), that is, the complete cessation of interaction. In contrast, "increased binding" refers to an increase in the binding affinity for the respective interaction.

In the context of the present invention, the term "activating T-cell antigen" refers to an antigenic determinant expressed on the surface of a T-lymphocyte, in particular a cytotoxic T-lymphocyte, which is capable of inducing T-cell activation upon interaction with an antigen-binding molecule. In particular, interaction of an antigen-binding molecule with an activating T cell antigen can induce T cell activation by inducing a T cell receptor complex signaling cascade. In one embodiment of the present invention, the activating T cell antigen is CD3, specifically the CD3 epsilon subunit (see UniProt no. P07766 (version 144), NCBI RefSeq no. NP_000724.1, SEQ ID NO: 40 for the human sequence; or UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, SEQ ID NO: 41 cynomolgus macaque [Macaca fascicularis] sequences).

"T cell activation" in the context of the present invention refers to one or more cellular responses of T lymphocytes, in particular cytotoxic T lymphocytes, selected from: proliferation, differentiation, secretion of cytokines, release of cytotoxic effector molecules, cytotoxic activity and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and are described in the present invention.

In the context of the present invention, the term "target cell antigen" refers to an antigenic determinant on the surface of a target cell, eg a cell in a tumor, eg a cancer cell or tumor stromal cell. In one embodiment of the present invention, the target cell antigen is GPRC5D, in particular human GPRC5D according to SEQ ID NO: 45.

In the present invention, the terms "first", "second", and "third" in relation to Fab molecules, etc. are used to make it convenient to separate when there is more than one fragment of each type. The use of these terms is not intended to define a particular order or orientation of a bispecific antigen-binding molecule unless explicitly indicated.

The term "fused" means that the components (eg, the Fab molecule and the subunit of the Fc domain) are connected by peptide bonds, either directly or through one or more peptide linkers.

The term “Fab molecule” refers to a protein consisting of the VH and CH1 domain of a heavy chain (“Fab of a heavy chain”) and the VL and CL domain of a light chain (“Fab of a light chain”) of an immunoglobulin.

By "crossover" Fab molecule (also referred to as "Crossfab") is meant a Fab molecule in which the variable domains or constant domains of the Fab heavy and light chains are exchanged (i.e. replace each other), i.e. the crossover Fab molecule includes a peptide chain consisting of a VL light chain variable domain and a CH1 heavy chain constant domain 1 (VL-CH1, N- to C-terminal direction), and a peptide chain consisting of a VH heavy chain variable domain and a CL light chain constant domain (VH-CL , in the direction from the N- to the C-terminus). To clarify, in a Fab crossover molecule in which the Fab light chain and Fab heavy chain variable domains are exchanged, the peptide chain containing CH1 heavy chain constant domain 1 is referred to herein as the "heavy chain" (crossover) of the Fab molecule. In contrast, in a crossover Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain VH variable domain is referred to in the present invention as the "heavy chain" of the (crossover) Fab molecule.

In contrast, by "regular" Fab molecule is meant the Fab molecule in its natural format, i.e. containing a heavy chain consisting of the variable and constant domains of the heavy chain (VH-CH1, in the direction from N- to C-terminus), and a light chain consisting of the variable and constant domains of the light chain (VL-CL, in the direction from N- towards the C-terminus).

The term "immunoglobulin molecule" refers to a protein having the structure of a natural antibody. For example, IgG class immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 daltons in size, consisting of two light chains and two heavy chains linked by a disulfide bond. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a heavy chain variable domain or a heavy chain variable region, followed by three constant domains (CH1, CH2 and CH3), also called a heavy chain constant region. Similarly, from the N- to the C-terminus, each light chain has a variable domain (VL), also called a light chain variable domain or a light chain variable region, followed by a light chain constant domain (CL), also called a light chain constant region. An immunoglobulin heavy chain can be assigned to one of five types called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into subtypes, for example, γ ₁ (IgG ₁ ), γ ₂ (IgG ₂ ), γ ₃ (IgG ₃ ), γ ₄ (IgG ₄ ), α ₁ (IgA ₁ ) and α ₂ (IgA ₂ ). An immunoglobulin light chain can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. An immunoglobulin actually consists of two Fab molecules and an Fc domain linked through the immunoglobulin hinge region.

The term "antibody" in the present invention is used in the broadest sense, and it covers various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) and antibody fragments, if they show the required antigen-binding activity.

The term "monoclonal antibody" in the context of the present invention refers to an antibody derived from a population of essentially homogeneous antibodies, i.e. individual antibodies in a population are identical and/or bind to the same epitope, with the exception of possible variants of antibodies, for example, containing natural mutations or arising from the preparation of a monoclonal antibody, such variants are usually present in small quantities. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the term "monoclonal" indicates the property of an antibody as derived from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of an antibody by any particular method. For example, monoclonal antibodies that can be used in accordance with the present invention can be obtained by various methods, including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the immunoglobulin loci. human, such methods and other acceptable methods for obtaining monoclonal antibodies are described in the present invention.

An "isolated" antibody is an antibody that has been separated from components of its natural environment, i.e. an antibody that is not in its natural environment. No special level of cleaning is required. For example, an isolated antibody can be removed from its native or natural environment. Recombinantly produced antibodies expressed in host cells and isolated for the purposes of the present invention are native or recombinant antibodies that have been isolated, fractionated, partially or highly purified by any suitable method. The antibodies and bispecific antigen-binding molecules of the present invention are thus isolated. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, IEF, capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. In 848, 2007, 79-87.

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

The term "antibody fragment" refers to a molecule, other than an intact antibody, which includes a part of the intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabodies, linear antibodies, single chain antibody molecules (eg scFv) and single domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 2003, 129-134. For a review of scFv fragments, see, for example, Pluckthun in: The Pharmacology of Monoclonal Antibodies, vol. 113, 1994, ed. Rosenburg and Moore, Springer-Verlag, New York, 269-315; WO 93/16185; US 5,571,894, US 5,587,458. For a discussion of Fab and F(ab')2 fragments containing reutilization receptor binding epitope residues and having an extended in vivo half-life, see US 5,869,046. Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, for example, EP 404097; WO 1993/01161; Hudson et al., Nat Med 9, 2003, 129-134; Hollinger et al., Proc Natl Acad Sci USA 90, 1993, 6444-6448. Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 2003, 129-134. Single domain antibodies are antibody fragments containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In some embodiments, the implementation of the present invention single-domain antibody is a single-domain human antibody (see, for example, US 6248516 B1). Antibody fragments can be obtained by various methods, including, but not limited to, proteolytic cleavage of intact antibodies, as well as production by recombinant host cells (eg, E. coli or phage), as described in the present invention.

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

The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody typically have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 2007, ^6th ed., WH Freeman and Co., p. 91. A single VH or VL domain may be sufficient to confer antigen-binding specificity. In the present invention, in relation to sequences of variable regions, the term "Kabat numbering" refers to the numbering system developed by Kabat et al., Sequences of Proteins of Immunological Interest, 1991, 5e ed., Public Health Service, National Institutes of Health, Bethesda , Maryland.

In the present invention, the amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 1991, 5e ed., Public Health Service, National Institutes of Health, Bethesda, Maryland, referred to in the present invention as "Kabat numbering" or "Kabat numbering". In particular, the Kabat numbering system (see pages 647-660 in Kabat et al., Sequences of Proteins of Immunological Interest, 1991, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland) is used to light chain constant domain CL of the kappa and lambda isotypes and the Kabat numbering system of the EU index (see pages 661-723) are used for the heavy chain constant domains (CH1, hinge, CH2 and CH3), which in the present invention is here further explained by reference to "numbering according to Kabat index EU".

The term "hypervariable region" or "HVR" in the context of the present invention refers to each of the regions of the variable domain of an antibody having a hypervariable sequence ("complementarity determining region" or "CDR"; CDR region/heavy chain domain are, for example, HCDR1, HCDR2 and HCDR3; light chain CDR region/domain are, for example, LCDR1, LCDR2 and LCDR3) and/or form structurally expressed loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigenic contacts"). Typically, antibodies include six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

Examples of HVRs in the present invention are:

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

(b) CDR regions arising 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, 1991, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD);

(c) antigenic contacts arising at amino acid residues 27 c-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, 1996, 732-745); And

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

Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered here in accordance with Kabat et al., referenced above.

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

The term "humanized" antibody refers to a chimeric antibody containing amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments of the present invention, a humanized antibody may include substantially all of at least one, usually two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to those HVRs that are not related to the human antibody, and all or substantially all FRs that match the FR from the human antibody. In the present invention, such variable domains are referred to as "humanized variable regions". The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, certain FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody. A "humanized form" of an antibody, such as "non-human antibodies", refers to an antibody that has been humanized. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been further modified or altered from that of the parent antibody to impart the properties of the present invention, especially in relation to C1q and/or Fc receptor (FcR) binding. .

A "human antibody" is an antibody that has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cells, or derived from another non-human source that uses the human antibody repertoire or other sequences encoding human antibodies. This definition of a human human antibody specifically excludes a humanized antibody containing non-human antigen-binding residues. In some embodiments, the human antibody is from a transgenic mammal other than a human, such as a mouse, rat, or rabbit. In some embodiments, the human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from human antibody libraries are also considered to be human antibodies or human antibody fragments in the present invention.

The "class" of an antibody or immunoglobulin is determined by the type of constant domain or constant region that its heavy chain possesses. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains that correspond to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "Fc domain" or "Fc region" in the present invention is used to define the C-terminal region of the heavy chain of an immunoglobulin, which contains at least part of the constant region. The term encompasses native sequences of Fc regions and variants of Fc regions. Although the boundaries of the IgG heavy chain Fc region may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expressing a particular nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or may include a cleaved variant of the full-length heavy chain (also referred to in the present invention as a "cleaved variant of the heavy chain"). It is possible that the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447) (numbered according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present.

Heavy chain amino acid sequences, including Fc domains (or a subunit of an Fc domain according to the present invention), are referred to herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one embodiment of the present invention, the heavy chain comprising the Fc domain subunit of the present invention contained in the antibody or bispecific antigen-binding molecule of the present invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of the present invention, the chain comprising the Fc domain subunit of the present invention contained in the antibody or bispecific antigen-binding molecule of the present invention includes an additional C-terminal glycine residue (G446, EU Kabat numbering). The compositions of the present invention, for example, the pharmaceutical compositions described in the present invention, contain a population of antibodies or bispecific antigen-binding molecules of the present invention. The population of antibodies or bispecific antigen-binding molecules may include molecules having a full length heavy chain and molecules having a cleaved version of the heavy chain. The population of antibodies or bispecific antigen-binding molecules may consist of a mixture of molecules having a full-length heavy chain and molecules having a cleaved version of the heavy chain, where 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 heavy chain variant. In one embodiment of the present invention, a composition comprising a population of antibodies or bispecific antigen-binding molecules of the present invention comprises an antibody or a bispecific antigen-binding molecule comprising a heavy chain comprising an Fc domain subunit of the present invention with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the EU index Kabat). In one of the embodiments of the present invention, a composition containing a population of antibodies or bespecifically antigennegative molecules of the present invention includes an antibody or bespecifically antigennegative molecule containing a heavy chain, comprising a subunit of the Fc domain of the present invention with an additional C-terminal glycine residue (G446, numbered according to EU index Kabat). In one of the embodiments of the present invention, such a composition includes a population of antibodies or bispecific antigen-binding molecules, consisting of molecules containing a heavy chain, including a subunit of the Fc domain according to the present invention; molecules containing a heavy chain comprising a subunit of the Fc domain according to the present invention with an additional C-terminal glycine residue (G446, numbering according to the Kabat EU index); and molecules containing a heavy chain comprising an Fc domain subunit of the present invention with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). Unless otherwise indicated in the present invention, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also referred to as the EU index and described in Kabat et al., Sequences of Proteins of Immunological Interest, 1991, 5e ed., ed. Public Health Service, National Institutes of Health, Bethesda, MD (also see above). The term "subunit" of the Fc domain in the context of the present invention refers to one of the two polypeptides that form a dimeric Fc domain, ie. a polypeptide containing constant C-terminal regions of the immunoglobulin heavy chain capable of stable self-association. For example, an IgG Fc domain subunit includes an IgG CH2 constant domain and an IgG CH3 constant domain.

The term "modification to promote association of the first and second subunits of the Fc domain" means manipulation of the peptide backbone or post-translational modifications of the subunit of the Fc domain that reduces or prevents the association of a polypeptide containing an Fc domain subunit with an identical polypeptide to form a homodimer. As used in the present invention, an association-promoting modification specifically includes separate modifications made in each of the two Fc domain subunits desired to associate (i.e., the first and second Fc domain subunits), the modifications being complementary to each other to promote association of the two subunits. Fc domain. For example, a modification promoting association may alter the structure or charge of one or both Fc domain subunits in such a way as to render their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing the first Fc domain subunit and a polypeptide containing the second Fc domain subunit, which may not be identical in the sense that the additional components fused to each of the subunits (e.g., antigen-binding fragments) are different. . In some embodiments, the association-enhancing modification comprises an amino acid mutation in the Fc domain, in particular an amino acid substitution. In one embodiment of the present invention, the association-enhancing modification comprises a single amino acid mutation, in particular an amino acid substitution, in each of the two subunits of the Fc domain.

The term "effector functions" refers to those biological activities that are inherent in the Fc region of the antibody, which vary depending on the isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex mediated uptake of antigen antigen- presenting cells, downregulation of cell surface receptors (eg, B cell receptor) and activation of B cells.

As used herein, "engineered" means any manipulation of the peptide backbone or post-translational modification of a natural or recombinant polypeptide or fragment thereof. Design includes modifications to the amino acid sequence, glycosylation pattern, or side chain group of individual amino acids, as well as combinations of these approaches.

The term "amino acid mutation" in the present invention means amino acid substitutions, deletions, insertions and modifications. Any combination of substitution, deletion, insertion, and modification can be made to produce the final construct, provided that the final construct has the desired properties, such as reduced binding to the Fc receptor or increased association with another peptide. Deletions and insertions in amino acid sequences include deletions at the amino and/or carboxy terminus and insertions of amino acids. Certain amino acid mutations are amino acid substitutions. In order to change, for example, the binding properties of the Fc region, non-conservative amino acid substitutions, ie. replacement of one amino acid with another amino acid having different structural and/or chemical properties is particularly preferred. Amino acid substitutions include substitution with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (eg, such as 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. The authors suggest that methods of altering the amino acid side chain group other than genetic engineering, such as chemical modification, for example, may also be useful. The present invention may use different designations for the same amino acid mutation. For example, a change from proline at position 329 of the Fc domain to glycine can be designated as 329G, G329, G329, P329G, or Pro329Gly.

"Percent (%) Amino Acid Sequence Identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the sequence of a reference polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and without taking into account any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways that are known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA software package. . Suitable parameters for sequence alignment can be determined by those skilled in the art, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences. However, for the purposes of the present invention, percent amino acid sequence identity values are generated using the ggsearch program of FASTA version 36.3.8 with or later with the BLOSUM50 comparison matrix. The FASTA software package developed by W.R. Pearson and D.J. Lipman, PNAS 85, 1988, 2444-2448; W.R. Pearson, Meth. Enzymol. 266, 1996, 227-258; Pearson et al., Genomics 46, 1997, 24-36, available publicly at:

http://fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml. Alternatively, you can use the public server: http://fasta.bioch.virginia.edu/fasta_www2/index.cgi to compare sequences using the ggsearch program (global protein: protein) and the default parameters (BLOSUM50 ; open: -10; ext: -2; Ktup=2) to ensure global rather than local alignment. The degree of amino acid identity, expressed as a percentage, is indicated in the title of the alignment results.

The term "polynucleotide" refers to a single nucleic acid molecule or construct, such as messenger RNA (mRNA), viral RNA or plasmid DNA (pDNA). A polynucleotide may contain a conventional phosphodiester bond or an unconventional bond (e.g., an amide bond, such as that found in peptide nucleic acids (PNA). The term "nucleic acid molecule" refers to any one or more segments of a nucleic acid, such as DNA or RNA fragments present in the polynucleotide.

By "single" nucleic acid molecule or polynucleotide is meant a nucleic acid, DNA or RNA molecule that has been isolated from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered to be isolated for the purposes of the present invention. Additional examples of an isolated polynucleotide include recombinant polynucleotides contained in heterologous host cells, or purified polynucleotides (partially or substantially) in solution. An isolated polynucleotide includes a polynucleotide molecule contained in cells that normally contain the polynucleotide molecule, but the polynucleotide molecule is present outside the chromosome or at a location on the chromosome that differs from its natural location. Isolated RNA molecules include the in vivo or in vitro RNA transcripts of the present invention, as well as positive, negative and double stranded forms. The isolated polynucleotides or nucleic acids of the present invention further include such synthetically produced molecules. In addition, the polynucleotide or nucleic acid may be or may include a regulatory element, such as a promoter, a ribosome binding site, or a transcription terminator.

The term "isolated polynucleotide (or nucleic acid) encoding [for example, an antibody or a bispecific antigen-binding molecule of the present invention]" refers to one or more polynucleotide molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such polynucleotide molecule(s). ) in one vector or in separate vectors, and such nucleic acid molecules (molecule) are present in one or more places in the host cell.

The term "expression cassette" refers to a polynucleotide, obtained recombinantly or synthetically, with a number of specific nucleic acid elements that provide transcription of a specific nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant portion of the expression cassette of the expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some embodiments of the present invention, the expression cassette contains polynucleotide sequences that encode antibodies or bispecific antigen-binding molecules of the present invention, or fragments thereof.

The term "vector" or "expression vector" refers to a DNA molecule that is used to introduce and direct the expression of a particular gene to which it is operatively linked in a cell. The concept includes a vector as a self-replicating nucleic acid structure, as well as a vector included in the genome of the host cell into which it has been introduced. The expression vector of the present invention contains an expression cassette. Expression vectors allow the transcription of large amounts of stable mRNA. Once the expression vector is inside the cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and/or translation machinery. In one of the embodiments of the present invention, the expression vector of the present invention includes an expression cassette that contains polynucleotide sequences encoding antibodies or bispecific antigen-binding molecules of the present invention, or fragments thereof.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, as well as to the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and its progeny, regardless of the number of passages. The offspring may not be completely identical in nucleic acid content to the parent cell and may contain mutations. This concept in the present invention also includes subsequent generations of mutant cells that have the same function or biological activity as originally identified or selected transformed cells. A host cell is any type of cell system that can be used to generate the antibodies or bispecific antigen-binding molecules of the present invention. Host cells include cultured cells, e.g. cultured mammalian cells such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridomas, yeast cells, insect cells and plant cells, to name but a few, but also cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues.

The term "activating Fc receptor" refers to an Fc receptor that, upon interaction with the Fc domain of an antibody, causes signaling that stimulates the receptor-bearing cell to perform effector functions. Activating human Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32) and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to lysis by immune effector cells of antibody-coated target cells. Target cells are cells to which antibodies or derivatives thereof containing an Fc region specifically bind, usually through that portion of the protein which is N-terminal to the Fc region. In the context of the present invention, the term "reduced ADCC" means a decrease in the number of target cells that are lysed at a given time and at a given concentration of antibody in the environment surrounding the target cells, according to the ADCC mechanism described above, and / or an increase in the concentration of antibodies in the environment surrounding target cells required to achieve lysis of a given number of target cells in a given time according to the ADCC mechanism. The reduction in ADCC is relative to ADCC mediated by the same antibody produced by the same host cell type using the same standard methods of preparation, purification, processing and storage (which are known to those skilled in the art) but have not been developed. For example, lowering ADCC mediated by an antibody containing an amino acid substitution in the Fc domain that lowers ADCC relative to ADCC mediated by the same antibody without that amino acid substitution in the Fc domain. Appropriate methods for measuring ADCC are known in the art (see, for example, WO 2006/082515 or WO 2012/130831).

An "effective amount" of an agent refers to the amount required to cause physiological changes in cells or tissues when administered.

The term "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the deleterious effects of the disease.

The terms "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domestic animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and other primates, such as monkeys), rabbits, and rodents (eg, mice and rats). In particular, the individual or subject is a person.

The term "pharmaceutical composition" refers to a preparation that is in a form that provides the effectiveness of the biological activity of the active ingredient contained therein, and which does not contain additional components that are unacceptably toxic to the subject to whom the composition can be administered.

A "pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical composition, other than the active ingredient, that is non-toxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, "treatment" (and its grammatical variations, e.g., "treat") refers to clinical intervention in an attempt to alter the natural course of a disease in the person being treated, and may be done either for prevention or during the course of a clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of the onset or recurrence of a disease, relief of symptoms, reduction of any direct or indirect pathological consequences of a disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or temporary mitigation of the consequences of a disease or disease state, and remission or improved prognosis. In some embodiments, the antibodies or bispecific antigen-binding molecules of the present invention are used to delay the development of a disease or to slow the progression of a disease.

The term "package insert" means instructions commonly included in commercial packages of therapeutic products that contain information about the indications, use, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

Detailed description of embodiments of the present invention

The present invention provides antibodies and bispecific antigen-binding molecules that bind GPRC5D, especially human GPRC5D. In addition, the molecules have other favorable properties for therapeutic use, eg in terms of efficacy and/or safety, and it is also important that they can be prepared.

Antibody against GPRC5D

In a first aspect of the present invention, an antibody is provided that binds to GPRC5D, wherein the antibody comprises (i) 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: 84, and HCDR 3 of SEQ ID NO: 86, and a light chain variable region (VL) including light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 of SEQ ID NO: 88 and LCDR 3 sequences 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 variable a light chain region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, a 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 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 a 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; (iv) a heavy chain variable region (VH) comprising a 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 a 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; or (v) a heavy chain variable region (VH) comprising a 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 a 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.

In some embodiments, the implementation of the present invention, the antibody is humanized. In one embodiment of the present invention, the VH is a humanized VH and/or the VL is a humanized VL. In one embodiment of the present invention, the antibody comprises CDRs similar to those described in any of the above embodiments of the present invention and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework.

In one embodiment of the present invention, (i) VH contains 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 VL contains an amino acid a 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) VH contains 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 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: 16; or (iii) VH contains 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 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: 53; or (iv) VH contains 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 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: 52; or (v) VH contains 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 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: 64; or (vi) VH contains 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 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.

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

In another embodiment of the present invention, the antibody is IgG, in particular IgG1. In one of the embodiments of the present invention, the antibody is a full length antibody. In another embodiment of the present invention, the antibody is an antibody fragment selected from the group consisting of an Fv molecule, an scFv molecule, a Fab molecule, and an F(ab') ₂ molecule. In one of the embodiments of the present invention, the antibody is a multispecific antibody.

In some embodiments of the present invention, the VH or VL sequence is at least 95%, 96%, 97%, 98%, or 99% identical and contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the compared sequence, but an antibody comprising such a sequence retains the ability to bind to GPRC5D. In some embodiments of the present invention, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in the sequence of SEQ ID NO: 13, and/or a total of 1 to 10 amino acids are substituted, inserted and/or deleted in the sequence of SEQ ID NO: 14, and/or a total of 1 to 10 amino acids are substituted, inserted and/or deleted in the sequence of SEQ ID NO: 15, and/or a total of 1 to 10 amino acids are substituted, inserted and/or deleted in sequence of SEQ ID NO: 16, and/or a total of 1 to 10 amino acids are substituted, inserted and/or deleted in the sequence of SEQ ID NO: 48, and/or a total of 1 to 10 amino acids are substituted, inserted and/or deleted in the sequence of SEQ ID NO: 53, and/or a total of 1 to 10 amino acids are substituted, inserted and/or deleted in the sequence of SEQ ID NO: 49, and/or a total of 1 to 10 amino acids are substituted, inserted and /or deleted in the sequence of SEQ ID NO: 52, and/or a total of 1 to 10 amino acids substituted, inserted and/or deleted in the sequence of SEQ ID NO: 57, and/or a total of 1 to 10 amino acids substituted, inserted and/or deleted in the sequence of SEQ ID NO: 64, and/or a total of 1 to 10 amino acids substituted, inserted and/or deleted in the sequence of SEQ ID NO: 58, and/or a total of 1 to 10 amino acids substituted, inserted and/or deleted in the sequence of SEQ ID NO: 63.

In some embodiments of the present invention, the substitutions, insertions, or deletions are contained in regions outside the HVR (ie, in the FR regions). 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 this sequence. 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 this sequence. Optionally, the antibody comprises the VH sequence of SEQ ID NO: 48 and/or the VL sequence of SEQ ID NO: 53, including post-translational modifications of this sequence. 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 this sequence. 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 this sequence. Optionally, the antibody comprises the VH sequence of SEQ ID NO: 58 and/or the VL sequence of SEQ ID NO: 63, including post-translational modifications of this sequence.

In one embodiment of the present invention, the antibody comprises a VH sequence comprising an amino acid sequence selected from the group of SEQ ID NO: 13 and SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 14.

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 of SEQ ID NO: 16.

In one embodiment, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 13 and a VL region comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 13 and the sequence VL SEQ ID NO: 14.

In one embodiment, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 15 and a VL region comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 15 and the sequence VL SEQ ID NO: 16.

In one embodiment, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 48 and a VL region comprising the amino acid sequence of SEQ ID NO: 53. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 48 , and the sequence VL SEQ ID NO: 53.

In one of the embodiments of the present invention, the antibody includes a VH region containing the amino acid sequence of SEQ ID NO: 49, and a VL region containing the amino acid sequence of SEQ ID NO: 52. In one of the embodiments of the present invention, the antibody includes a VH region containing the sequence of SEQ ID NO: 49, and the sequence VL SEQ ID NO: 52.

In one embodiment, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 57 and a VL region comprising the amino acid sequence of SEQ ID NO: 64. In one embodiment, the antibody comprises the VH sequence of SEQ ID NO: 57 , and the sequence VL SEQ ID NO: 64.

In one embodiment, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 58 and the VL amino acid sequence of SEQ ID NO: 63. In one embodiment of the present invention, the antibody comprises the VH sequence of SEQ ID NO: 58, and the sequence VL SEQ ID NO: 63.

In one embodiment, the antibody comprises a human constant region. In one embodiment of the present invention, the antibody is an immunoglobulin molecule comprising a human constant region, in particular an IgG class immunoglobulin molecule containing a human CHI, CH2, CH3 and/or CL domain. Examples of human constant domain sequences are shown in SEQ ID NOs 37 and 38 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 39 (human IgG1 heavy chain CH1-CH2-CH3 constant domains). 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, especially the amino acid sequence of SEQ ID NO: 38. In some embodiments, the implementation of the present invention, the antibody contains a heavy chain constant region containing 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, the heavy chain constant region may contain amino acid mutations in the Fc domain of the present invention.

In one of the embodiments of the present invention, the antibody is a monoclonal antibody.

In one embodiment of the present invention, the antibody is an IgG, especially an IgG ₁ antibody. In one of the embodiments of the present invention, the antibody is full length.

In one of the embodiments of the present invention, the antibody contains an Fc domain, in particular an IgG Fc domain, especially an IgG ₁ Fc domain. In one embodiment of the present invention, the Fc domain is a human Fc domain. The Fc domain of an antibody may have any of the features, alone or in combination, described herein in relation to the Fc domain of the bispecific antigen binding molecule of the present invention.

In another embodiment of the present invention, the antibody is an antibody fragment selected from the group consisting of Fv molecules, scFv molecules, Fab molecules and F(ab') _{2 molecules} . In another embodiment of the present invention, the antibody is a diabody, triabody, or tetrabody.

In yet another aspect, an antibody in accordance with any of the described embodiments of the present invention may include any of the features, alone or in combination, as described in the sections below.

Glycosylation Variants

In some embodiments, implementation of the present invention provide an antibody that is modified to increase or decrease the degree of glycosylation of the antibody. The addition or removal of glycosylation sites in an antibody can be easily accomplished by changing the amino acid sequence such that one or more glycosylation sites are created or removed.

If the antibody includes an Fc region, the oligosaccharide attached thereto may be changed. Native antibodies produced by mammalian cells typically contain a branched bianthenary oligosaccharide that is typically N-linked to the Asn297 CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15, 1997, 26-32. Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the bianthenary oligosaccharide structure. In some embodiments, implementation of the present invention can be made modifications to the oligosaccharide in the antibody of the present invention to create variants of antibodies with certain improved properties.

In one embodiment, the present invention provides antibody variants having non-fucosylated oligosaccharides, i.e. oligosaccharide structures that have lost fucose attached (directly or indirectly) to the Fc region. Such a non-fucosylated oligosaccharide (also referred to as an "afucosylated" oligosaccharide) is specifically an N-linked oligosaccharide that lacks a fucose residue attached to the first GlcNAc at the base of the oligosaccharide bianthenary structure. In one of the embodiments of the present invention, antibody variants are provided that have an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to the native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (ie, no fucosylated oligosaccharides). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues relative to the sum of all oligosaccharides attached to Asn 297 (e.g. complex, hybrid and high mannose structures) measured by MALDI-TOF mass spectrometry as described in WO 2006/082515, for example, Asn297 refers to an asparagine residue located approximately at position 297 in the Fc region (EU residue numbering of the Fc region); however, Asn297 can also be located about ±3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300, due to minor sequence changes in the antibodies. Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and/or improved effector function, in particular improved ADCC function. See, for example, US 2003/0157108; US 2004/0093621.

Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249, 1986, 533-545; US 2003/0157108; and WO 2004/056312 , especially example 11), knockout cell lines, e.g., alpha-1,6-fucosyltransferase gene knockout, FUT8, CHO knockout cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87, 2004, 614- 622; Kanda Y. et al., Biotechnol. Bioeng., 94(4), 2006, 680-688; WO 2003/085107) or cells with reduced or impaired activity in the synthesis of GDP-fucose or transport protein (see, e.g. , US 2004259150, US 2005031613, US 2004132140, US 2004110282).

In another embodiment, the present invention provides antibody variants that contain bisected oligosaccharides, in which, for example, a bianthenary oligosaccharide attached to the Fc region of an antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation function and/or improved ADCC function as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 1999, 176-180; Ferrara et al., Biotechn Bioeng 93, 2006, 851-861; W099/54342; WO 2004/065540, WO 2003/011878.

Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also contemplated. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

Cysteine engineered antibody variants

In some embodiments, implementation of the present invention, it may be desirable to create antibodies designed on the basis of cysteine, "thioMAb", in which one or more antibody residues are replaced by cysteine residues. In some embodiments, implementation of the present invention, the substituted residues are in accessible places of the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create the immunoconjugate of the present invention. Cysteine-engineered antibodies can be prepared as described, for example, in US 7521541, US 830930, US 7855275, US 9000130 or WO 2016040856.

Antibody derivatives

In some embodiments, implementation of the present invention, the antibody can be further modified to contain additional non-protein fragments that are known in the art and are readily available. Fragments suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, copolymer ethylene/maleic anhydride, polyamino acids (either homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propylene glycol homopolymers, prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol and mixtures thereof. Propionaldehyde polyethylene glycol may have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the amount and/or type of polymers used for derivatization may be determined based on factors such as, but not limited to, the specific properties or functions of the antibody being improved, depending on if the antibody derivative is to be used in therapy for certain conditions, etc.

In another embodiment, the present invention provides that the conjugates of the antibody and non-protein fragment can be selectively heated by irradiation. In one embodiment of the present invention, the non-protein fragment is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102, 2005, 11600-11605). The irradiation may be of any wavelength and includes, among others, wavelengths that do not damage normal cells but heat the non-protein fragment to a temperature at which cells proximal to the antibody-non-protein fragment die.

Immunoconjugants

The present invention also provides immunoconjugates comprising an anti-GPRC5D antibody of the present invention conjugated (chemically linked) to one or more therapeutic agents such as cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g. protein toxins , enzymatically active toxins of bacterial, fungal, plant or animal origin or their fragments) or radioactive isotopes.

In one embodiment of the present invention, the immunoconjugate is an antibody-drug conjugate (ADC) wherein the antibody is conjugated to one or more of the therapeutic agents mentioned above. The antibody is usually associated with one or more therapeutic agents using linkers. An overview of ADC technology, including examples of therapeutic agents, drugs, and linkers, is provided in Pharmacol Review 68, 2016, 3-19.

In another embodiment of the present invention, the immunoconjugate comprises an antibody of the present invention conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria toxin A chain, exotoxin, non-binding active diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diantine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes.

In another embodiment of the present invention, the immunoconjugate comprises an antibody of the present invention conjugated to a radioactive atom to form a radioconjugate. Various radioactive isotopes are available for the production of radioconjugates.

Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu radioactive isotopes. If the radioconjugate is used for detection, it may contain a radioactive atom for scintigraphy, such as tc99m or I123, or a spin label 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.

Antibody-cytotoxic agent conjugates can be prepared using various bifunctional protein binding agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate.( SMCC), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, the ricin immunotoxin can be prepared as described by Vitetta et al., Science 238, 1987, 1098. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelating agent for conjugation of a radionucleotide to an antibody. See WO 94/11026. The linker may be a "cleavable linker" to promote release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52, 1992, 127-131; US 5208020).

The immunoconjugates or ADCs of the present invention are, but are not limited to, such conjugates prepared with cross-linker reagents including but not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB , SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone) benzoate), which are commercially available (eg, from Pierce Biotechnology, Inc., Rockford, IL, USA).

Multispecific antibodies

In some embodiments, implementation of the present invention provided by the antibody is a multispecific antibody, for example, bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites, ie different epitopes on different antigens or different epitopes on the same antigen. In some embodiments, the multispecific antibody has three or more binding specificities. In some embodiments of the present invention, one of the binding specificities causes binding to GPRC5D, and the other two (two or more) specificities cause binding to some other antigen. In some embodiments, the bispecific antibodies can bind to two (or more) different GPRC5D epitopes. Multispecific antibodies (eg, bispecific) can also be used to localize cytotoxic agents or cells to cells that express GPRC5D. Multispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305, 1983, 537), as well as the construction of "protrusion" in the hollow" (see, for example, US 5731168; Atwell et al., J. Mol. Biol. 270, 1997, 26). Multispecific antibodies can also be generated by electrostatically driven effects engineering to create Fc-heterodimeric antibody molecules (see, for example, WO 2009/089004); cross-linking of two or more antibodies or fragments (see, for example, US 4676980, Brennan et al., Science, 229, 1985, 81); the use of leucine zippers to obtain bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5), 1992, 1547-1553, and WO 2011/034605); the use of common light chain technology to circumvent the problem of light chain mismatch (see, for example, WO 98/50431); the use of the "diabody method" to obtain bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90, 1993, 6444-6448); the use of single-chain Fv dimers (single-chain, sFv) (see, for example, Gruber et al., J. Immunol., 152, 1994, 5368); ai and obtaining trispecific antibodies as described, for example, in the publication of Tutt et al.J. Immunol. 147, 1991, 60.

The present invention also includes engineered antibodies with three or more antigen binding sites, including, for example, "octopus" or DVD-Ig antibodies (see, for example, WO 2001/77342 and WO 2008/024715). Other multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792 and WO 2013/026831. A bispecific antibody or binding fragment thereof refers to a "dual-acting Fab" or "DAF" comprising an antigen binding site that binds to GPRC5D and another different antigen, or two different GPRC5D epitopes (see, for example, US 2008/0069820 and WO 2015/095539).

Multispecific antibodies can also be produced in an asymmetric form with a crossover domain in one or more binding arms of the same antigen specificity, i.e. by exchanging VH/VL domains (see e.g. WO 2009/080252 and WO 2015/150447), CH1/CL domains (see e.g. WO 2009/080253) or complete Fab branches (see e.g. WO 2009 /080251, WO 2016/016299, and also see Schaefer et al., PNAS, 108, 2011, 1187-1191; Klein et al., MAbs 8, 2016, 1010-1020). Asymmetric Fab branches can also be engineered by introducing charged or uncharged amino acid mutations into domain interfaces for targeted correction by proper Fab pairing. See, for example, WO 2016/172485.

Various other molecular formats of multispecific antibodies are known in the art and are included within the scope of the present invention (see, for example, Spiess et al., Mol Immunol 67, 2015, 95-106).

A specific type of multispecific antibody also included in the present invention are bispecific antibodies designed to simultaneously bind to a surface antigen on a target cell, such as a tumor cell, and to an activating invariant component of the T cell receptor (TCR) complex, such as CD3 , to retarget T cells to destroy target cells. Therefore, in some embodiments, the antibody provided is a multispecific antibody, in particular a bispecific antibody, where one of the binding specificities is for GPRC5D and the other is for CD3.

Exp Cell Res 317, 201 1, 1255-1260); диатела (Holliger с соавт., Prot Eng 9, 1996, 299-305) и их производные, например, тандемные антитела («TandAb»; Kipriyanov с соавт., JMol Biol 293, 1999, 41-56); молекулы «DART» (перенацеливание двойной афинностью - dual affinity retargeting), которые основаны на формате диатела, но характерной особенностью которых является С-концевой дисульфидный мост для дополнительной стабилизации (Johnson с соавт., J Mol Biol 399, 2010, 436-449), и так называемые триомабсы, являющиеся полным гибридом молекул мышиного/крысиного IgG (обзор в публикации Seimetz с соавт., Cancer Treat Rev 36, 2010, 458-467). В частности, Т-клеточные биспецифические форматы антител описаны в WO 2013/026833, WO 2013/026839, WO 2016/020309; Васас с соавт., Oncoimmunology 5(8), 2016, е1203498.Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, so-called "BiTE" (bispecific T cell engager) molecules, in which two scFv molecules are fused using a flexible linker ( see for example WO 2004/106381, WO 2005/061547, WO 2007/042261 and WO 2008/119567, Nagorsen and

Exp Cell Res 317, 201 1, 1255-1260); diabodies (Holliger et al., Prot Eng 9, 1996, 299-305) and their derivatives, for example, tandem antibodies ("TandAb"; Kipriyanov et al., JMol Biol 293, 1999, 41-56); "DART" (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 2010, 436-449) , and the so-called triomabs, which are full hybrids of mouse/rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 2010, 458-467). In particular, T-cell bispecific antibody formats are described in WO 2013/026833, WO 2013/026839, WO 2016/020309; Vasas et al., Oncoimmunology 5(8), 2016, e1203498.

Bispecific antigen-binding molecules that bind to GPRC5D and to a second antigen

The present invention also relates to a bespecifically antigen-binding molecule, that is, an antigen-binding molecule that contains at least two antigen-binding fragments capable of specifically binding to two different antigenic determinants (a first and a second antigen).

In some embodiments, the antigen-binding fragments contained in the bispecific antigen-binding molecule are Fab molecules (ie, antigen-binding domains consisting of heavy and light chains, each containing a variable and a constant domain). In one of the embodiments of the present invention, the first and/or second antigennegative fragment is a Fab molecule. In one embodiment of the present invention, said Fab is a human molecule. In one embodiment of the present invention, said Fab molecule is humanized. In yet another embodiment of the present invention, said Fab molecule comprises human heavy and light chain constant domains.

Preferably, at least one of the antigen-binding fragments is a Fab crossover molecule. Such a modification reduces mismatch between heavy and light chains of different Fab molecules, thereby improving the yield and purity of the bispecific antigen-binding molecule of the present invention in the production of recombinants. In a particular Fab crossover molecule suitable for the bispecific antigen-binding molecule of the present invention, the Fab light chain and Fab heavy chain variable domains (VL and VH, respectively) are exchanged. However, even with this domain swap, the bispecific antigen-binding molecule preparation may include certain by-products due to the so-called Bence-Jones-type interaction between mismatched heavy and light chains (see Schaefer et al., PNAS, 108, 2011, 11187-11191 ). To further reduce mismatch between heavy and light chains from different Fab molecules and thus increase the purity and yield of the desired bispecific antigen-binding molecule, charged amino acids with opposite charges can be introduced at specific amino acid positions in the CH1 and CL domains of either the Fab molecule(s). , which binds to the first antigen (GPRC5D), or a Fab molecule that binds to the second antigen (for example, an activating T-cell antigen such as CD3), as further described in the present invention. Charge modifications are made either in conventional Fab molecules (molecule) contained in a bispecific antigen-binding molecule (such as shown, for example, in Fig.1 A-B, G-K), or in the VH / VL crossover Fab molecule (s), contained in a bispecific antigen-binding molecule (for example, shown in Figure 1 D-F, L-O) (but not both). In some embodiments of the present invention, charge modifications are made in the normal Fab molecule(s) contained in the bispecific antigen-binding molecule (which in certain embodiments of the present invention binds to the first antigen, i.e., GPRC5D).

In some embodiments, the bispecific antigen-binding molecule is capable of simultaneously binding to a first antigen (ie, GPRC5D) and a second antigen (eg, an activating T-cell antigen, particularly CD3). In one embodiment of the present invention, the bispecific antigen-binding molecule is capable of cross-breeding T cells and target cells by simultaneously binding GPRC5D and an activating T cell antigen. In another preferred embodiment of the present invention, such simultaneous binding leads to the lysis of target cells, in particular tumor cells expressing GPRC5D. In one of the embodiments of the present invention, such simultaneous binding leads to the activation of T cells. In another embodiment of the present invention, such simultaneous binding results in a cellular response of T lymphocytes, especially cytotoxic T lymphocytes, selected from the group consisting of: proliferation, differentiation, secretion of cytokines, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one embodiment of the present invention, binding of the bispecific antigen-binding molecule to an activating T cell antigen, especially CD3, without simultaneous binding to GPRC5D does not result in T cell activation.

In one embodiment of the present invention, the bispecific antigen-binding molecule is capable of redirecting the cytotoxic activity of T cells to target cells. In one embodiment of the present invention, said retargeting is independent of presentation of the MHC-mediated peptide antigen by target cells and/or T cell specificity.

In particular, the T cells of any of the embodiments of the present invention are cytotoxic T cells. In some embodiments of the present invention, the T cells are CD4 ⁺ or CD8 ⁺ T cells, especially CD8 ⁺ T cells.

First antigen binding fragment

The bispecific antigen-binding molecule of the present invention contains at least one antigen-binding fragment, in particular a Fab molecule that binds to GPRC5D (first antigen). In some embodiments, the bispecific antigen-binding molecule comprises two antigen-binding moieties, specifically Fab molecules that bind to GPRC5D. In one such embodiment of the present invention, each of these antigen-binding fragments binds to the same antigenic determinant. In an even more preferred embodiment of the present invention, all of these antigen-binding fragments are identical, ie. they contain the same amino acid sequences, including the same amino acid substitutions in the CH1 and CL domains as described herein (if any). In one of the embodiments of the present invention, the bispecific antigen-binding molecule of the present invention contains no more than two antigen-binding fragments, in particular Fab molecules that bind to GPRC5D.

In some embodiments of the present invention, the antigen-binding fragment(s) that bind to GPRC5D is a conventional Fab molecule. In such embodiments, the antigen-binding fragment(s) that bind to the second antigen are the Fab crossover molecule described herein, i.e. a Fab molecule in which the variable domains VH and VL or the consensus domains CH1 and CL of the heavy and light chains of the Fab are exchanged/replaced with each other.

In other alternative embodiments of the present invention, the antigen binding fragment(s) that bind to GPRC5D is a Fab crossover molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or the consensus domains CH1 and CL of the heavy and light chains of the Fab are exchanged/replaced with each other. In other similar embodiments of the present invention, the antigen-binding fragment(s) that bind the second antigen is a conventional Fab molecule.

The GPRC5D-binding fragment is capable of directing the bispecific antigen-binding molecule to a target site, such as a certain type of tumor cell that expresses GPRC5D.

The first antigen-binding fragment of a bispecific antigen-binding molecule may have any of the specific characteristics, alone or in combination, described herein in connection with an antibody that binds GPRC5D, except where it is scientifically clear that this is clearly unreasonable or impossible. .

Thus, in one aspect of the present invention, a bispecific antigen-binding molecule is provided, comprising (a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding fragment comprises a heavy chain variable region (VH) comprising a heavy chain region , complementarity determining (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 complementarity determining light chain region ( LCDR) 1 of SEQ ID NO: 87, LCDR 2 of SEQ ID NO: 88 and LCDR 3 of SEQ ID NO: 89, and (b) a second antigen binding fragment that binds to the second antigen. In a second aspect of the present invention, a bispecific antigen-binding molecule is provided, comprising (a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding fragment comprises a heavy chain variable region (VH) comprising a complementarity-determining heavy chain 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) including complementarity determining light chain region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 of SEQ ID NO: 88 and LCDR 3 of SEQ ID NO: 89, and (b) a second antigen-binding fragment that binds to the second antigen. In another aspect of the present invention, a bispecific antigen-binding molecule is provided, comprising (a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding fragment comprises a heavy chain variable region (VH) comprising a complementarity-determining heavy chain region (HCDR) 1 sequences of SEQ ID NO: 90, HCDR 2 sequences of SEQ ID NO: 91 and HCDR 3 sequences of SEQ ID NO: 93, and a light chain variable region (VL) including a light chain complementarity determining region (LCDR) 1 sequence 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 fragment that binds to the second antigen. In another aspect of the present invention, a bispecific antigen-binding molecule is provided, comprising (a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding fragment comprises a heavy chain variable region (VH) comprising a complementarity-determining heavy chain region (HCDR) 1 sequences of SEQ ID NO: 90, HCDR 2 sequences of SEQ ID NO: 91 and HCDR 3 sequences of SEQ ID NO: 93, and a light chain variable region (VL) including a light chain complementarity determining region (LCDR) 1 sequence SEQ ID NO: 94, LCDR 2 of SEQ ID NO: 96 and LCDR 3 of SEQ ID NO: 97, and (b) a second antigen binding fragment that binds to the second antigen. In another aspect of the present invention, a bispecific antigen-binding molecule is provided, comprising (a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding fragment comprises a heavy chain variable region (VH) comprising a complementarity-determining heavy chain region (HCDR) 1 sequences of SEQ ID NO: 90, HCDR 2 sequences of SEQ ID NO: 92 and HCDR 3 sequences of SEQ ID NO: 93, and a light chain variable region (VL) including a light chain complementarity determining region (LCDR) 1 sequence 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 fragment that binds to the second antigen. In another aspect of the present invention, a bispecific antigen-binding molecule is provided, comprising (a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding fragment comprises a heavy chain variable region (VH) comprising a complementarity-determining heavy chain region (HCDR) 1 sequences of SEQ ID NO: 1, HCDR 2 sequences of SEQ ID NO: 2 and HCDR 3 sequences of SEQ ID NO: 3, and a light chain variable region (VL) including a light chain complementarity determining region (LCDR) 1 sequence 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 fragment that binds to the second antigen. In another aspect of the present invention, a bispecific antigen-binding molecule is provided, comprising (a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D, and the first antigen-binding fragment comprises a heavy chain variable region (VH) comprising a complementarity-determining heavy chain region (HCDR) 1 sequences of SEQ ID NO: 7, HCDR 2 sequences of SEQ ID NO: 8 and HCDR 3 sequences of SEQ ID NO: 9, and a light chain variable region (VL) including a light chain complementarity determining region (LCDR) 1 sequence 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 fragment that binds to the second antigen.

In some embodiments of the present invention, the first antigen-binding fragment is (derived from) a humanized antibody. In one embodiment of the present invention, the VH is a humanized VH and/or the VL is a humanized VL. In one embodiment of the present invention, the first antigen-binding fragment contains a CDR as in any of the above embodiments of the present invention, and further comprises a human acceptor region, for example, a human immunoglobulin framework region or a human consensus framework region.

In one embodiment of the present invention, the VH of the first antigen-binding fragment contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group of sequences 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 fragment contains an amino acid sequence that is at least about 95% 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of sequences 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.

In one embodiment of the present invention, the first antigen-binding fragment comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group of sequences 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 an amino acid sequence selected from the group of sequences 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.

In one embodiment of the present invention, the first antigen-binding fragment comprises a VH sequence comprising an amino acid sequence selected from the group 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 comprising an amino acid sequence selected from the group of sequences 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.

In one embodiment, the first antigen-binding fragment comprises a VH sequence selected from the group 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 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.

In one embodiment of the present invention, the first antigen-binding fragment comprises a VH sequence comprising the amino acid sequence of SEQ ID NO: 13 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment of the present invention, the first antigen-binding fragment comprises the VH sequence of SEQ ID NO: 13 and the sequence VL SEQ ID NO: 14.

In one embodiment of the present invention, the first antigen-binding fragment comprises a VH sequence comprising the amino acid sequence of SEQ ID NO: 15 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment of the present invention, the first antigen-binding fragment comprises the VH sequence of SEQ ID NO: 15 and the sequence VL SEQ ID NO: 16.

In one embodiment of the present invention, the first antigen-binding fragment comprises a VH sequence comprising the amino acid sequence of SEQ ID NO: 48 and a VL sequence comprising the amino acid sequence of SEQ ID NO: 53. In one embodiment of the present invention, the first antigen-binding fragment comprises the VH sequence of SEQ ID NO: 48 and the sequence VL SEQ ID NO: 53.

In one embodiment of the present invention, the first antigen-binding fragment comprises a VH region containing the amino acid sequence of SEQ ID NO: 49 and a VL region containing the amino acid sequence of SEQ ID NO: 52. In one embodiment of the present invention, the first antigen-binding fragment comprises the VH sequence of SEQ ID NO: 49 and VL sequence SEQ ID NO: 52.

In one embodiment of the present invention, the first antigen-binding fragment comprises a VH region containing the amino acid sequence of SEQ ID NO: 57 and a VL region containing the amino acid sequence of SEQ ID NO: 64. In one embodiment of the present invention, the first antigen-binding fragment comprises the VH sequence of SEQ ID NO: 57 and the sequence of SEQ ID NO: 64.

In one embodiment of the present invention, the first antigen-binding fragment comprises a VH region containing the amino acid sequence of SEQ ID NO: 58 and a VL region containing the amino acid sequence of SEQ ID NO: 63. In one embodiment of the present invention, the first antigen-binding fragment comprises the VH sequence of SEQ ID NO: 58 and the sequence of SEQ ID NO: 63. In one embodiment of the present invention, the first antigen-binding fragment comprises a human constant region. In one embodiment of the present invention, the first antigen-binding fragment is a Fab molecule comprising a human constant region, especially a human CH1 and/or CL domain. Examples of human constant domain sequences are SEQ ID NOs 37 and 38 (human CL kappa and lambda domains) and SEQ ID NO: 39 (human IgG1 heavy chain CH1-CH2-CH3 constant domains). In some embodiments, the first antigen-binding fragment includes a constant region; the first antigen-binding fragment includes a light chain constant region containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% amino acid identical. sequence of SEQ ID NO: 37 or SEQ ID NO: 38, particularly the amino acid sequence of SEQ ID NO: 37. In particular, the light chain constant region may contain amino acid mutations described in the present invention in the section "charge modification", and/ or may include deletion or substitutions of one or more (especially two) N-terminal amino acids in the case of a Fab crossover molecule. In some embodiments of the present invention, the first antigen-binding fragment comprises a heavy chain constant region containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 39. In particular, the heavy chain constant region (especially the CH1 domain) may include the amino acid mutations described in the present invention in the "charge modifications" section.

Second antigen binding fragment

The bispecific antigen-binding molecule of the present invention comprises at least one antigen-binding fragment, in particular a Fab molecule that binds to a second antigen (other than GPRC5D).

In some embodiments, the antigen-binding fragment that binds the second antigen is a Fab crossover molecule of the present invention, i. e. a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged/replaced with each other. In such embodiments, the antigen-binding fragment(s) that binds to the first antigen (ie, GPRC5D) is preferably a conventional Fab molecule. In embodiments of the present invention, there is more than one antigen-binding fragment, in particular a Fab molecule that binds to GPRC5D, which is part of a bispecific antigen-binding molecule, an antigen-binding fragment that binds to a second antigen, preferably a Fab crossover molecule, and antigen-binding fragments that bind to GPRC5D are regular Fab molecules.

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

In some embodiments, the second antigen is an activating T cell antigen (also referred to herein as "activating T cell antigen binding fragment or activating T cell antigen binding Fab"). In one embodiment of the present invention, the bispecific antigen-binding molecule contains no more than one antigen-binding moiety capable of specifically binding to an activating T-cell antigen. In one embodiment of the present invention, the bispecific antigen-binding molecule provides monovalent binding to an activating T-cell antigen.

In some embodiments, the second antigen is CD3, in particular human CD3 (SEQ ID NO: 40) or cynomolgus monkey CD3 (SEQ ID NO: 41), most preferably human CD3. In one embodiment of the present invention, the second antigen-binding fragment exhibits cross-reactivity (ie, specific binding) against human and cynomolgus CD3. In some embodiments of the present invention, the second antigen is the CD3 epsilon subunit (CD3 epsilon).

In one embodiment, the second antigen-binding fragment 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: 32 SEQ ID NO: 33 and LCDR 3 sequences of SEQ ID NO: 34.

In one embodiment of the present invention, the second antigen-binding fragment comprises a VH containing HCDR 1 of SEQ ID NO: 29, HCDR 2 of SEQ ID NO: 30 and HCDR 3 of SEQ ID NO: 31, and a VL containing LCDR 1 of SEQ ID NO: : 32, LCDR 2 sequences of SEQ ID NO: 33 and LCDR 3 sequences of SEQ ID NO: 34.

In some embodiments, implementation of the present invention, the second antigennegative fragment is (derived from) a humanized antibody. In one embodiment of the present invention, the VH region is a humanized VH and/or the VL is a humanized VL. In one embodiment of the present invention, the second antigen binding fragment comprises CDR regions as in any of the above embodiments of the present invention and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework.

In one embodiment, the second antigen-binding fragment 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, embodiment of the present invention, the second antigen-binding fragment 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 of the present invention, the second antigen-binding fragment 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, which 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 of the present invention, the VH of the second antigen binding fragment 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 the antigen-binding fragment 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 of the present invention, the second antigen binding fragment comprises a VH sequence containing the amino acid sequence of SEQ ID NO: 35 and a VL containing the amino acid sequence of SEQ ID NO: 36.

In one embodiment of the present invention, the second antigen-binding fragment comprises the VH sequence of SEQ ID NO: 35, and the VL sequence of SEQ ID NO: 36.

In one embodiment of the present invention, the second antigen-binding fragment comprises a human constant region. In one embodiment of the present invention, the second antigen-binding moiety is a Fab molecule, a molecule containing a human constant region, in particular a human CH1 and/or CL domain. Exemplary human constant domain sequences are shown in SEQ ID NO 37, SEQ ID NO 38 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 39 (human IgG1 heavy chain CH1-CH2-CH3 chain constant domains). In some embodiments, the second antigen-binding fragment 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: 38, especially the amino acid sequence of SEQ ID NO: 37. In particular, the light chain constant region may contain amino acid mutations, as described in the present invention in the "charge modification" section, and/or may include a deletion or substitution of one or several (especially two) N-terminal amino acids if they are in the Fab crossover molecule. In some embodiments, the second antigen-binding fragment comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 39. In particular, the constant region of the heavy chain (in particular, the CH1 domain) may contain amino acid mutations, as described in the "charge modification" section of this description.

In some embodiments, the second antigen-binding moiety is a Fab molecule in which the VL and VH variable domains or the CL and CH1 constant domains, especially the VL and VH variable domains, of the Fab light chain and the Fab heavy chain are swapped (i.e. according to such an embodiment of the present invention, the second antigen-binding moiety is a Fab crossover molecule in which the variable or constant domains of the Fab light chain and the Fab heavy chain are swapped). In one such embodiment of the present invention, the first (and third, if any) antigen-binding fragment is a conventional Fab molecule.

In one embodiment of the present invention, no more than one antigen-binding moiety is present in the bispecific antigen-binding molecule that binds to a second antigen (eg, an activating T-cell antigen such as CD3), i.e. the bispecific antigen-binding molecule provides monovalent binding to the second antigen.

Charge modifications

The bispecific antigen binding molecules of the present invention may contain amino acid substitutions in Fab molecules that are particularly effective in reducing light chain mismatch with non-complementary heavy chains (Bence-Jones type by-products) that can occur in the production of bi-/multispecific antigen binding molecules with VH exchange. /VL in one (or more, in the case of molecules containing more than two antigen-binding Fab molecules) of the binding branches (see also WO 2015/150447, especially the examples given therein, the essence of which is given in the present invention by reference). The proportion of the desired bispecific antigen-binding molecule compared to undesired by-products, in particular Bence-Jones-type by-products, occurring among bispecific antigen-binding molecules with an exchange of VH/VL domains in one of their binding branches, can be improved by introducing charged amino acids with opposite charges in certain amino acid positions in the CHI and CL domains (the term "charge modification" is sometimes used in the present invention).

Thus, in some embodiments of the present invention, where the first and second antigen-binding fragment of the bispecific antigen-binding molecule are both Fab molecules, and in one of the antigen-binding fragments (in particular, in the second antigen-binding fragment), the VL and VH variable domains of the Fab light chain and heavy chain Fab are replaced by each other,

i) in the CL constant domain of the first antigen-binding fragment, the amino acid at position 124 is replaced with a positively charged amino acid (Kabat numbering), and where in the CH1 constant domain of the first antigen-binding fragment, the amino acid at position 147 or the amino acid at position 213 is replaced with a negatively charged amino acid (Kabat EU numbering index); or

ii) in the CL constant domain of the second antigen binding fragment, the amino acid at position 124 is replaced with a positively charged amino acid (Kabat numbering), and where in the CH1 constant domain of the second antigen binding fragment, the amino acid at position 147 or the amino acid at position 213 is replaced with a negatively charged amino acid (Kabat EU numbering index).

The bispecific antigen-binding molecule does not contain both of the modifications mentioned in i) and ii). CL and CH1 constant domains of an antigen-binding fragment in which VH/VL are not interchanged (i.e., remain unchanged).

In a more specific embodiment of the present invention

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

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

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

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

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

In one embodiment of the present invention, in the CL constant domain of the first antigen-binding fragment, the amino acid at position 124 is replaced by lysine (K) (numbering according to the Kabat EU index) and the amino acid at position 123 is replaced by lysine (K) (numbering according to the Kabat EU index), and in in the CH1 constant domain of the first antigen-binding fragment, the amino acid at position 147 is independently substituted with glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is independently substituted with glutamic acid (E) (Kabat EU index numbering).

In yet another embodiment of the present invention, in the CL constant domain of the first antigen-binding fragment, the amino acid at position 124 is replaced by lysine (K) (Kabat EU numbering) and the amino acid at position 123 is replaced by arginine (R) (Kabat numbering), and in the constant in the CH1 domain of the first antigen-binding fragment, the amino acid at position 147 is independently substituted with glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is independently substituted with glutamic acid (E) (Kabat EU index numbering).

In some embodiments of the present invention, if the amino acid substitutions in accordance with the above embodiments of the present invention are made in the CL constant domain and the CH1 constant domain of the first antigen-binding fragment, the CL constant domain of the first antigen-binding fragment is kappa isotype.

In another alternative embodiment of the present invention, the amino acid substitutions according to the above embodiments may be made in the CL constant domain and the CH1 constant domain of the second antigen-binding fragment instead of the CL constant domain and the CH1 constant domain of the first antigen-binding fragment. In certain such embodiments, the CL constant domain of the second antigen-binding fragment is kappa isotype.

Thus, in one of the embodiments of the present invention, in the CL constant domain of the second antigen-binding fragment, the amino acid at position 124 is independently replaced by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the CH1 constant domain of the second antigen-binding fragment fragment, the amino acid at position 147 or the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

In another embodiment of the present invention, in the CL constant domain of the second antigen-binding fragment, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the CH1 constant domain of the second antigen-binding fragment, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU numbering).

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

In one embodiment of the present invention, in the CL constant domain of the second antigen-binding fragment, the amino acid at position 124 is independently replaced by lysine (K) (Kabat numbering) and the amino acid at position 123 is replaced by lysine (K) (Kabat numbering), and in the constant domain CH1 of the second antigen-binding fragment, the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the Kabat EU index).

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

In one embodiment of the present invention, the bispecific antigen binding molecule of the present invention comprises

(a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89, and

(b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the Fab heavy chain are swapped;

where in the CL constant domain of the first antigen-binding fragment, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (Kabat numbering) (in some embodiments of the present invention, independently replaced by lysine (K) or arginine (R )) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering) (in one embodiment of the present invention, independently substituted with lysine (K) or arginine (R)), and in the CH1 constant domain of the first antigen-binding fragment, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (Kabat EU numbering) and the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) ( numbering according to Kabat EU index).

In one embodiment of the present invention, the bispecific antigen binding molecule of the present invention comprises

(a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89, and

(b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the Fab heavy chain are swapped;

where in the CL constant domain of the first antigen-binding fragment, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (Kabat numbering) (in some embodiments of the present invention, independently replaced by lysine (K) or arginine (R )) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering) (in one embodiment of the present invention, independently substituted with lysine (K) or arginine (R)), and in the CH1 constant domain of the first antigen-binding fragment, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (Kabat EU numbering) and the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) ( numbering according to Kabat EU index).

In one embodiment of the present invention, the bispecific antigen binding molecule of the present invention comprises

(a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97, and

(b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the Fab heavy chain are swapped;

where in the CL constant domain of the first antigen-binding fragment, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (Kabat EU index numbering) (in some embodiments of the present invention, independently replaced by lysine (K) or arginine (R)) and the amino acid at position 123 is independently replaced by lysine (K), arginine (R), or histidine (H) (Kabat EU index numbering) (in one embodiment of the present invention, independently replaced by lysine (K) or arginine (R )), and in the CH1 constant domain of the first antigen-binding fragment, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering) and the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index).

In one embodiment of the present invention, the bispecific antigen binding molecule of the present invention comprises

(a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 96 and LCDR 3 sequences of SEQ ID NO: 97, and

(b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the Fab heavy chain are swapped;

where in the CL constant domain of the first antigen-binding fragment, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (Kabat EU index numbering) (in some embodiments of the present invention, independently replaced by lysine (K) or arginine (R)) and the amino acid at position 123 is independently replaced by lysine (K), arginine (R), or histidine (H) (Kabat EU index numbering) (in one embodiment of the present invention, independently replaced by lysine (K) or arginine (R )), and in the CH1 constant domain of the first antigen-binding fragment, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering) and the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index).

In one embodiment of the present invention, the bispecific antigen binding molecule of the present invention comprises

(a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97, and

(b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the Fab heavy chain are swapped;

where in the CL constant domain of the first antigen-binding fragment, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (Kabat EU index numbering) (in some embodiments of the present invention, independently replaced by lysine (K) or arginine (R)) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering) (in one embodiment of the present invention, independently substituted with lysine (K) or arginine (R)) , and in the CH1 constant domain of the first antigen-binding fragment, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering) and the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D ) (numbered according to the Kabat EU index).

In one embodiment of the present invention, the bispecific antigen binding molecule of the present invention comprises

(a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a heavy chain complementarity determining region (HCDR) 1, 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) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 4, LCDR 2 sequences of SEQ ID NO: 5 and LCDR 3 sequences of SEQ ID NO: 6, and

(b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the Fab heavy chain are swapped;

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

In one embodiment of the present invention, the bispecific antigen binding molecule of the present invention comprises

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

(b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen-binding fragment is a Fab molecule, wherein the VL and VH variable domains of the Fab light chain and the Fab heavy chain are swapped; where in the CL constant domain of the first antigen-binding fragment, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering) (in one embodiment of the present invention, independently with lysine (K) or arginine (R )) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering) (in one embodiment of the present invention, independently with lysine (K) or arginine (R)), and in constant domain CH1 of the first antigen-binding fragment, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU numbering) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

Formats of bispecific antigen-binding molecules

The components of the bispecific antigen-binding molecule of the present invention may be fused to each other in various configurations. Configuration examples are shown in Fig. 1A-E.

In some embodiments, the antigen-binding molecule fragments included in the bispecific antigen-binding molecule are Fab molecules. In such embodiments, implementation of the present invention, the first, second, third and other antigennegative fragments can be referred to as the first, second, third and other Fab molecules, respectively.

In one embodiment of the present invention, the first and second antigen-binding fragments of the bispecific antigen-binding molecule are fused to each other, optionally via a peptide linker. In some embodiments of the present invention, the first and second antigen-binding fragments are each a Fab molecule. In one such embodiment of the present invention, the second antigen-binding fragment 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 fragment. In another such embodiment of the present invention, the first antigen-binding fragment 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 fragment. In embodiments of the present invention, where either (i) the second antigen-binding fragment 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 fragment, or (ii) the first antigen-binding fragment is fused at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment, additionally the light chain of the Fab of the first antigen-binding fragment and the light chain of the Fab of the second antigen-binding fragment can be fused to each other, optionally via a peptide linker.

A bispecific antigen-binding molecule with a single antigen-binding moiety (e.g., such as a Fab molecule) is capable of specifically binding to a target cell antigen, such as GPRC5D (e.g., shown in FIGS. 1A, 1D, 1G, 13, 1L, 1M), particularly useful in in cases where internalization of the target cell antigen is to be expected after binding of a high affinity antigen binding fragment. In such cases, the presence of more than one antigen-binding fragment specific for the target cell antigen may enhance the internalization of the target cell antigen, thereby reducing its availability.

However, in other cases, it may be useful to have a bispecific antigen-binding molecule containing two or more antigen-binding fragments (for example, Fab molecules) specific for the antigen of target cells (see examples presented in Fig. 1B, 1C, 1E, 1E, 1I , IK, 1H or 1O), for example, to optimize targeting at the target site or to allow cross-linking of target cell antigens.

Thus, in some embodiments of the present invention, the bispecific antigen-binding molecule of the present invention comprises a third antigen-binding moiety.

In one of the embodiments of the present invention, the third antigen-binding fragment binds to the first antigen, i. GPRC5D. In one embodiment of the present invention, the third antigen-binding moiety is a Fab molecule.

In one of the embodiments of the present invention, the third antigennegative fragment is identical to the first antigennegative fragment.

The third antigen-binding fragment of the bispecific antigen-binding molecule may include any of the features, alone or in combination, described herein in relation to the first antigen-binding fragment and/or antibody that binds GPRC5D, except where this is clearly unfounded or scientifically impossible. points of view.

In one embodiment, the third antigen binding fragment comprises 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: 84, and HCDR 3 of SEQ ID NO: 84, and HCDR 3 of SEQ ID NO: 83. SEQ ID NO: 86, and a light chain variable region (VL) containing 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 .

In one embodiment, the third antigen binding fragment comprises 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: 85, and HCDR 3 of SEQ ID NO: 83. SEQ ID NO: 86, and a light chain variable region (VL) containing 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 .

In one embodiment, the third antigen-binding fragment comprises a heavy chain variable region (VH) comprising a 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: 91 SEQ ID NO: 93, and a light chain variable region (VL) containing a 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 .

In one embodiment, the third antigen-binding fragment comprises a heavy chain variable region (VH) comprising a 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: 91 SEQ ID NO: 93, and a light chain variable region (VL) containing a 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 .

In one embodiment, the third antigen-binding fragment comprises a heavy chain variable region (VH) comprising a 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: 92 SEQ ID NO: 93, and a light chain variable region (VL) containing a 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 .

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

In one embodiment, the third antigen binding fragment comprises a heavy chain variable region (VH) comprising a 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: 8, and HCDR 3 of SEQ ID NO: 8. SEQ ID NO: 9, and a light chain variable region (VL) containing a 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 .

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

In one embodiment of the present invention, the VH of the third antigen-binding fragment contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group of sequences 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 fragment contains an amino acid sequence that is at least about 95% 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of sequences 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.

In one embodiment of the present invention, the third antigen binding fragment comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group of sequences 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 an amino acid sequence selected from the group of sequences 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.

In one of the embodiments of the present invention, the third antigen-binding fragment includes a VH region containing an amino acid sequence selected from the group of sequences 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 containing an amino acid sequence selected from the group of sequences 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.

In one embodiment, the third antigen-binding fragment comprises a VH sequence selected from the group 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 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.

In one embodiment, the third antigen-binding fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 13 and a VL region comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment of the present invention, the third antigen-binding fragment comprises the VH sequence of SEQ ID NO: 13 and the sequence VL SEQ ID NO: 14.

In one embodiment, the third antigen-binding fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 15 and a VL region comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment of the present invention, the third antigen-binding fragment comprises the VH sequence of SEQ ID NO: 15 and the sequence VL SEQ ID NO: 16.

In one embodiment, the third antigen-binding fragment comprises a VH region containing the amino acid sequence of SEQ ID NO: 48 and a VL region containing the amino acid sequence of SEQ ID NO: 53. In one embodiment of the present invention, the third antigen-binding fragment comprises the sequence of SEQ ID NO: : 48 and the sequence VL SEQ ID NO: 53.

In one embodiment, the third antigen-binding fragment comprises a VH region containing the amino acid sequence of SEQ ID NO: 49 and a VL region containing the amino acid sequence of SEQ ID NO: 52. In one embodiment of the present invention, the third antigen-binding fragment comprises the sequence of SEQ ID NO: : 49 and the sequence VL SEQ ID NO: 52.

In one embodiment, the third antigen-binding fragment comprises a VH region containing the amino acid sequence of SEQ ID NO: 57 and a VL region containing the amino acid sequence of SEQ ID NO: 64. In one embodiment of the present invention, the third antigen-binding fragment comprises the sequence of SEQ ID NO: : 57 and the sequence VL SEQ ID NO: 64.

In one of the embodiments of the present invention, the third antigen-binding fragment includes a VH region containing the amino acid sequence of SEQ ID NO: 58, and a VL containing the amino acid sequence of SEQ ID NO: 63. In one of the embodiments of the present invention, the third antigen-binding fragment includes the sequence of SEQ ID NO: : 58 and the sequence VL SEQ ID NO: 63.

In one embodiment of the present invention, the third antigen binding fragment comprises a human constant region. In one embodiment of the present invention, the third antigen binding fragment is a Fab molecule containing a human constant region, especially a human CH1 and/or CL domain. Examples of human constant domain sequences are SEQ ID NOs: 37 and 38 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 39 (human IgG ₁ heavy chain CH1-CH2-CH3 constant domains). In some embodiments, the third antigen-binding fragment 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: 38, especially the amino acid sequence of SEQ ID NO: 37. In particular, the light chain constant region may include amino acid mutations as described herein under the term "charge modifications" and/or may include deletion or substitutions of one or several (in particular two) N-terminal amino acids in the case of a crossover Fab molecule. In some embodiments, the third antigen-binding fragment comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the CH1 domain contained in the amino acid sequence. SEQ ID NO: 39. In particular, the heavy chain constant region (particularly the CH1 domain) may include amino acid mutations as described herein under the term "charge modifications".

In some embodiments, the implementation of the present invention every third and first antigennegative fragment is a Fab molecule, and the third antigennegative fragment is identical to the first antigennegative fragment. Thus, in these embodiments, the implementation of the present invention, the first and third antigennegative fragment have the same amino acid sequences of the heavy and light chains, as well as the same arrangement of domains (ie, normal or crossover). In addition, in these embodiments, implementation of the present invention, the third antigennegative fragment contains the same amino acid substitutions, if any, as the first antigennegative fragment. For example, the amino acid substitutions described herein under the term "charge modifications" can be made in the CL constant domain and the CH1 constant domain of each of both the first antigen-binding fragment and the third antigen-binding fragment. In another embodiment, said amino acid substitutions can be made in the CL constant domain and in the CH1 constant domain of the second antigen-binding fragment (which in specific embodiments of the present invention is also a Fab molecule), but not in the CL constant domain and the CH1 constant domain of the first antigen-binding fragment and the third antigen binding fragment.

Like the first antigen-binding fragment, the third antigen-binding fragment, in particular, is a conventional Fab molecule. However, the present invention also contemplates those embodiments in which the first and third antigen-binding fragments are Fab crossover molecules (and the second antigen-binding fragment is a regular Fab molecule). Thus, in some embodiments of the present invention, the first and third antigen-binding fragments are a regular Fab molecule, and the second antigen-binding fragment is a crossover Fab molecule, as described in the present invention, that is, a Fab molecule in which the VH and VL variable domains or the CL constant domains and The CH1s of the Fab heavy and light chains are exchanged/replaced with each other. In other embodiments, the first and third antigen-binding fragments are a crossover Fab molecule and the second antigen-binding fragment is a normal Fab molecule.

If a third antigen binding fragment is present, in one embodiment of the present invention, the first and third antigen binding fragments bind to GPRC51), and the second antigen binding fragment binds to a second antigen, in particular an activating T cell antigen, especially CD3, most preferably CD3 epsilon.

In some embodiments, the implementation of the present invention bespecifically antigennegative molecule contains an Fc domain consisting of the first and second subunits. The first and second subunits of the Fc domain are able to stably associate.

The bispecific antigen-binding molecule of the present invention may have different configurations, ie. the first, second (and optionally third) antigen-binding fragments can be fused to each other and to the Fc domain by various methods. The components may be fused to each other directly, or preferably via one or more suitable peptide linkers. The fusion of the Fab molecule occurs to the N terminus of the Fc domain subunit, this usually occurs through the immunoglobulin hinge region.

In some embodiments, the first and second antigen-binding fragments are Fab molecules, and the second antigen-binding fragment is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In such embodiments, the first antigen-binding fragment may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding fragment, or to the N-terminus of another subunit of the Fc domain. In specific such embodiments of the present invention, said first antigen-binding fragment is a conventional Fab molecule and the second antigen-binding fragment is a crossover Fab molecule of the present invention, i.e. a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the heavy and light chains of the Fab are exchanged/replaced with each other. In other similar embodiments of the present invention, said first Fab molecule is a crossover Fab molecule and the second Fab molecule is a regular Fab molecule.

In one embodiment of the present invention, the first and second antigen-binding fragments are Fab molecules, and the second antigen-binding fragment 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 fragment. In a specific embodiment of the present invention, the bispecific antigen-binding molecule essentially consists of a first and a second Fab molecule, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the heavy chain Fab to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Such a configuration is shown schematically in Fig. 1G and 1L (with the second antigen-binding domain in these examples being the VH/VL Fab crossover molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

In another embodiment of the present invention, both the first and second antigen-binding fragment are a Fab molecule, and both the first and second antigen-binding fragment are 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 of the present invention, the bispecific antigen-binding molecule essentially consists of a first and second Fab molecule, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, with both the first and second Fab molecule fused at C -end of the Fab heavy chain with the N-terminus of one of the subunits of the Fc domain. Such a configuration is shown schematically in Fig. 1A and 1D (in these examples, the second antigen binding domain is a VH/VL Fab crossover molecule and the first antigen binding fragment is a regular Fab molecule). The first and second Fab molecules can be fused to the Fc domain directly or through a peptide linker. In one embodiment of the present invention, both the first and second Fab molecules are fused to the Fc domain through the immunoglobulin hinge region. In a specific embodiment of the present invention, the immunoglobulin hinge region is a human IgG ₁ hinge region, in particular if the Fc domain is an IgG ₁ Fc domain.

In some embodiments, both the first and second antigen-binding fragment is a Fab molecule, and the first antigen-binding fragment is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In such embodiments, the second antigen-binding fragment may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding fragment, or (as described above) to the N-terminus of another subunit of the Fc domain. In particular embodiments of the present invention, said first antigen-binding fragment is a normal Fab molecule and the second antigen-binding fragment is a Fab crossover 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 heavy and light chains of the Fab are exchanged/replaced with each other. In other such embodiments of the present invention, said first Fab molecule is a crossover Fab molecule and the second Fab molecule is a regular Fab molecule.

In one embodiment of the present invention, both the first and second antigen-binding fragment is a Fab molecule, and the first antigen-binding fragment 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, and the second antigen-binding fragment is fused at the C-terminus Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen binding fragment. In a specific embodiment of the present invention, the bispecific antigen binding molecule essentially consists of a first and a second Fab molecule, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, with the second Fab molecule fused at the C-terminus of the heavy chain Fab to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Such a configuration is shown schematically in Fig. 13 and 1M (in these examples, the second antigen binding domain is a VH/VL Fab crossover molecule and the first antigen binding fragment is a regular Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

In some embodiments of the present invention, the third antigen-binding fragment, in particular 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 specific such embodiments of the present invention, both the first and third said Fab molecules are a regular Fab molecule, and the second Fab molecule is a crossover Fab molecule as described in the present invention, i.e., a Fab molecule in which the VH and VL variable domains or the CL constant domains and CH1 heavy and light chains of Fab are exchanged/replaced for each other. In other such embodiments, both the first and third Fab molecules are crossover Fab molecules, and the second Fab molecule is a regular Fab molecule.

In one such embodiment of the present invention, each second and third antigen-binding fragment is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits, and the first antigen-binding fragment is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain the second Fab molecule. In one embodiment of the present invention, the bispecific antigen-binding molecule essentially consists of first, second, and third Fab molecules, an Fc domain consisting of first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at C- end 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 N -end of the second subunit of the Fc domain. Such a configuration is schematically shown in Fig. 1B and 1E (in these examples, the second antigen-binding fragment is the VH/VL of a Fab crossover molecule, and the first and third antigen-binding fragments are a regular Fab molecule), and also in FIG. 1K and 10 (in these examples, the second antigen-binding fragment is a regular Fab molecule, and the first and third antigen-binding fragments are the VH/VL of the Fab crossover molecule). The second and third Fab molecules can be fused to the Fc domain directly or through a peptide linker. In one embodiment of the present invention, the second and third Fab molecules are both fused to the Fc domain through the immunoglobulin hinge region. In another embodiment of the present invention, the immunoglobulin hinge region is a human IgG ₁ hinge region, especially if the Fc domain is an IgG ₁ Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

In another similar embodiment of the present invention, the first and third antigen-binding fragment is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits, and the second antigen-binding fragment 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 fragment. In one embodiment of the present invention, the bispecific antigen-binding molecule consists of a first, second, and third Fab molecule, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, with the second Fab molecule 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 where the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunits of the Fc domain. Such a configuration is schematically shown in Fig. 1B and 1E (in these examples, the second antigen-binding fragment is the VH/VL of a Fab crossover molecule and the first and third antigen-binding fragments are a normal Fab molecule) and FIG. 1I and 1H (in these examples, the second antigen binding fragment is a regular Fab molecule and the first and third antigen binding fragment is the VH/VL crossover Fab molecule). The first and third Fab molecules can be fused to the Fc domain directly or via a peptide linker. In one of the embodiments of the present invention, the first and third Fab molecules can be fused to the Fc domain through the hinge region of the immunoglobulin. In a certain embodiment of the present invention, the immunoglobulin hinge region is a human IgG ₁ hinge region, especially if the Fc domain is an IgG ₁ Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

In bispecific antigen-binding molecule configurations in which a Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of each of the Fc domain subunits via the immunoglobulin hinge regions, two Fab molecules, the hinge regions and the Fc domain essentially form an immunoglobulin molecule. In one of the embodiments of the present invention, the immunoglobulin molecule belongs to the class of immunoglobulin IgG. In yet another more specific embodiment of the present invention, the immunoglobulin is an immunoglobulin of the IgG ₁ subclass. In another embodiment of the present invention, the immunoglobulin is an immunoglobulin of the IgG ₄ subclass. In yet another embodiment of the present invention, 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, especially a human Fc region.

In some of the bispecific antigen binding molecules of the present invention, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are fused to each other, optionally via a peptide linker. Depending on the configuration of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused at the C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at the C-terminus to N the Fab light chain end of the first Fab molecule. Fusion of the Fab light chains of the first and second Fab molecules further reduces the mismatch between mismatched Fab heavy and light chains, and also reduces the number of plasmids required to express some of the bispecific antigen-binding molecules of the present invention.

The antigen binding fragments can be fused to the Fc domain or to each other directly or through a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described in the present invention. Useful non-immunogenic linkers include, for example, peptide linkers (G ₄ S) _n , (SG ₄ ) _n , (G ₄ S) _n or G ₄ (SG ₄ ) _n . Usually "n" is an integer from 1 to 10, usually from 2 to 4. In one embodiment of the present invention, the specified peptide linker is at least 5 amino acids in length, in one embodiment of the present invention from 5 to 100, and in another embodiment of the present invention, from 10 to 50 amino acids. In one of the embodiments of the present invention, the specified peptide linker is (GxS) _n or (GxS) _n G _m where G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m =0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3), in one embodiment of the present invention x=4 and n=2 or 3, in another embodiment of the present invention x=4 and n=2. In one of the embodiments of the present invention, the specified peptide linker is (G ₄ S) ₂ . A particularly suitable peptide linker for fusing the Fab light chains of the first and second Fab molecules to each other is (G ₄ S) ₂ . An exemplary peptide linker suitable for joining the Fab heavy chains of the first and second Fab fragments comprises the sequence (D)-(G ₄ S) ₂ (SEQ ID NOS 43 and 44). Another similar useful linker contains the sequence (G ₄ S) ₄ . Additionally, the linkers may include (part of) the hinge region of the immunoglobulin. In particular, if a Fab molecule is fused to the N-terminus of an Fc domain subunit, it may be fused using an immunoglobulin hinge or portion thereof, with or without an additional peptide linker.

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

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

In some embodiments, the bispecific antigen binding molecule comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover heavy chain Fab in which the heavy chain variable region is replaced by a light chain variable region) which in turn has a carboxy-terminal peptide bond to the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond to 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 carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond end with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule includes the Fab crossover heavy chain where the heavy chain variable region is replaced by the light chain variable region), which in turn has a carboxy-terminal peptide bond to the subunit Fc domain (VH ₍₁₎ -CH1 ₍₁₎ -VL ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4)).

In some of these embodiments, the bispecific antigen binding molecule comprises a Fab crossover light chain polypeptide of a second Fab molecule, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH _{(2 )} -CL ₍₂₎ ), and the Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ). In other such embodiments, the bispecific antigen binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, which in turn shares a peptide bond at the carboxy-terminus to the Fab light chain polypeptide of the first Fab molecule (VH ₍₂₎ -CL ₍₂₎ -VL ₍₁₎ -CL ₍₁₎ ), or a polypeptide in which the Fab light chain polypeptide of the first Fab molecule has a common carboxy -terminal peptide bond to the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond to the Fab light chain constant region of the second Fab molecule (VL ₍₁₎ -CL ₍₁₎ -VH ₍₂₎ -CL ₍₂₎ ), depending on the situation.

The bispecific antigen-binding molecule of such embodiments of the present invention 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 has a carboxy-terminal peptide bond with the Fc domain subunit (VH ₍₃₎ -CH1 ₍₃₎ -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL ₍₃₎ -CL ₍₃₎ ). In some embodiments, implementation of the present invention, the polypeptides are covalently linked, for example, by a disulfide bond.

In certain embodiments, the bispecific antigen-binding molecule comprises a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover heavy chain Fab, where the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the subunit Fc domain (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 carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond end with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule includes the heavy chain of the Fab crossover, where the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxy-terminal common peptide bond with the domain subunit Fc (VH ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CL ₍₂₎ -CH2-CH3(-CH4)).

In some such embodiments, the bispecific antigen binding molecule further comprises a crossover Fab light chain polypeptide of the second Fab molecule, wherein the Fab light chain variable region of the second Fab molecule has a carboxy-terminal peptide bond to the Fab heavy chain constant region of the second Fab molecule (VL ₍₂₎ -CH1 ₍₂₎ ), and the Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ). In other such embodiments, the bispecific antigen-binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, which in turn shares a peptide bond at the carboxy-terminus to the Fab light chain polypeptide of the first Fab molecule (VL ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CL ₍₁₎ ), or a polypeptide in which the Fab light chain polypeptide of the first Fab molecule has a common carboxy -terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VL ₍₁₎ -CL ₍₁₎ -VL _{(2 )} -CH1 ₍₂₎ ), as needed.

The bispecific antigen-binding molecule of such embodiments of the present invention 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 has a carboxy-terminal peptide bond to the Fc domain subunit Fc (VH ₍₃₎ -CH1 ₍₃₎ -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL ₍₃₎ -CL ₍₃₎ ). In certain embodiments of the present invention, the polypeptides are covalently linked, for example, by a disulfide bond.

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

In certain such embodiments, the bispecific antigen-binding molecule essentially consists of a first and a second antigen-binding moiety and optionally one or more peptide linkers, wherein the first and second antigen-binding moiety are Fab molecules and the first antigen-binding moiety is fused at the C-terminus of the heavy chain Fab with the N-terminus of the Fab heavy chain of the second antigen binding fragment. Such a configuration is shown schematically in Fig. 1P and 1U (in these examples with the second antigen-binding domain being a VH/VL Fab crossover molecule and the first antigen-binding fragment being a regular Fab molecule).

In some embodiments of the present invention, 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 fragment, in particular, the third Fab molecule, in which the specified third molecule The Fab 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 essentially consists of first, second, and third Fab molecules and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain. of the second Fab molecule, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. Such a configuration is schematically depicted in Figures 1C and 1X (in these examples, the second antigen-binding domain is a VH/VL Fab crossover molecule, and the first and antigen-binding fragment are conventional Fab molecules) or in Figures 1S and 1E (in these examples, the second antigen-binding domain is a conventional a Fab molecule, and each of the first and third antigen binding fragments is a VH/VL crossover Fab molecule).

In some embodiments, the second Fab molecule is fused at the C-terminus of the heavy chain to the N-terminus of the heavy chain of the first Fab molecule, and the bispecific antigen-binding molecule further comprises a third antigen-binding fragment, in particular a third Fab molecule, the third Fab molecule being fused at N- end of the heavy chain with the C-terminus of the heavy chain of the first Fab molecule. In some such embodiments, the bispecific antigen-binding molecule essentially consists of first, second, and third Fab molecules and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the heavy chain to the N-terminus of the heavy chain of the first molecule. Fab, and the third Fab molecule is fused at the N-terminus of the heavy chain to the C-terminus of the heavy chain of the first Fab molecule. Such a configuration is schematically depicted in Figures 1T and II (in these examples, the second antigen-binding domain is a VH/VL_Fab crossover molecule, and the first domain and antigen-binding fragment are conventional Fab molecules) or in Figures 1H and 1R (in these examples, the second antigen-binding domain is a conventional Fab molecule). Fab, and the first domain and the third antigen-binding fragment are VH/VL_Fab crossover molecules).

In some embodiments, the bispecific antigen binding molecule of the present invention comprises a polypeptide wherein the heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond. -the end with the heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule includes the heavy chain of the crossover Fab molecule, where 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 the heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the light chain constant region of the second Fab molecule (VH ₍₂₎ -CL ₍₂₎ ) and the polypeptide light chain of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ).

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

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

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

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

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

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

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

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

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

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

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

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits; Where

(i) the first antigen-binding fragment of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding fragment of b) and the third antigen-binding fragment of c) are all fused at the C-terminus heavy chain Fab with the N-terminus of the subunits of the Fc domain according to item d), or

(ii) the second antigen-binding fragment of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding fragment of a) and the first antigen-binding fragment of a) the third antigen-binding fragment of c. ) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of claim d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits; Where

(i) the first antigen-binding fragment of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding fragment of b), the second antigen-binding fragment of b), and the third antigen-binding fragment of b) c) all fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d), or

(ii) the second antigen-binding fragment according to 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 fragment according to paragraph a), and the first antigen-binding fragment according to paragraph a) and the third antigen-binding fragment according to paragraph c) all fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits; Where

(i) the first antigen-binding fragment according to 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 fragment according to b), and the second antigen-binding fragment according to b), as well as the third antigen-binding fragment c) all fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c) or

(ii) the second antigen-binding fragment according to 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 fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of claim d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 96 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits; Where

(i) the first antigen-binding fragment according to 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 fragment according to b), and the second antigen-binding fragment according to b), as well as the third antigen-binding fragment c) all fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c) or

(ii) the second antigen-binding fragment according to 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 fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of claim d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits; Where

(i) the first antigen-binding fragment according to 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 fragment according to b), and the second antigen-binding fragment according to b), as well as the third antigen-binding fragment c) all fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c) or

(ii) the second antigen-binding fragment according to 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 fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of claim d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

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

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits; Where

(i) the first antigen-binding fragment according to 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 fragment according to b), and the second antigen-binding fragment according to b), as well as the third antigen-binding fragment c) all fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c) or

(ii) the second antigen-binding fragment according to 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 fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of claim d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, LCDR 2 sequences of SEQ ID NO: 11 and LCDR 3 sequences of SEQ ID NO: 12;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits; Where

(i) the first antigen-binding fragment according to 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 fragment according to b), and the second antigen-binding fragment according to b), as well as the third antigen-binding fragment c) all fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c) or

(ii) the second antigen-binding fragment according to 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 fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of claim d).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits; Where

(i) the first antigen-binding fragment of a) and the second antigen-binding fragment of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits; Where

(i) the first antigen-binding fragment of a) and the second antigen-binding fragment of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits;

Where

(i) the first antigen-binding fragment of a) and the second antigen-binding fragment of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 96 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits; Where

(i) the first antigen-binding fragment of a) and the second antigen-binding fragment of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits; Where

(i) the first antigen-binding fragment of a) and the second antigen-binding fragment of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

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

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits; Where

(i) the first antigen-binding fragment of a) and the second antigen-binding fragment of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, LCDR 2 sequences of SEQ ID NO: 11 and LCDR 3 sequences of SEQ ID NO: 12;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits;

Where

(i) the first antigen-binding fragment of a) and the second antigen-binding fragment of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c).

In all the various configurations of the bispecific antigen-binding molecule of the present invention, the amino acid substitutions described herein, if any, may be located either in the CH1 and CL domains of the first and (if any) third antigen-binding fragment/Fab molecule, or in the CH1 and CL domains of the second antigen-binding Fab fragment/molecule. Preferably they are in the CH1 and CL domains of the first and (if present) third antigen-binding fragment/Fab molecule. In accordance with the purpose of the present invention, if the amino acid substitutions of the present invention are made in the first (and, if any, third) antigen-binding fragment/Fab molecule, such amino acid substitutions are not made in the second antigen-binding fragment/Fab molecule. Conversely, if the amino acid substitutions of the present invention are made in the second antigen-binding fragment/Fab molecule, such amino acid substitutions are not made in the first (and, if present, third) antigen-binding fragment/Fab molecule. Amino acid substitutions, in particular, are made in bispecific antigen-binding molecules containing a Fab molecule in which the variable domains VL and VH1 of the Fab light chain and the Fab heavy chain are exchanged for each other.

In certain embodiments of the present invention, the bispecific antigen-binding molecule, in particular, when the amino acid substitutions of the present invention are made in the first (and, if any, third) antigen-binding fragment/Fab molecule, the CL constant domain of the first (and, if present, third) Fab molecule belongs to the kappa isotype. In other embodiments of the present invention, the bispecific antigen binding molecule of the present invention, in particular, where the amino acid substitutions of the present invention are made in the second antigen binding fragment/Fab molecule, the CL constant domain of the second antigen binding fragment/Fab molecule is kappa isotype. In some embodiments, the CL constant domain of the first (and, if present, third) Fab/antigen-binding fragment molecule is of the kappa isotype.

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant region of the first antigen-binding fragment according to a) the amino acid at position 124 is replaced by lysine (K) (Kabat numbering) and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)), and where in the CH1 constant domain of the first antigen-binding fragment according to a), the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the Kabat EU index) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering po Kabat EU index; and

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to c), or

(ii) the second antigen-binding fragment of b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment of a), and the first antigen-binding fragment of a) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to item c).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a) the amino acid at position 124 is replaced by lysine (K) (Kabat numbering) and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)), and where in the CH1 constant domain of the first antigen-binding fragment according to a), the amino acid at position 147 is replaced by glutamic acid (E) (Kabat EU numbering) and the amino acid at position 213 is replaced by glutamic acid (E) (numbered by Kabat EU index); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to c), or

(ii) the second antigen-binding fragment of b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment of a), and the first antigen-binding fragment of a) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to item c).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)); numbering according to Kabat EU index); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to c), or

(ii) the second antigen-binding fragment of b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment of a), and the first antigen-binding fragment of a) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to item c).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 96 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)); numbering according to Kabat EU index); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to c), or

(ii) the second antigen-binding fragment of b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment of a), and the first antigen-binding fragment of a) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to item c).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)); numbering according to Kabat EU index); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to c), or

(ii) the second antigen-binding fragment of b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment of a), and the first antigen-binding fragment of a) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to item c).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

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

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R));

the amino acid at position 213 is replaced by glutamic acid (E) (po Kabat EU index numbering); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to c), or

(ii) the second antigen-binding fragment of b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment of a), and the first antigen-binding fragment of a) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to item c).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, LCDR 2 sequences of SEQ ID NO: 11 and LCDR 3 sequences of SEQ ID NO: 12;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more specifically CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains or the CL constant domains and CH1 light chain Fab and heavy chain Fab are exchanged for each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)); numbering according to Kabat EU index); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to c), or

(ii) the second antigen-binding fragment of b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment of a), and the first antigen-binding fragment of a) is hybridized at the C-terminus of the heavy Fab chains with the N-terminus of one of the subunits of the Fc domain according to item c).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to item a) and the third antigen-binding fragment according to item c), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine ( R) (Kabat numbering) (usually arginine (R)), and where in the CH1 constant domain of the first antigen-binding fragment according to a) and the third antigen-binding fragment according to c), the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the Kabat EU index); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) and the third antigen-binding fragment according to p c) hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d), or

(ii) the second antigen-binding fragment according to b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to item a) and the third antigen-binding fragment according to item c), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine ( R) (Kabat numbering) (usually arginine (R)), and where in the CH1 constant domain of the first antigen-binding fragment according to a) and the third antigen-binding fragment according to c), the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the Kabat EU index); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) and the third antigen-binding fragment according to p c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of c), or

(ii) the second antigen-binding fragment according to b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to item a) and the third antigen-binding fragment according to item c), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine ( R) (Kabat numbering) (usually arginine (R)), and where in the CH1 constant domain of the first antigen-binding fragment according to a) and the third antigen-binding fragment according to c), the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the Kabat EU index); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) and the third antigen-binding fragment according to p c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d), or

(ii) the second antigen-binding fragment according to b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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) containing the region

light chain complementarity determining (HCDR) 1 sequences of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 96 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to item a) and the third antigen-binding fragment according to item c), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine ( R) (Kabat numbering) (usually arginine (R)), and where in the CH1 constant domain of the first antigen-binding fragment according to a) and the third antigen-binding fragment according to c), the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the Kabat EU index); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) and the third antigen-binding fragment according to p c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d), or

(ii) the second antigen-binding fragment according to b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to item a) and the third antigen-binding fragment according to item c), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine ( R) (Kabat numbering) (usually arginine (R)), and where in the CH1 constant domain of the first antigen-binding fragment according to a) and the third antigen-binding fragment according to c), the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the Kabat EU index); And

Where:

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) and the third antigen-binding fragment according to p c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d), or

(ii) the second antigen-binding fragment according to b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

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

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to item a) and the third antigen-binding fragment according to item c), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine ( R) (Kabat numbering) (usually arginine (R)), and where in the CH1 constant domain of the first antigen-binding fragment according to a) and the third antigen-binding fragment according to c), the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the Kabat EU index); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) and the third antigen-binding fragment according to p c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d), or

(ii) the second antigen-binding fragment according to b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d).

In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, LCDR 2 sequences of SEQ ID NO: 11 and LCDR 3 sequences of SEQ ID NO: 12;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) a third antigen-binding fragment that binds to the first antigen and is identical to the first antigen-binding fragment; And

d) Fc domain, consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to item a) and the third antigen-binding fragment according to item c), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine ( R) (Kabat numbering) (usually arginine (R)), and where in the CH1 constant domain of the first antigen-binding fragment according to a) and the third antigen-binding fragment according to c), the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 is replaced by glutamic acid (E) (numbering according to the Kabat EU index); And

Where

(i) the first antigen-binding fragment according to a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to b), and the second antigen-binding fragment according to b) and the third antigen-binding fragment according to p c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d), or

(ii) the second antigen-binding fragment according to b) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding fragment according to a), the first antigen-binding fragment according to a) and the third antigen-binding fragment according to p. c) each hybridized at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits of d).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to item a) and the third antigen-binding fragment according to item c), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine ( R) (Kabat numbering) (usually arginine (R)), and where in the CH1 constant domain of the first antigen-binding fragment according to a) the amino acid at position 147 is replaced by glutamic acid (E) (Kabat EU index numbering), and the amino acid in position 213 is replaced by glutamic acid (E) (numbering according to the Kabat EU index); And

where the first antigen-binding fragment according to paragraph a) and the second antigen-binding fragment according to paragraph 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 according to paragraph c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 87, LCDR 2 sequences of SEQ ID NO: 88 and LCDR 3 sequences of SEQ ID NO: 89;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)); numbering according to Kabat EU index); And

where the first antigen-binding fragment according to paragraph a) and the second antigen-binding fragment according to paragraph 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 according to paragraph c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)); numbering according to Kabat EU index); And

where the first antigen-binding fragment according to paragraph a) and the second antigen-binding fragment according to paragraph 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 according to paragraph c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 96 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)); numbering according to Kabat EU index); And

where the first antigen-binding fragment according to paragraph a) and the second antigen-binding fragment according to paragraph 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 according to paragraph c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 94, LCDR 2 sequences of SEQ ID NO: 95 and LCDR 3 sequences of SEQ ID NO: 97;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)); numbering according to Kabat EU index); And

where the first antigen-binding fragment according to paragraph a) and the second antigen-binding fragment according to paragraph 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 according to paragraph c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

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

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)); numbering according to Kabat EU index); And

where the first antigen-binding fragment according to paragraph a) and the second antigen-binding fragment according to paragraph 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 according to paragraph c).

In another embodiment, the present invention provides a bispecific antigen binding molecule comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment is a Fab molecule containing a heavy chain variable region (VH) containing a 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 light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, LCDR 2 sequences of SEQ ID NO: 11 and LCDR 3 sequences of SEQ ID NO: 12;

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is an activating T-cell antigen, in particular CD3, more precisely CD3 epsilon, and the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the heavy Fab chains are replaced with each other;

c) an Fc domain consisting of the first and second subunits;

where in the CL constant domain of the first antigen-binding fragment according to a), the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) (usually arginine (R)); numbering according to Kabat EU index); And

where the first antigen-binding fragment according to paragraph a) and the second antigen-binding fragment according to paragraph 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 according to paragraph c).

According to any of the above embodiments of the present invention, the components of the bispecific antigen-binding molecule (e.g., Fab molecules, Fc domain) can be hybridized directly or through various linkers, in particular peptide linkers containing one or more amino acids, usually about 2-20 amino acids, which are described in the present invention or known in the art. Suitable non-immunogenic peptide linkers include, for example, peptide linkers (G ₄ S) _n , (SG ₄ ) _n , (G ₄ S) _n or G ₄ (SG ₄ ) _n , where n is usually an integer from 1 to 10, usually 2 to 4.

In one aspect of the present invention, a bispecific antigen-binding molecule is provided, comprising

a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first and second antigen-binding fragments are a (conventional) Fab molecule containing a heavy chain variable region containing the amino acid sequences of SEQ ID NO: 13 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 14.

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is CD3, the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the Fab heavy chain, interchanged, contain a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 36;

c) an Fc domain consisting of the first and second subunits;

Where

in the CL constant domain of the first and third antigen-binding fragments according to paragraph a) the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) ( usually arginine (R)), and where in the CH1 constant domain of the first and third antigen-binding fragment according to a) the amino acid at position 147 is replaced by glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is replaced by glutamic acid (E ) (numbering according to Kabat EU index);

and additionally

the first antigen-binding fragment according to paragraph a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to paragraph b), the second antigen-binding fragment according to paragraph b) and the third antigen-binding fragment according to paragraph a) hybridized at the C-terminus of the Fab heavy chain with the N-terminus of one of the subunits of the Fc domain according to item c).

In one aspect of the present invention, a bispecific antigen-binding molecule is provided, comprising

a) first and third antigen-binding fragments that bind to the first antigen, wherein the first antigen is GPRC5D and the first and second antigen-binding fragments are a (conventional) Fab molecule containing a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and variable a light chain region containing the amino acid sequence of SEQ ID NO: 16.

b) a second antigen-binding fragment that binds to a second antigen, wherein the second antigen is CD3, the second antigen-binding fragment is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the Fab heavy chain, interchanged, contain a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 36;

c) an Fc domain consisting of the first and second subunits; Where

in the CL constant domain of the first and third antigen-binding fragments according to paragraph a) the amino acid at position 124 is replaced by lysine (K) (Kabat numbering), and the amino acid at position 123 is replaced by lysine (K) or arginine (R) (Kabat numbering) ( usually arginine (R)), and where in the CH1 constant domain of the first and third antigen-binding fragment according to a) the amino acid at position 147 is replaced by glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is replaced by glutamic acid (E ) (numbering according to Kabat EU index);

and additionally

the first antigen-binding fragment according to paragraph a) is hybridized at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding fragment according to paragraph b), the second antigen-binding fragment according to paragraph b) and the third antigen-binding fragment according to paragraph a) hybridized at the C-terminus of the Fab heavy chain with the N-terminus of one of the subunits of the Fc domain according to item c).

In one of the embodiments of the present invention, with respect to one of the objects, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V) and optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (Kabat EU index numbering).

In another embodiment of the present invention with respect to one of the objects, in the first subunit of the Fc domain, the serine residue at position 354 is additionally replaced by a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced by a cysteine residue), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is additionally replaced by a cysteine residue (Y349C) (numbering according to the Kabat EU index).

In another embodiment of the present invention, in one of the objects of the present invention, in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbered according to the Kabat EU index).

In yet another embodiment of the present invention with respect to one of the objects, Fc is a human IgGi Fc domain.

In one embodiment of the present invention, the bispecific antigen-binding molecule comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 17, a polypeptide comprising an amino acid sequence, which is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 18, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 19, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 20.

In yet another embodiment of the present invention, the bispecific antigen-binding molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 17, a polypeptide comprising the amino acid sequence of SEQ ID NO: 18, a polypeptide comprising the amino acid sequence of SEQ ID NO: 19, and a polypeptide comprising the amino acid sequence the sequence of SEQ ID NO: 20.

In another specific embodiment of the present invention, the bispecific antigen-binding molecule comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 21, a polypeptide comprising an amino acid sequence, which is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 22, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 23, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 24. In another specific embodiment, The bispecific antigen-binding molecule of the present invention comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 21, a polypeptide comprising the amino acid sequence of SEQ ID NO: 22, a polypeptide comprising the amino acid sequence of SEQ ID NO: 23, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 24 .

Fc domain

In some embodiments, the implementation of the present invention bespecifically antigennegative molecule contains an Fc domain consisting of the first and second subunits. Obviously, the features of the Fc domain described in the present invention in relation to the bispecific antigen-binding molecule can equally apply to the Fc domain that is part of the antibody of the present invention.

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

In one embodiment of the present invention, the Fc domain of the bispecific antigen-binding molecule is an IgG Fc domain. In one embodiment of the present invention, the Fc domain is an IgG ₁ Fc domain. In another embodiment of the present invention, the Fc domain is an IgG ₄ Fc domain. In one specific embodiment of the present invention, the Fc domain is an L3G4 Fc domain containing an amino acid substitution at position S228 (Kabat EU index numbering), in particular the S228P amino acid substitution. This amino acid substitution reduces in vivo turnover of the Fab arm of IgG ₄ antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 2010, 84-91). In another specific embodiment of the present invention, the Fc domain is a human Fc domain. In yet another more specific embodiment of the present invention, the Fc domain is a human IgG ₁ Fc domain. As an example, the sequences of the Fc region of human IgG ₁ SEQ ID NO: 42 are given.

Fc Domain Modifications Activating Heterodimerization

The bispecific antigen-binding molecules of the present invention comprise various antigen-binding moieties that can be hybridized to one or the other of two Fc domain subunits such that the two Fc domain subunits are typically contained in two non-identical polypeptide chains. Co-recombinant expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. Thus, in order to increase the yield and purity of bispecific antigen-binding molecules, it is advisable to introduce a modification into the Fc domain of the bispecific antigen-binding molecule that promotes the association of the corresponding polypeptides.

Accordingly, in certain embodiments of the implementation of the present invention, the Fc domain of the bispecific antigen-binding molecule of the present invention contains a modification that activates the association of the first and second subunits of the Fc domain. The location of most of the intense protein-protein interactions between the two subunits of the human IgG Fc domain is contained in the CH3 domain of the Fc domain. Thus, in one of the embodiments of the present invention, the specified modification is contained in the CH3 domain of the Fc domain.

There are several approaches to modifying the CH3 domain of the Fc domain to enhance heterodimerization, which are described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such methods, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are constructed elementary, resulting in each domain CH3 (or the heavy chain containing it) can no longer homodimerize with itself, but are forced to form a heterodimer with a complementary engineered other CH3 domain (resulting in the first and second CH3 domains being heterodimerized rather than homodimerizing between the first two or the two second CH3 domains) . These different approaches to improve heavy chain heterodimerization are considered as different alternatives in combination with heavy-light chain modifications (e.g. exchange/substitution between VH and VL at the same binding arm and introduction of charged amino acid substitutions with opposite charges at the CH1/CL interface) in a bispecific antigen-binding molecule that reduce heavy/light chain mismatch and the formation of Bence-Jones type by-products.

In one embodiment of the present invention, said modification to promote association of the first and second subunits of the Fc domain is a so-called "trough-to-trough" modification, comprising a "trough" modification in one of the two Fc domain subunits and a "trough" modification to the other of the Fc domain subunits. two subunits of the Fc domain.

The method "protrusion in the cavity" is described, for example, in US 5731168; US 7695936; Ridgway et al., Prot Eng 9, 1996, 617-621; Carter, J Immunol Meth 248, 2001, 7-15). The method usually involves the introduction of a bulge ("protrusion") at the interface in the first polypeptide and a corresponding cavity ("cavity") at the interface in the second polypeptide, so that the protrusion can be located in the cavity, which promotes the formation of a heterodimer and prevents the formation of a homodimer. Overhangs are formed by replacing small amino acid side chains at the interface in the first polypeptide with larger side chains (eg tyrosine or tryptophan). Compensatory pits corresponding to or similar in size to the protrusions are created at the interface of the second polypeptide by replacing large amino acid side chains with smaller chains (eg, alanine or threonine).

Thus, in one embodiment of the present invention, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen-binding molecule, an amino acid residue is replaced with an amino acid residue having a larger side chain, thereby creating an overhang in the CH3 domain of the first subunit that is located in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain, thereby creating a trough in the CH3 domain of the second subunit that can accommodate the overhang of the CH3 domain of the first subunit.

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

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

The ridge and pit can be produced by altering the nucleic acid encoding the polypeptides, for example by site-directed mutagenesis or by peptide synthesis.

In one of the embodiments of the present invention, in the (CH3 domain) of the first subunit of the Fc domain (the "lump" subunit), the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit (in the CH3 domain) of the Fc domain (the "trough" subunit ) the tyrosine residue at position 407 is replaced by a valine residue (Y407V). In one embodiment of the present invention, in the second subunit of the Fc domain, the threonine residue at position 366 is additionally replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (Kabat EU index numbering).

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

In one embodiment of the present invention, the first subunit of the Fc domain contains the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain contains the amino acid substitutions Y349C, T366S, L368A and Y407V (Kabat EU index numbering).

In a specific embodiment of the present invention, an antigen-binding fragment that binds to a second antigen (e.g., an activating T-cell antigen) is hybridized (optionally through a first antigen-binding fragment that binds to GPRC5D and/or a peptide linker) to a first subunit of the Fc domain (containing the modification "protrusion"). Without wishing to be bound by theory, it is noted that fusion of an antigen-binding moiety that binds a second antigen, e.g., an activating T-cell antigen, to an overhang-containing Fc domain subunit will (additionally) minimize the formation of antigen-binding molecules containing two antigen-binding groups that bind to an activating T-cell antigen (steric collision of two polypeptides containing projections).

The present invention considers as an alternative other methods of CH3 modification to enhance heterodimerization, as described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

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

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

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

In one of the embodiments of the present invention, the method described in WO 2013/157953 is used as an alternative. In another embodiment of the present invention, the first CH3 domain contains the amino acid mutation T366K and the second domain contains the amino acid mutation L351D (Kabat EU index numbering). In another embodiment of the present invention, the first CH3 domain further comprises the amino acid mutation L351K. In another embodiment of the present invention, the second domain further comprises an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (Kabat EU index numbering).

In one embodiment of the present invention, the heterodimerization approach described in WO 2012/058768 is used alternatively. In one embodiment of the present invention, the first CH3 domain contains the amino acid mutations L351Y, Y407A, and the second CH3 domain contains the amino acid mutations T366A, K409F. In another embodiment of the present invention, the second CH3 domain contains an additional amino acid mutation at position T411, D399, S400, F405, N390 or K392, for example selected from a) T41 IN, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M , K392R, K392L, K392F or K392E (numbering according to Kabat EU index). In another embodiment of the present invention, the first CH3 domain contains the amino acid mutations L351Y, Y407A, and the second CH3 domain contains the amino acid mutations T366V, K409F. In another embodiment of the present invention, the first CH3 domain contains the amino acid mutation Y407A and the second CH3 domain contains the amino acid mutations T366A, K409F. In another embodiment of the present invention, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (Kabat EU numbering index).

In one embodiment of the present invention, the heterodimerization approach described in WO 2011/143545 is applied alternatively, for example, with modification of the amino acid at a position selected from the group consisting of amino acid positions 368 and 409 (Kabat EU index numbering).

In one embodiment of the present invention, alternatively, the heteromerization method described in WO 2011/090762 is used, which also uses the ridge-to-trough approach described above. In one embodiment, the first CH3 domain contains the T366W amino acid mutation and the second CH3 domain contains the Y407A amino acid mutation. In one embodiment of the present invention, the first CH3 domain contains the T366Y amino acid mutation and the second CH3 domain contains the Y407T amino acid mutation (Kabat EU index numbering).

In one embodiment of the present invention, the bispecific antigen binding molecule or Fc domain thereof belongs to the IgG ₂ subclass and the heterodimerization approach is described in WO 2010/129304 and is used as an alternative.

In an alternative embodiment of the present invention, an association that promotes modification of the first and second subunits of the Fc domain includes a modification that mediates the effects of electrostatic guidance, for example, as described in PCT publication WO 2009/089004. Typically, this method involves replacing one or more amino acid residues at the interface between two Fc domain subunits with charged amino acid residues such that homodimer formation becomes electrostatically unfavorable but electrostatically favorable for heterodimerization. In one such embodiment of the present invention, the first CH3 domain comprises the replacement of the amino acid K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain comprises the amino acid substitution D399 , E356, D356 or E357 to a positively charged amino acid (eg lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, more preferably D399K and E356K). In yet another embodiment of the present invention, the first CH3 domain further comprises a replacement of amino acid K409 or R409 with a negatively charged amino acid (eg, glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In yet another embodiment of the present invention, the first CH3 domain further or alternatively comprises replacing the amino acid K439 and/or K370 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) (Kabat EU index numbering).

In yet another embodiment of the present invention, the heterodimerization method described in WO 2007/147901 is alternatively used. In one embodiment of the present invention, the first CH3 domain contains the amino acid mutations K253E, D282K and K322D, and the second CH3 domain contains the amino acid mutations D239K, E240K and K292D (Kabat EU index numbering).

In another embodiment of the present invention, the heterodimerization method described in WO 2007/110205 can be used alternatively.

In one embodiment of the present invention, the first subunit of the Fc domain includes the amino acid changes K392D and K409D, and the second subunit of the Fc domain contains the amino acid changes D356K and D399K (Kabat EU index numbering).

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

The Fc domain confers on the bispecific antigen-binding molecule (or antibody) favorable pharmacokinetic properties, including a long serum half-life, which promotes good uptake in the target tissue and a favorable tissue-to-blood distribution ratio. However, at the same time, this may undesirably target the bispecific antigen-binding molecule (or antibody) to cells expressing Fc receptors rather than the preferred antigen-bearing cells. Moreover, co-activation of Fc receptor signaling metabolic pathways can result in the release of cytokines which, in combination with T-cell activating properties (e.g., in embodiments of a bispecific antigen-binding molecule in which a second antigen-binding moiety binds to an activating T-cell antigen) and long-lasting The half-life of the bispecific antigen-binding molecule leads to excessive activation of cytokine receptors and serious side effects when administered systemically. Activation of immune cells (bearing the Fc receptor) other than T cells can even reduce the effectiveness of the bispecific antigen binding molecule (in particular, the bispecific antigen binding molecule in which the second antigen binding fragment binds to the activating T cell antigen) due to the possible destruction of T cells such as NK cells.

Thus, in some embodiments of the present invention, the Fc domain of the bispecific antigen-binding molecule of the present invention exhibits reduced Fc receptor binding affinity and/or reduced effector function compared to a native IgG ₁ Fc domain. In one such embodiment of the present invention, an Fc domain (or a bispecific antigen-binding molecule comprising said Fc domain) exhibits a binding affinity for an Fc receptor that is less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% compared to the native IgG ₁ Fc domain (or bespecifically antigen-binding molecule containing the native IgG ₁ Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% compared to the native Fc domain of IgG ₁ (or a bispecific antigen-binding molecule comprising the Fc domain of native IgG ₁ ). In one of the embodiments of the present invention, the Fc domain (or bispecific antigennegative molecule containing the specified Fc domain) does not significantly bind to the Fc receptor and/or does not induce effector function. In one embodiment of the present invention, the Fc receptor is an Fcγ receptor. In one embodiment of the present invention, the Fc receptor is a human Fc receptor. In one embodiment of the present invention, the Fc receptor is an activating Fc receptor. In one embodiment of the present invention, the Fc receptor is an activating human Fcγ receptor, more specifically a human FcγRIIIa, FcγRI or FcγRIIa receptor, especially a human FcγRIIIa receptor. In one embodiment of the present invention, the effector function is one or more of the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In one embodiment of the present invention, the effector function is ADCC. In one embodiment of the present invention, the Fc domain exhibits substantially equal binding affinity for the neonatal Fc receptor (FcRn) compared to the native IgG1 Fc domain. Substantially equal binding to FcRn is achieved when the Fc domain (or a bispecific antigen-binding molecule comprising said Fc domain) exhibits a binding affinity greater than about 70%, especially about 80%, especially about 90%, relative to the binding affinity of the native Fc domain. IgG ₁ (or a bispecific antigen-binding molecule comprising a native IgG ₁ Fc domain) with FcRn.

In some embodiments of the present invention, the Fc domain is designed to have reduced Fc receptor binding affinity and/or reduced effector function compared to an unengineered Fc domain. In some embodiments, the Fc domain of the bispecific antigen-binding molecule contains one or more amino acid mutations that reduce the Fc domain's binding affinity for the Fc receptor and/or effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment of the present invention, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor. In one embodiment of the present invention, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments of the present invention, if there is more than one amino acid mutation that reduces the binding affinity of the Fc domain for the Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain for the Fc receptor by at least 10-fold, at least 20-fold, or even at least 50 times. In one embodiment of the present invention, the bespecifically antigen-binding molecule comprising the engineered Fc domain exhibits a binding affinity for the Fc receptor that is less than 20%, especially less than 10%, more preferably less than 5% compared to the bispecific antigen-binding molecule. , containing the Fc domain, not subjected to construction. In a specific embodiment of the present invention, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In one embodiment of the present invention, the Fc receptor is an activating human Fey receptor, more specifically a human FcγRIIIa, FcγRI or FcγRIIa receptor, especially a human FcγRIIIa receptor. Preferably, binding to each of these receptors is reduced. In some embodiments of the present invention, the binding affinity for the complement component specifically binding to C1q is also reduced. In one embodiment of the present invention, the binding affinity for the neonatal Fc receptor (FcRn) is not reduced. Essentially similar binding to FcRn, ie. retention of the binding affinity of an Fc domain for said receptor is achieved if the Fc domain (or a bispecific antigen-binding molecule containing said Fc domain) exhibits a binding affinity that is about 70% higher than the unengineered (or bispecific) form of the Fc domain. an antigen-binding molecule comprising the unengineered form of the Fc domain) with FcRn. The Fc domain or bispecific antigen-binding molecules of the present invention comprising said Fc domain may exhibit said binding affinity, which may be greater by about 80% and even greater by about 90%. In some embodiments of the present invention, the Fc domain of the bispecific antigen-binding molecule is designed to have a reduced effector function compared to an undesigned Fc domain. Decreased effector function may include, but is not limited to, one or more of the following: decreased complement-dependent cytotoxicity (CDC), decreased antibody-dependent cell-mediated cytotoxicity (ADCC), decreased antibody-dependent cellular phagocytosis (ADCP), decreased secretion of cytokines, reduced immune complex mediated antigen uptake by antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced forward apoptosis signaling, reduced cross-linking of target-bound antibodies , reduced dendritic cell maturation, or reduced T cell priming. In one embodiment of the present invention, reduced effector function is one or more effector functions selected from the group consisting of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In one embodiment of the present invention, the reduced effector function is reduced ADCC. In one embodiment of the present invention, the reduced ADCC is less than 20% of the ADCC induced by an unengineered Fc domain (or a bispecific antigen-binding molecule comprising an unengineered Fc domain).

In one embodiment of the present invention, the amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor and/or effector function is an amino acid substitution. In one embodiment of the present invention, the Fc domain contains an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331 and P329 (Kabat EU index numbering). In a more specific embodiment of the present invention, the Fc domain contains an amino acid substitution at a position selected from the group consisting of L234, L235 and P329 (Kabat EU index numbering). In some embodiments of the present invention, the Fc domain contains amino acid substitutions at positions L234A and L235A (Kabat EU index numbering). In some embodiments, the Fc domain is an IgG ₁ Fc domain, especially a human IgG ₁ Fc domain. In one embodiment of the present invention, the Fc domain contains an amino acid substitution at position P329. In a more specific embodiment of the present invention, the amino acid substitution is P329A or P329G, especially P329G (Kabat EU index numbering). In one embodiment of the present invention, the Fc domain comprises an amino acid substitution at position P329 and an additional amino acid substitution at position selected from E233, L234, L235, N297, and P331 (Kabat EU index numbering). In one embodiment of the present invention, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In some embodiments of the present invention, the Fc domain contains amino acid substitutions at positions P329, L234, and L235 (Kabat EU index numbering). In more preferred embodiments, the Fc domain contains the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). Specifically, in certain embodiments of the present invention, each Fc domain subunit contains amino acid substitutions at positions L234A, L235A, and P329G (Kabat EU index numbering), i.e. in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (Kabat EU numbering index).

In one such embodiment of the present invention, the Fc domain is an IgG ₁ Fc domain, especially a human IgG ₁ Fc domain. The combination of amino acid substitutions "P329G LALA" almost completely abolishes the binding of the Fcγ receptor (as well as complement) to the Fc domain of human IgG ₁ as described in PCT publication WO 2012/130831, the spirit of which is incorporated herein by reference. WO 2012/130831 also describes methods for producing such mutant Fc domains and methods for determining their properties such as Fc receptor binding or effector functions.

IgG ₄ antibodies exhibit reduced binding affinity for Fc receptors and reduced effector functions compared to IgG ₁ antibodies. Therefore, in some embodiments of the present invention, the Fc domain of the bispecific antigen-binding molecules of the present invention is an IgG ₄ Fc domain, in particular a human IgG ₄ Fc domain. In one embodiment of the present invention, the IgG ₄ Fc domain includes amino acid substitutions at position S228, especially the S228P amino acid substitution (Kabat EU index numbering). To further reduce its Fc receptor binding affinity and/or its effector function, in one embodiment of the present invention, the IgG ₄ Fc domain includes an amino acid substitution at position L235, in particular the L235E amino acid substitution (Kabat EU index numbering). In another embodiment of the present invention, the IgG ₄ Fc domain contains an amino acid substitution at position P329, especially an amino acid substitution at position P329G (Kabat EU index numbering). In one embodiment of the present invention, the IgG ₄ Fc domain includes amino acid substitutions at positions S228, L235 and P329, in particular amino acid substitutions S228P, L235E and P329G (Kabat EU index numbering). Such IgG ₄ Fc domain mutants and their ability to bind to the Fcγ receptor are described in PCT publication WO 2012/130831, the spirit of which is incorporated herein by reference.

In a specific embodiment of the present invention, an Fc domain showing reduced Fc receptor binding affinity and/or reduced effector function compared to a native IgG1 Fc domain is a human IgG ₁ Fc domain containing amino acid substitutions L234A, L235A and optionally P329G, or a domain Human IgG ₄ Fc containing amino acid substitutions S228P, L235E and optionally P329G (Kabat EU index numbering).

In some embodiments of the present invention, N-glycosylation of the Fc domain has been eliminated. In one such embodiment of the present invention, the Fc domain contains an amino acid mutation at position N297, in particular an amino acid substitution that changes asparagine to alanine (N297A) or aspartic acid (N297D) (Kabat EU index numbering).

In addition to the Fc domains described herein above and also in PCT publication WO 2012/130831, Fc domains with reduced Fc receptor binding and/or reduced effector function also include those Fc domains in which one or more residues are changed to 238 , 265, 269, 270, 297, 327 and 329 (US 6737056) (Kabat EU numbering). Such Fc mutants include Fc mutants with substitutions at two or more positions of amino acids 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with the replacement of residues 265 and 297 with alanine (US 7332581).

Mutant Fc domains can be obtained by deletion, substitution, insertion or modification of amino acids using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of the DNA coding sequence, PCR, gene synthesis, and the like. The correctness of the change in nucleotides can be checked, for example, by sequencing.

Binding to Fc receptors can be easily determined, for example, by ELISA or Surface Plasmon Resonance (SPR) using standard instruments such as the BIAcore instrument (GE Healthcare) and Fc receptors that can be generated by recombinant expression . Alternatively, the binding affinity of Fc domains or Fc domain-containing bispecific antigen-binding molecules for Fc receptors can be assessed using cell lines known to express certain Fc receptors, e.g., human NK cells expressing the FcγIIIa receptor.

The effector function of an Fc domain or a bispecific antigen-binding molecule containing an Fc domain can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in US 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 1986, 7059-7063; Hellstrom et al., Proc Natl Acad Sci USA 82, 1985, 1499-1502; US 5821337; Bruggemann et al., J Exp Med 166, 1987, 1351-1361. Alternatively, non-radioactive assays can be used (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); CytoTox ^96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model such as that described in Clynes et al., Proc Natl Acad Sci USA 95, 1998, 652-656.

In some embodiments, implementation of the present invention, the binding of the Fc domain to the complement component, especially C1q, is reduced. Alternatively, in some embodiments of the present invention, where the Fc domain is designed to have reduced effector function, said reduced effector function includes reduced CDC. C1q binding assays can be performed to determine if an Fc domain, or a bespecifically antigen-binding molecule containing an Fc domain, is capable of binding to C1q and therefore having CDC activity. See, for example, C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. The CDC assay can be used to evaluate complement activation (see, e.g., Gazzano-Santoro et al., J Immunol Methods 202, 1996, 163; Cragg et al., Blood 101, 2003, 1045-1052; Cragg and Glennie, Blood 103, 2004, 2738-2743).

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

Polynucleotides

The present invention is also directed to isolated polynucleotides encoding an antibody or a bispecific antigen-binding molecule of the present invention, or a fragment thereof.

In some embodiments of the present invention, the specified fragment is antigennegative fragment. The polynucleotides encoding the antibodies or bispecific antigen-binding molecules of the present invention are expressed as a single polynucleotide encoding an entire antibody or antigen-binding molecule, or multiple (eg, two or more) expression polynucleotides. The polypeptides encoded by the polynucleotides that are coexpressed can be linked, for example, via disulfide bonds or otherwise, to form a functional antibody or a bispecific antigen-binding molecule. For example, the light chain portion of an antibody or bispecific antigen binding molecule may be encoded by a separate polynucleotide from the antibody or bispecific antigen binding molecule portion containing the heavy chain of the antibody or bispecific antigen binding molecule. When co-expressed, the heavy chain polypeptides will bind to the light chain polypeptides to form an antibody or a bispecific antigen-binding molecule. In another example, an antibody or bispecific antigen binding molecule portion comprising one of two Fc domain subunits and optionally (part of) one or more Fab molecules may be encoded by a separate polynucleotide from an antibody or antigen binding molecule portion containing the other of the two Fc domain subunits and optionally (part of) a Fab molecule. When co-expressed, the Fc domain subunits will associate to form the Fc domain.

In some embodiments, the implementation of the present invention selected polynucleotide encodes a whole antibody or bespecifically antigennegative molecule of the present invention. In other embodiments, the implementation of the present invention selected polynucleotide encodes a polypeptide in the antibody or bespecifically antigennegative molecule of the present invention.

In some embodiments of the present invention, the polynucleotide or nucleic acid is DNA. In other embodiments, the implementation of the present invention, the polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention may be single stranded or double stranded.

Recombination methods

The antibodies or bispecific antigen-binding molecules of the present invention can be prepared, for example, by solid phase peptide synthesis (eg, solid phase Merrifield synthesis) or by recombination. To be produced by recombination, one or more polynucleotides encoding an antibody or a bispecific antigen-binding molecule (fragment), such as those described above, for example, are isolated and inserted into one or more vectors for subsequent cloning and/or expression in host cells. Such a polynucleotide can be easily isolated and sequenced using conventional methods. In one of the embodiments of the present invention provide a vector, preferably an expression vector containing one or more polynucleotides of the present invention. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence for an antibody or a bispecific antigen-binding molecule (fragment) along with appropriate transcription/translation control signals. These methods include in vitro recombinant DNA methods, in vivo synthesis and recombination/genetic recombination methods. See, for example, the methods described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector may be part of a plasmid, a virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette in which a polynucleotide encoding an antibody or a bispecific antigen-binding molecule (fragment) (ie, coding region) is cloned in operative association with a promoter and/or other transcription or translation control elements. In the context of the present invention, the term "coding region" means the part of the nucleic acid, which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered part of the coding region if present, but any flanking sequences such as promoters, ribosome binding sites, transcription terminators, introns, The 5' and 3' untranslated regions, and the like, are not part of the coding region. The two or more coding regions may be present in a single polynucleotide construct, eg in a single vector, or in separate polynucleotide constructs, eg in separate (different) vectors. In addition, any vector may contain a single coding region or may contain two or more coding regions, for example, the vector of the present invention may encode one or more polypeptides that are post- or co-translationally cleaved into final proteins by proteolytic cleavage. In addition, the vector, polynucleotide or nucleic acid of the present invention may encode heterologous coding regions, either hybridized or not hybridized with a polynucleotide encoding an antibody or bispecific antigen-binding molecule (fragment) of the present invention, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs such as a secretory signal peptide or a heterologous functional domain. The term "functional association" means that the coding region of a gene product, such as a polypeptide, is associated with one or more regulatory sequences in such a way as to place the expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (e.g., a polypeptide coding region and its associated promoter) are "operably associated" if induction of promoter function results in transcription of an mRNA encoding the desired gene product, and if the nature of the association between the two DNA fragments does not interfere with the ability of the regulatory expression sequences to drive gene product expression or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region will be operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of influencing the transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs a particular DNA transcription only in predetermined cells. Other transcriptional control elements besides the promoter, such as enhancers, operators, repressors, and transcription termination signals, may be operably linked to the polynucleotide to direct cell-specific transcription. The present invention describes useful promoters and other regions of transcriptional control. Many areas of transcriptional control are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g., the immediate early promoter in combination with intron-A), simian virus 40 ( eg early promoter) and retroviruses (eg Rous sarcoma virus). Other areas of transcriptional control include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional useful transcriptional control regions include tissue-specific promoters and enhancers, as well as inducible promoters (eg, tetracycline-inducible promoters). Similarly, various broadcast controls are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (in particular, the internal ribosome entry site or IRES, also referred to as the CITE sequence). The expression cassette may also include other functions such as origins of replication and/or chromosomal integration elements such as long terminal repeats (LTRs) or inverted terminal repeats (ITRs) of adeno-associated viral , AAV).

The polynucleotides and nucleic acid coding regions of the present invention may be linked to additional coding regions that encode secretory or signal peptides that direct secretion of the polypeptide encoded by the polynucleotide of the present invention. For example, if secretion of an antibody or a bispecific antigen-binding molecule is required, the DNA encoding the signal sequence can be placed upstream of the nucleic acid encoding the antibody or bispecific antigen-binding molecule of the present invention, or a fragment thereof. According to the signaling hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein as soon as the export of the growing protein chain through the granular endoplasmic reticulum begins. It is known to those skilled in the art that polypeptides secreted by vertebrate cells typically contain a signal peptide hybridized to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to form the secreted or "mature" form of the polypeptide. In some embodiments of the present invention, a native signal peptide, for example, an immunoglobulin heavy chain or light chain signal peptide, or a functional derivative of that sequence, is used that retains the ability to control the secretion of a polypeptide that is operably linked to it. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the leader sequence of the wild type can be replaced by the leader sequence of the human tissue plasminogen activator (tissue plasminogen activator, TP A) or mouse β-glucuronidase.

DNA encoding a short protein sequence that can be used to facilitate subsequent purification (e.g., a histidine tag) or to aid in the labeling of an antibody or bispecific antigen-binding molecule can be included within or at the ends of a polynucleotide encoding an antibody or bispecific antigen-binding molecule (molecular fragment) .

In another embodiment, the present invention provides a host cell containing one or more polynucleotides of the present invention. In some embodiments, implementation of the present invention provide a host cell containing one or more vectors of the present invention. Polynucleotides and vectors may include individually or in combination any of the features described in the present invention in relation to polynucleotides and vectors, respectively. In one such embodiment of the present invention, the host cell contains (eg, transformed or transfected) one or more vectors containing one or more polynucleotides that encode (part of) an antibody or a bispecific antigen-binding molecule of the present invention. In the context of the present invention, the term "host cell" refers to any type of cellular system that can be designed to produce an antibody or bispecific antigen-binding molecule of the present invention, or fragments thereof. Suitable host cells for replication and support for the expression of antibodies or bispecific antigen-binding molecules are well known in the art. Such cells can be transfected or transduced, as appropriate, with a particular expression vector, and large numbers of cells containing the vector can be grown to seed large scale fermenters to produce sufficient amounts of antibody or bispecific antigen binding molecule for clinical applications. Suitable host cells include prokaryotic microorganisms such as E. coli, or various eukaryotic cells such as Chinese hamster ovary cell (CHO), insect cells, and others. For example, polypeptides can be produced in bacteria, in particular when glycosylation is not required. After expression, the polypeptide can be isolated from the bacterial cell paste into a soluble fraction and can be further purified. In addition to prokaryotes, useful hosts for cloning or expression of polypeptide-encoding vectors are eukaryotic microbes such as filamentous fungi or yeasts, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of a polypeptide with partially or fully humanized glycosylation. See Gerngross, Nat Biotech 22, 2004, 1409-1414, Li et al., Nat Biotech 24, 2006, 210-215. Host cells that can be used to express (glycosylated) polypeptides also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, US 5959177, US 6040498, US 6420548, US 7125978, US 6417429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic animals). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the CV1 monkey kidney cell line transformed with the SV40 virus (COS-7), the human embryonic kidney cell line (293 or 293T as described, for example, Graham et al., J Gen Virol 36, 1977, 59), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., Mather, Biol Reprod 23, 1980, 243-251), monkey kidney cells (CV1), kidney cells African green monkey (VERO-76), human cervical carcinoma (HELA), dog kidney (MDCK), gray rat liver (BRL 3A), human lung (W138), human liver (Hep G2), tumor cells mouse mammary gland cells (MMT 060562), TRI cells (as described, for example, by Mather et al., Annals NY Acad Sci 383, 1982, 44-68), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including dhfr ^- CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 1980, 4216); myeloma cell lines such as YO, NS0, P3X63 and Sp2/0. For a review of some mammalian host cell lines useful for protein production, see, for example, Yazaki and Wu, in: Methods in Molecular Biology, Volume 248 (ed. B.K.C. Lo, Humana Press, Totova, New Jersey), 2003, 255-268. Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells, and plant cells, as well as cells contained in a transgenic animal, transgenic plant, or cultured plant or animal tissue cells. In one embodiment of the present invention, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20 cells).

Standard technologies for the expression of foreign genes in such systems are known in the art. Cells expressing a polypeptide containing either the heavy or light chain of an antigen-binding domain, such as an antibody, can be engineered to also express the other of the antibody chains such that the product expressed is an antibody that has both a heavy and a light chain. In one embodiment, the present invention provides a method for producing an antibody or a bispecific antigen-binding molecule of the present invention, which includes culturing host cells containing a polynucleotide encoding an antibody or a bispecific antigen-binding molecule, as provided in the present invention, under conditions suitable for expressing the antibody or a bispecific antigen-binding molecule; and optionally isolating the antibody or the bispecific antigen-binding molecule from the host cell (or from the host cell culture medium).

The components of the bispecific antigen-binding molecule (or antibody) of the present invention can be genetically hybridized with each other. A bispecific antigen binding molecule can be designed such that its components can be fused directly to each other or indirectly via a linker sequence. The composition and length of the linker can be determined according to methods known in the art and can be tested for effectiveness. Examples of linker sequences between different components of the bispecific antigen binding molecules are provided in the present invention. Additional sequences may also be mentioned to include a cleavage site to separate individual fusion components, if desired, such as endopeptidase recognition sequences.

The antibody or bispecific antigen-binding molecule of the present invention typically contains at least an antibody variable region capable of binding an antigenic determinant. Variable regions may form part of and be derived from natural or non-natural antibodies and fragments thereof. Methods for making polyclonal antibodies and monoclonal antibodies are well known in the art (see, for example, Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-natural antibodies can be constructed by solid phase peptide synthesis, can be made by recombination (eg, as described in US 4,186,567), or can be made, for example, by screening combinatorial libraries containing variable heavy chains and variable light chains (see, for example, , US 5969108).

Any kind of animal-derived antibodies, antibody fragments, antigen-binding domains or variable regions can be used in the antibody or bispecific antigen-binding molecule of the present invention. Antibodies, antibody fragments, antigen-binding domains or variable regions useful in the present invention may be from mice, primates, humans, but are not limited to. If the antibody or bispecific antigen-binding molecule is intended for use in humans, a chimeric form of the antibody may be used in which the antibody constant regions are derived from a human. A humanized form of an antibody or a fully human antibody can also be prepared according to methods known in the art (see, for example, US 5,565,332). Humanization can be achieved by various methods, including, but not limited to, (a) transplantation of a non-human derived CDR region (e.g., from a donor antibody) into a human framework (e.g., a recipient antibody) and constant regions while maintaining or without retaining critical framework region residues (e.g., those that are important for maintaining good antigen binding affinity or antibody functions), (b) transplanting only non-human-derived regions that determine specificity (SDR or a-CDR; residues critical for interaction antibody-antigen) onto human framework and constant regions, or (c) transplantation of entire non-human variable domains derived from non-humans, but "masking" them with a region similar to that from a human by replacing surface residues. Considered humanized antibodies and methods for their production, for example, in the publication of Almagro and Fransson, Front. biosci. 13, 2008, 1619-1633, as well as those described, for example, in Riechmann et al., Nature 332, 1988, 323-329; Queen et al., Proc. Nat'l Acad. sci. USA 86, 1989, 10029-10033; US 5821337, US 7527791, US 6982321, US 7087409; Kashmiri et al., Methods 36, 2005, 25-34 (description of the specificity determining region (SDR) of transplantation); Padlan, Mol. Immunol. 28, 1991, 489-498 (description of domain swapping); Dall'Acqua et al., Methods 36, 2005, 43-60 (description of domain flipping); Osbourn et al., Methods 36, 2005, 61-68; Klimka et al., Br. J. Cancer, 83, 2000, 252-260 (description of a "directed selection" approach to domain switching). Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (see, for example, Sims et al. J. Immunol. 151, 1993, 2296); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89, 1992, 4285; Presta et al. J. Immunol ., 151, 1993, 2623); mature (somatically mutated) human framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13, 2008, 1619-1633); framework regions derived from FR screening libraries (see, e.g., Vasa et al., J. Biol. Chem. 272, 1997, 10678-10684; Rosok et al., J. Biol. Chem. 271, 1996, 22611- 22618).

Human antibodies can be obtained by various methods known in this field. Human antibodies are described in detail in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 2001, 368-374, and Lonberg, Curr Opin Immunol 20, 2008, 450-459. Human antibodies can be generated by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact human variable region antibodies in response to an antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or that are present outside the chromosomes or are randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23, 2005, 1117-1125. See also, for example, US 6075181 and US 6150584 which describe XENOMOUSE™ technology; US 5770429, which describes the HuMab® technology; US 7041870, which describes the K-M MOUSE® technology; and patent application US 2007/0061900, which describes the VelociMouse® technology). Human variable regions from intact antibodies produced by such animals can be further modified, for example by combining with another human constant region.

Human antibodies can also be obtained by hybridoma-based methods. Cell lines of human myeloma and mouse-human heteromyeloma are described for obtaining human monoclonal antibodies. (See, for example, Kozbor J. Immunol., 133, 1984, 3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, 1987, 51-63, Marcel Dekker, Inc., New York, Boerner et al., J. Immunol., 147, 1991, 86.) Human antibodies generated by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. sci. USA, 103, 2006, 3557-3562. Additional methods are also described in, for example, US 7189826 (description of the production of human IgM monoclonal antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4), 2006, 265-268 (description of human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3), 2005, 927-937 and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3), 2005, 185-191.

Human antibodies can also be obtained by isolation from human antibody libraries as described herein.

Antibodies useful in the present invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. Methods for screening combinatorial libraries are considered, for example, in the work of Lerner et al. Nature Reviews 16, 2016, 498-508. For example, many methods are known in the art for creating phage display libraries and screening such libraries for antibodies having the desired binding parameters. Such methods are considered, for example, in the works of Frenzel et al. in mabs 8, 2016, 1177-1194; Bazan et al. Human Vaccines and Immunotherapeutics 8, 2012, 1817-1828; Zhao et al. Critical Reviews in Biotechnology 36, 2016, 276-289; Hoogenboom et al. in: Methods in Molecular Biology 178, 2001, 1-37 (ed. O'Brien et al., Human Press, Totova, New Jersey); Marks and Bradbury in: Methods in Molecular Biology 248, 2003, 161-175 (ed. Lo, Human Press, Totova, New Jersey).

In some phage display methods, VH and VL gene repertoires are individually cloned by polymerase chain reaction (PCR) and recombined randomly into phage libraries, which can then be screened for antigen-binding phage as described by Winter et al. in Annual Review of Immunology 12, 1994, 433-455. The phage typically displays antibody fragments, either as single chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide antibodies with high affinity for the immunogen without the need for hybridoma construction. Alternatively, the naive repertoire can be cloned (eg from a human) to provide a single source of antibodies to a wide range of foreign as well as self antigens without any immunization, as described by Griffiths et al., EMBO Journal 12, 1993, 725-734. Finally, naive libraries can also be generated synthetically by cloning unrearranged V-gene segments from stem cells and using random PCR primers to encode highly variable CDR3 regions and to perform in vitro rearrangement as described by Hoogenboom and Winter in the Journal of Molecular Biology 227, 1992, 381-388). Patents that describe human antibody phage libraries include, for example, US 5750373, US 7985840, US 7785903, US 8679490, as well as patent publications US 2005/0079574, US 2007/0117126, US 2007/0237764 and US 2007/029293 6 . Additional examples of methods known in the art for screening combinatorial libraries of antibodies with the desired activity or activities include ribosome and mRNA display, as well as methods for displaying and selecting antibodies on bacteria, mammalian cells, insect cells, or yeast cells. Methods for displaying on the surface of yeasts are considered, for example, in Scholler et al. Methods in Molecular Biology 889, 2012, 73-84. Ribosome display methods are described in, for example, He et al., Nucleic Acids Research 25, 1997, 5132-5134, and Hanes et al. PNAS 94, 1997, 4937-4942.

Antibodies or bispecific antigen-binding molecules obtained according to the description of the present invention can be purified by methods known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as overall charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. Affinity chromatography purification can use an antibody, ligand, receptor, or antigen to which the antibody or bispecific antigen-binding molecule binds. For example, a protein A or protein G matrix can be used to purify antibodies or bispecific antigen-binding molecules of the present invention using affinity chromatography. description in examples. The purity of an antibody or bispecific antigen-binding molecule can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.

Analyzes

Antibodies or bispecific antigen-binding molecules of the present invention can be identified, tested or characterized for their physical/chemical properties and/or biological activity using various assays known in the art.

Affinity (affinity) assays

The affinity of an antibody or bispecific antigen-binding molecule for an Fc receptor or target antigen can be determined, for example, by surface plasmon resonance (SPR) using standard instruments such as the BIAcore instrument (GE Healthcare) and receptors or target proteins, e.g. recombinant expression. Alternatively, the binding of antibodies or bispecific antigen-binding molecules to various receptors or target antigens can be assessed using cell lines expressing a particular receptor or target antigen, such as by fluid cytometry (FACS). A specific exemplary embodiment for measuring binding affinity is described below.

According to one embodiment of the present invention, Co is measured by surface plasmon resonance using a BIACORE® T100 apparatus (GE Healthcare) at 25°C.

To analyze the interaction between the Fc moiety and Fc receptors, the His-tagged recombinant Fc receptor was captured by an anti-Penta His antibody (Qiagen) immobilized on CM5 chips, and the bispecific constructs were used as analytes. Briefly, carboxymethylated dextran (CM5, GE Healthcare) biosensor chips are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The anti-Penta-His antibody is diluted with 10 mM sodium acetate, pH 5.0, to 40 μg/ml before injection at a rate of 5 μl/min to achieve approximately 6500 response units (response units, RU) of the attached protein. After ligand injection, 1 M ethanolamine is injected to block unreacted groups. Subsequently, the Fc receptor is captured within 60 sec at 4 or 10 nM. For kinetic measurements, four-fold serial dilutions of the antibody or bispecific antigen-binding molecule (range 500 nM to 4000 nM) are injected into HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05%). Surfactant P20, pH 7.4) at 25°C with a flow rate of 30 µl/min for 120 sec.

To determine affinity for a target antigen, antibodies or bispecific antigen-binding molecules are captured by a specific anti-human Fab antibody (GE Healthcare) that is immobilized on the surface of an activated CM5 sensor chip as described for an anti-Penta-His antibody. The final amount of bound protein is approximately 12,000 RU. Antibodies or bispecific antigen-binding molecules are captured within 90 seconds at 300 nM. Target antigens are passed through flow cuvettes for 180 sec in the concentration range from 250 to 1000 nM at a flow rate of 30 μl/min. Dissociation is monitored for 180 sec.

Differences in the cumulative refractive index are corrected for by subtracting the response from the reference flow cell. _The steady state reaction was used to obtain the dissociation constant KD by fitting a non-linear Langmuir binding isotherm curve _. 1.1.1) by simultaneously fitting association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio k _off /k _on . See, for example, Chen et al., J Mol Biol 293, 1999, 865-881.

Activity analysis

The biological activity of the bispecific antigen-binding molecules (or antibodies) of the present invention can be measured using the various assays described in the examples. Biological activities may, for example, include induction of T cell proliferation, induction of signaling in T cells, induction of expression of activation markers in T cells, induction of cytokine secretion by T cells, induction of lysis of target cells such as tumor cells, and induction tumor regression and/or improved survival.

Compositions, formulations and routes of administration

In an additional aspect, the present invention relates to pharmaceutical compositions containing any of the antibodies or bispecific antigen-binding molecules presented in the present description, for example, for use in any of the following therapeutic methods. In one of the embodiments of the present invention, the pharmaceutical composition includes any of the antibodies or bispecific antigen-binding molecules provided in the present invention, and a pharmaceutically acceptable carrier. In another embodiment of the present invention, the pharmaceutical composition contains any of the antibodies or bispecific antigen-binding molecules provided herein and at least one additional therapeutic agent, for example, as described below.

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

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of an antibody or a bispecific antigen-binding molecule dissolved or dispersed in a pharmaceutically acceptable carrier. The term "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations used, i.e. do not cause adverse, allergic or other undesirable reactions when administered to an animal, such as a human, as the case may be. The preparation of a pharmaceutical composition that contains an antibody or a bispecific antigen-binding molecule and optionally an additional active ingredient is known to those skilled in the art in light of the present invention as exemplified in: Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, incorporated herein by reference. Moreover, for administration to animals (eg, humans), it should be understood that formulations must meet the standards of sterility, pyrogenicity, general safety, and purity as required by the FDA's Biological Standards Office or relevant authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. In the context of the present invention, the term "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, agents that delay absorption , salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, colorants, like materials, and combinations thereof, as known to those skilled in the art (see ., for example, in the book: Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, 1289-1329, incorporated herein by reference). Except where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

The immunoconjugate of the present invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and, if necessary, for topical treatment, by intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration. Dosing may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic.

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

The active ingredients can be incorporated into microcapsules prepared, for example, by coacervation or interfacial polymerization methods, e.g. , nanoparticles and nanocapsules) or in macroemulsions. Such methods are disclosed in: Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990). Sustained release formulations may be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the polypeptide, the matrices being in the form of shaped articles such as films or microcapsules. In some embodiments of the present invention, sustained release of the injectable composition can be achieved by using absorption delaying agents in the compositions, such as, for example, aluminum monostearate, gelatin, or a combination thereof.

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

Pharmaceutical compositions containing the antibodies or bispecific antigen-binding molecules of the present invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, trapping, or lyophilizing processes. Pharmaceutical compositions can be formulated in the usual way using one or more physiologically acceptable carriers, diluents, excipients or excipients that facilitate the processing of proteins into drugs that can be used in pharmaceuticals. The correct composition depends on the chosen route of administration.

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

Therapeutic Methods and Formulations

Any of the antibodies or bispecific antigen-binding molecules of the present invention can be used in therapeutic methods. The antibodies or bispecific antigen-binding molecules of the present invention can be used as immunotherapeutic agents, for example in the treatment of cancer.

For use in therapeutic methods, the antibodies or bispecific antigen-binding molecules of the present invention must be designed, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disease being treated, the particular mammalian patient, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to practitioners.

In one embodiment, the present invention provides antibodies or bispecific antigen-binding molecules of the present invention for use as a drug. In other embodiments, the present invention provides antibodies or bispecific antigen-binding molecules of the present invention for the treatment of a disease. In some embodiments, the present invention provides the antibodies or bispecific antigen-binding molecules of the present invention for use in methods of treating a disease. In one embodiment, the present invention provides the antibodies or bispecific antigen-binding molecules of the present invention for use in the treatment of a disease in an individual in need of such treatment. In some embodiments, the present invention provides the antibodies or the bispecific antigen binding molecule of the present invention for use in a method of treating a subject with a disease comprising administering to the subject a therapeutically effective amount of the antibody or the bispecific antigen binding molecule. In some embodiments of the present invention, the disease being treated is a proliferative disease. In one embodiment of the present invention, the disease is cancer. In some embodiments of the present invention, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent, such as an anticancer agent, if the disease being treated is cancer. In further embodiments, the present invention provides an antibody or a bispecific antigen-binding molecule of the present invention for use in inducing lysis of target cells, especially cancer cells. In some embodiments, the present invention provides an antibody or a bispecific antigen-binding molecule for use in a method of inducing lysis of target cells, in particular tumor cells, in an individual, comprising administering to the individual an effective amount of an antibody or a bispecific antigen-binding molecule to induce lysis of target cells. The "individual" according to any of the above embodiments of the present invention may be a mammal, preferably a human. In some embodiments of the present invention, the disease being treated is an autoimmune disease, in particular systemic lupus erythematosus and/or rheumatoid arthritis. The production of pathogenic autoantibodies by autoreactive plasma cells is a hallmark of autoimmune diseases. Therefore, GPRC5D can be used to target autoreactive plasma cells in autoimmune diseases.

In another embodiment, the present invention provides for the use of an antibody or a bispecific antigen-binding molecule of the present invention for the manufacture or formulation of a medicament. In one of the embodiments of the present invention, the medicinal product is intended for the treatment of a disease in an individual in need of such treatment. In yet another embodiment of the present invention, the medicament is for use in a method of treating a disease comprising administering a therapeutically effective amount of a medicament to a diseased individual. In some embodiments of the present invention, the disease being treated is a proliferative disorder. In one embodiment of the present invention, the disease is cancer. In one embodiment of the present invention, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent, such as an anti-cancer agent, if the disease being treated is cancer. In yet another embodiment of the present invention, the drug is intended to induce lysis of target cells, in particular cancer cells. In another embodiment of the present invention, the drug is for use in a method for inducing lysis of target cells in a subject, particularly cancer cells, comprising administering to the subject an effective amount of the drug to induce lysis of the target cells. The "individual" according to any of the above embodiments of the present invention may be a mammal, preferably a human.

In another aspect of the present invention, a method for treating a disease is provided. In one embodiment of the present invention, the method comprises administering to an individual with such a disease a therapeutically effective amount of an antibody or a bispecific antigen-binding molecule of the present invention. In one of the embodiments of the present invention, the specified individual is administered a composition containing the antibody or bispecific antigen-binding molecule of the present invention in a pharmaceutically acceptable form. In some embodiments of the present invention, the disease being treated is a proliferative disorder. In one embodiment of the present invention, the disease is cancer. In some embodiments of the present invention, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent, such as an anticancer agent, if the disease being treated is cancer. The "individual" according to any of the above embodiments of the present invention may be a mammal, preferably a human.

In another aspect of the present invention, a method is provided for inducing lysis of a target cell, in particular a tumor cell. In one of the embodiments of the present invention, the method involves contacting the target cell with the antibody or bispecific antigen-binding molecule of the present invention in the presence of T cells, especially cytotoxic T cells. In another aspect of the present invention, a method is provided for inducing lysis of a target cell, in particular a tumor cell, in an individual. In yet another embodiment of the present invention, the method comprises administering to an individual an effective amount of an antibody or a bispecific antigen-binding molecule to induce lysis of the target cell. In one embodiment of the present invention, the "individual" is a human.

In some embodiments of the present invention, the disease being treated is a proliferative disorder, in particular cancer. Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer , colorectal cancer, rectal cancer, stomach cancer, prostate cancer, blood cancer, skin cancer, squamous cell cancer, bone cancer and kidney cancer. Other cell proliferation disorders that can be treated using the antibody or bispecific antigen-binding molecule of the present invention include, but are not limited to, neoplasms located in the abdomen, bones, breast, digestive system, liver, pancreas, peritoneum, endocrine glands ( adrenal glands, parathyroid glands, pituitary gland, testicles, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissues, spleen, thoracic region and genitourinary system. Also included are precancerous conditions or lesions and metastases of cancer. In some embodiments, the cancer is selected from the group consisting of kidney cancer, bladder cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, and prostate cancer. In one embodiment of the present invention, the cancer is prostate cancer. One skilled in the art will appreciate that in many cases an antibody or bispecific antigen-binding molecule may not provide a cure, but may provide only a partial benefit. In some embodiments of the present invention, a physiological change that has some benefit is also considered to be therapeutically useful. Thus, in some embodiments of the present invention, the amount of an antibody or a bispecific antigen-binding molecule that provides a physiological change is referred to as an "effective amount" or "therapeutically effective amount". The subject, patient or individual in need of treatment is usually a mammal, more specifically a human.

In some embodiments of the present invention, an effective amount of an antibody or a bispecific antigen-binding molecule of the present invention is administered to a cell. In other embodiments of the present invention, an effective amount of an antibody or a bispecific antigen-binding molecule of the present invention is administered to a cell. In other embodiments of the present invention, a therapeutically effective amount of an antibody or a bispecific antigen-binding molecule of the present invention is administered to an individual for the treatment of a disease.

For the prevention or treatment of a disease, the appropriate dosage of the antibody or bispecific antigen-binding molecule of the present invention (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the route of administration, the body weight of the patient, the type of antibody, or the bispecific antigen-binding molecule, the severity and course of the disease, whether the antibody or the bispecific antigen-binding molecule is administered for prophylactic or therapeutic purposes, prior or concurrent therapeutic interventions, the patient's medical history and response to the antibody or the bispecific antigen-binding molecule, and the judgment of the attending physician. The practitioner responsible for administration will in any case determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for the individual. The present invention contemplates various dosing regimens, including, but not limited to, single or multiple administration at different time points, bolus administration, and pulse infusion.

The antibody or bispecific antigen-binding molecule is suitably administered to the patient in a single dose or as a series of injections. Depending on the type and severity of the disease, from about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg - 10 mg/kg) of an antibody or bispecific antigen-binding molecule may be an initial candidate dose for administration to a patient, e.g. , by one or more separate injections or by continuous infusion. One typical daily dose may vary from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. With repeated administrations over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of the symptoms of the disease occurs. One exemplary dosage of the antibody or bispecific antigen binding molecule can be in the range of about 0.005 mg/kg to about 10 mg/kg. In other examples to which the present invention is not limited, the dose may also be from about 1 µg/kg body weight, about 5 µg/kg body weight, about 10 µg/kg body weight, about 50 µg/kg body weight, about 100 µg /kg body weight, about 200 µg/kg body weight, about 350 µg/kg body weight, about 500 µg/kg body weight, about 1 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50 mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg body weight, about 350 mg/kg body weight, about 500 mg/kg body weight, up to about 1000 mg/kg body weight bodies or more in one injection and any derived range. In non-limiting examples, the resulting range is from the numbers listed in the present invention, the range is from about 5 mg/kg body weight to about 100 mg/kg body weight, from about 5 μg/kg body weight to about 500 mg /kg of body weight, etc. may be entered based on the numbers described above. Thus, one or more doses of approximately 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg, or 10 mg/kg (or some combination thereof) may be administered to the patient. Such doses can be administered intermittently, eg, every week or every three weeks (eg, such that the patient receives from about two to about twenty, or eg, about six doses of the antibody or bispecific antigen-binding molecule). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosing regimens may be useful. The progress of such treatment can be easily monitored using conventional methods and tests.

The antibodies or bispecific antigen-binding molecules of the present invention can generally be used in an amount effective to achieve the intended purpose. For use in the treatment or prevention of a disease state, the antibodies or bispecific antigen-binding molecules of the present invention, or pharmaceutical compositions thereof, are administered or used in a therapeutically effective amount. Determination of a therapeutically effective amount is within the ability of those skilled in the art, especially in light of the detailed description of the present invention.

For systemic administration, a therapeutically effective dose can be initially estimated using in vitro assays, for example by cell culture assay. The dose can then be formulated in animal models to achieve a range of circulating concentrations that includes the IC ₅₀ established in cell culture. Such information can be used to more accurately determine useful doses for humans.

Initial dosages can also be estimated based on in vivo data, eg, animal models, using methods known in the art. One skilled in the art can easily optimize administration to humans based on animal data.

Dose amounts and dosing intervals can be adjusted individually to provide plasma levels of antibodies or bispecific antigen-binding molecules sufficient to maintain a therapeutic effect. Typical patient doses for injection are in the range of about 0.1 to 50 mg/kg/day, typically about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by multiple doses daily. Plasma levels can be measured, for example, using HPLC.

In cases of topical administration or selective consumption, the effective local concentration of antibodies or bispecific antigen-binding molecules may not be related to plasma concentration. One skilled in the art will be able to optimize therapeutically effective topical doses without undue experimentation.

A therapeutically effective dose of the antibodies or bispecific antigen-binding molecules described in the present invention will generally provide a therapeutic effect without causing significant toxicity. The toxicity and therapeutic efficacy of an antibody or bispecific antigen-binding molecule can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine LD ₅₀ (lethal dose for 50% of the population) and ED ₅₀ (therapeutically effective dose for 50% of the population). The ratio of doses associated with toxic and therapeutic effects is a therapeutic index, which can be expressed as the ratio of LD ₅₀ /ED ₅₀ . Antibodies or bispecific antigen-binding molecules that exhibit large therapeutic indices are preferred. In one of the embodiments of the present invention, the antibody or bispecific antigen-binding molecule of the present invention shows a high therapeutic index. Data obtained from cell culture analysis and animal studies may be useful in determining the range of doses suitable for administration to humans. The dosage is preferably within a circulating concentration range that includes the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending on a variety of factors, such as the dosage form used, the route of administration, the condition of the subject, and the like. The exact composition, route of administration and dosage can be selected by the attending physician taking into account the condition of the patient, (see, for example, the publication of Fingl et al., 1975, in the book: "The Pharmacological Basis of Therapeutics)", chapter 1, page 1 included in the present invention by reference).

The attending physician using the antibodies or bispecific antigen-binding molecules of the present invention to treat a patient must know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the treating physician should also be aware to adjust treatment to higher levels if the clinical response has not been adequate (excluding toxicity). The amount of dose to be administered for a particular treatment will vary depending on the severity of the condition being treated, the route of administration, and the like. The severity of the condition may be partly assessed, for example, by standard predictive assessment methods. In addition, the dose and possibly the frequency of administration may also vary depending on the age, body weight and response of the individual patient.

Other agents and treatments

The antibodies and bispecific antigen-binding molecules of the present invention may be administered in combination with one or more other agents in treatment. For example, an antibody or bispecific antigen-binding molecule of the present invention may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" refers to any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agent may include any active ingredients applicable to the specific indication to be treated, preferably ingredients with additional activities that do not adversely affect each other. In some embodiments of the present invention, the additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, a cell apoptosis activator, or an agent that sensitizes cells to apoptosis inducers. In one embodiment of the present invention, the additional therapeutic agent is an anticancer agent, e.g., a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis activator, or an antiangiogenic agent. Such additional agents are usually present in combination in amounts effective for the intended purpose. The effective amount of such additional agents depends on the amount of antibody or bispecific antigen-binding molecule used, the type of disorder or treatment, and other factors discussed above. Antibodies or bispecific antigen-binding molecules are generally used at the same dosages and by the same routes of administration as described in the present invention, or from about 1 to 99% of the dosages described in the present invention, or in any dose and in any way, which are recommended empirically/clinically.

Such combined therapies as outlined above include combined administration (when two or more therapeutic agents are included in one or separate compositions) and separate administration, in which case administration of the antibody or bispecific antigen-binding molecule of the present invention may occur before, simultaneously and/or after administration of an additional therapeutic agent and/or adjuvant. The antibodies or bispecific antigen-binding molecules of the present invention can also be used in combination with radiation therapy.

Products received

In another aspect of the present invention, a product is provided that contains materials useful in the treatment, prevention and/or diagnosis of the disorders described above. The product includes a container and a label or leaflet on or in the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. Containers can be made from various materials such as glass or plastic. The container contains a composition that, alone or in combination with another composition effective for treating, preventing and/or diagnosing a condition, may have a sterile access port (for example, the container may be a packet of intravenous solution or a vial having a stopper, which can be pierced with a hypodermic needle). At least one active agent in the composition is an antibody or a bispecific antigen-binding molecule of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the product may include (a) a first container containing a composition that contains the antibody or bispecific antigen-binding molecule of the present invention; and (b) a second container containing a composition that contains an additional cytotoxic or other therapeutic agent. The product in this embodiment of the present invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively or additionally, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials that are commercially and user-desirable, including other buffers, diluents, filters, needles, and syringes.

Methods and formulations for diagnosis and detection

In some embodiments of the present invention, any of the anti-GPRC5D antibodies described in the present invention is useful for detecting the presence of GPRC5D in a biological sample. Used in the present invention, the term "detection" refers to quantitative or qualitative detection. In some embodiments, implementation of the present invention, the biological sample includes cells or tissue, for example, prostatitis tissue.

In one embodiment, the present invention provides an anti-GPRC5D antibody for use in a diagnostic or detection method. In a further aspect, a method for detecting GPRC5D in a biological sample is provided. In some embodiments of the present invention, the method comprises contacting a biological sample with an anti-GPRC5D antibody as described herein under conditions suitable for binding of the anti-GPRC5D antibody to GPRC5D, and detecting whether a complex is formed between the anti-GPRC5D antibody and GPRC5D. Such a method may be an in vitro or in vivo method. In one embodiment of the present invention, an anti-GPRC5D antibody is used to select subjects suitable for anti-GPRC5D antibody therapy, for example, wherein GPRC5D is a biomarker for patient selection.

Typical disorders that can be diagnosed using the antibody of the present invention include cancer, in particular multiple myeloma.

In some embodiments, the present invention provides labeled anti-GPRC5D antibodies. Labels include, but are not limited to, labels or moieties that are directly detectable (e.g., fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels) as well as moieties, such as enzymes or ligands, that are directly detectable, e.g., by enzymatic reaction or by molecular interaction. Examples of labels include, but are not limited to, ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I radioisotopes, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases , for example, firefly luciferase and bacterial luciferase (US 4737456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, for example, glucose oxidase, galactose oxidase, glucose -6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase combined with an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals and etc.

Another object of the present invention relates to an antibody (10B10) that binds GPRC5D containing a heavy chain variable region (VL), where VL may include an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99 % or 100% identical to the sequence of SEQ ID NO: 81. The antibody may contain a light chain variable region (VL), where VL contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 82. The antibody may include VH and VL, and VL may include an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identical. SEQ ID NO: 81, and wherein VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 82. Preferably, the antibody comprises a VH region, containing the amino acid sequence of SEQ ID NO: 81, and VL containing the amino acid sequence of SEQ ID NO: 82.

Another object of the present invention relates to an antibody (10B10-TSV). The antibody may include a first light chain that contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 67. The antibody may include a second light chain, which contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 68. The antibody may include a first heavy chain that contains an amino acid sequence that at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 69. The antibody may include a second heavy chain that contains an amino acid sequence that is at least about 95% 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 70. In a preferred embodiment of the present invention, the antibody comprises a first light chain containing the amino acid sequence of SEQ ID NO: 67, a second light chain containing the amino acid sequence SEQ ID NO: 68, a first heavy chain containing the amino acid sequence of SEQ ID NO: 69 and a second heavy chain containing the amino acid sequence of SEQ ID NO: 70.

Examples

The following are examples of the methods and compositions of the present invention. It should be appreciated that other embodiments of the present invention as described above in the general description may be applied.

Example 1 Expression of tumor targets.

To identify differential genes expressed by multiple myeloma cells compared to normal plasma cells, RNAseq was performed on 10 samples obtained from patients with multiple myeloma (MM) and 10 plasma cells (plasma cell, PC) obtained from the bone marrow of healthy donors. RNA was extracted using the RNeasy Micro kit (Qiagen) according to the manufacturer's instructions. Genomic DNA was removed using an RNase free DNase kit (Qiagen) in RNA extraction. The quality of the extracted RNA is monitored on Agilent Eukaryote Total RNA picochips (Agilent Technologies). The Illumina SMARTer Ultra Low RNA Sequencing Kit (Clontech) was used to generate and amplify cDNA from 1.6 ng of total RNA according to the manufacturer's instructions. Then, 1 ng of the amplified cDNA was processed by the Nextera XT library (Illumina) according to the manufacturer's instructions. Sequencing libraries were quantified using the Kapa Library Quantification Kit (Kapa Biosystems) and quality controlled by bioanalyzer capillary electrophoresis using High Sensitivity chips (Agilent Technologies). Libraries were sequenced on a HiSeq2500 sequencer (Illumina) for 2 x 50 cycles using version 4 clustering kits and version 4 sequencing reagents (Illumina).

B-cell maturation antigen (BCMA) is a cell surface protein that is expressed on malignant plasma cells and thus recognized as a target of multiple myeloma (Tai Y.T., Anderson K.C., Immunotherapy, 7(11 ), 2015, 1187-1199). Deep analysis using RNAseq technology showed that GPRC5D is expressed at the same high level as BCMA in the plasma cells of patients with multiple myeloma (Fig. 2). More importantly, GPRC5D expression in plasma cells from patients with multiple myeloma and healthy plasma cells differs by about 20-fold. In contrast, the differential expression of BCMA between plasma cells of patients with multiple myeloma and healthy plasma cells only differed by a factor of 2. In general, GPRC5D expression is much higher than that of other known multiple myeloma target molecules such as SLAM7, CD138 and CD38. In addition, GPRC5D expression is hindered by naive or memory B cells.

Example 2 Generation of GPRC5D binders and production of T-cell bispecific (TSV) antibodies.

GPRC5D binders are produced by immunizing rats with DNA followed by hybridoma preparation, screening, and sequencing of the hybridoma. Screening for specific binding is measured by ELISA by binding to a transfectant expressing GPRC5D. The two GPRC5D coupling agents are identified as 5E11 (SEQ ID NOs: 13 and 14) and 5F11 (SEQ ID NOs: 15 and 16) below. Once the specific binders have been identified, the IgG is converted into T-cell bispecific antibodies. The principles for converting binders into T-cell bispecific antibodies have been studied and described in the art, for example, in WO 2014/131712 A1, the substance of which is incorporated herein by reference. The anti-T cell bispecific antibodies comprise two GRKC5D binding fragments and one CD3 binding fragment (anti-GPRC5D/anti-CD3 T cell bispecific antibodies) shown in FIG. 3. The following anti-GRKC5D/anti-CD3 T cell bispecific antibodies are prepared: i) 5E11-TCB (SEQ ID NOs: 17, 18, 19 and 20); ii) 5F11-TCB (SEQ ID NOS: 21, 22, 23 and 24); iii) ET150-5-TCB (SEQ ID NOS: 25, 26, 27 and 28);

iv) B72-TCB (SEQ ID NOS: 73, 74, 75 and 76); and v) BCMA-TCB (SEQ ID NOS: 77, 78, 79 and 80). The ET150-5 GPRC5D linking fragment is described in WO 2016/090329 A2. The term "ET-150-5" is equivalent to the term "ET150-5" used in the present invention and vice versa. Untargeted DP47-TCB is obtained as a negative control. DP47-TCB is a non-targeted T cell bispecific antibody that only binds to CD3 but does not bind to GPRC5D. DP47-TCB is described in WO 2014/131712 A1, the gist of which is incorporated herein by reference. B72-TCB is a derivative of the GCDB72 antibody described in Table 1. 23 in WO 2018/0117786 A2 and contains the GPRC5D binding fragment of GCDB72. B72-TCB is generated in a 1+1 crossmab format (SEQ ID NOs: 73, 74, 75 and 76). BCMA-TCB is derived from that described in WO 2016/166629 A1 and contains the GPRC5D-binding fragment A02_Rd4_6nM_C01 as described in said patent. BCMA-TCB is generated in a crossmab 2+1 format (SEQ ID NOs: 77, 78, 79 and 80).

Example 3 Binding of T-cell bispecific antibodies to multiple myeloma cell lines.

To measure binding to GPRC5D, fluorescent cell sorting (FACS)-based binding was assayed (Lombardi et al., Genes Chromosomes Cancer. Mar; 46(3), 2007, 226-238.). Cell lines AMO-1, L363, and OPM-2 are cultured in RPMI 1640 + Glutamax (Gibco) supplemented with 20% heat-inactivated fetal calf serum (Fetal Bovine Serum, FBS; Gibco) and 1% penicillin-streptomycin 100X ( Gibco). The WSU-DLCL2 cell line (negative control) was cultured in the same medium but enriched with only 10% FBS. Cell lines NCI-H929 and RPMI-8226 were also cultured in the same medium supplemented with 50 μM mercaptoethanol (Gibco) and 1 mM sodium pyruvate (Gibco). The cell lines are cultured in 75 cm flasks (TPP) at two passages per week.

Binding of various anti-GPRC5D human-TSV antibodies (5E11-TSB, 5F11-TCB and ET 150-5 TCB) was assessed using indirect staining.

Cells are incubated with GPRC5D-TCB constructs (5E11-TCB, 5F11-TCB, or ET150-5 TCB) ranging from 10 µg/mL to 0.00064 µg/mL using a serial dilution factor of 0.2, or no construct at 100 µl of phosphate-buffered saline (PBS; Gibco) for 1 h at 4°C. Cells are stained with Live blue (Life Technologies) diluted 1:800 in PBS for 20 min at 4°C before staining with PE conjugated goat anti-human IgG, Fcy fragment specific (Jackson Laboratories) diluted 1/300 in buffer for staining for liquid cytometry (firm eBioscience), incubated for 30 min at 4°C. Liquid cytometry is performed on a custom designed BD Biosciences Fortessa cytometer and analyzed using FlowJo software (Tree Star, Ashland, Oregon) and GraphPad Prism software.

Fig. 4A-B show that both 5E11-TCB and 5F11-TCB bind all multiple myeloma cell lines tested in a dose-dependent manner. In contrast, ET150-5-TCB binds much less strongly to the tested cell lines. No binding to WSU-DLCL2 cells (GPRC5D non-Hodgkin's lymphoma cell line) of anti-GPRC5D-TCB was observed.

Example 4 Anti-GPRC5D-TSV mediated T-cell cytotoxicity.

To measure the functionality of anti-GPRC5D-TCB antibodies, an in-vitro T cell cytotoxicity assay is performed. Briefly, AMO-1, L363, and OPM-2 cell lines were cultured in RPMI 1640 + Glutamax (Gibco) supplemented with 20% heat-inactivated fetal calf serum (Fetal Bovine Serum, FBS; Gibco) and 1% penicillin-streptomycin 100X. (Gibco). Cell lines NCI-H929 and RPMI-8226 were cultured in the same medium supplemented with 50 μM mercaptoethanol (Gibco) and 1 mM sodium pyruvate (Gibco). The cell lines are cultured in 75 cm ² flasks (TPP) at two passages per week.

The cell lines were co-cultured at a target:effector ratio of 1:10 with 3.105 allogeneic T cells isolated from peripheral blood mononuclear cells (PBMCs) (buffy coat from Blutspende Schlieren) using a human Pan T cell isolation kit (Miltenyi Biotec). ) in IMDM (Gibco) supplemented with 10% PBS (Gibco) + 1% PS (Gibco). Human anti-GPRC5D-TCB antibodies (5E11-TCB, 5F11-TCB, ET150-5TCB or DP47-TCB) are added to the co-culture at different concentrations ranging from 1 µg/mL to 0.000001 µg/mL in serial dilution with a coefficient of 0.1 or 0 µg/ml. After 20 hours of incubation at 37°C with 5% CO ₂ , 75 μl of the supernatant was transferred into the wells of a 96-well plate (Greiner bio-one) with a 25 μl/well CytoTox-Glo Cytotoxicity Assay (Promega). The resulting luminescence was recorded using a PerkinElmer En Vision recorder after 15 min incubation at room temperature and analyzed using GraphPad Prism and XL fit software. The data is presented as a luminescence signal for LDH release.

Fig. 5A-E show that both 5E11-TCB and 5F11-TCB mediate strong T cell cytotoxicity against multiple myeloma cell lines, especially NCI-H929 (FIG. 5B), RPMI-8226 (FIG. 5C), L363, and (FIG. 5D) AMO-1 (FIG. 5A), while no death was observed in the WSU-DLCL2 negative control cell line (FIG. 5E). In contrast, ET150-5-TCB mediated little or much less killing of tested multiple myeloma cell lines. In table. 1 summarize the EC ₅₀ values obtained from the data presented in FIG. 5A-D. The EC ₅₀ value is calculated using the optional XLfit function in Excel by plotting the raw signal data against the titrated TCB.

Example 5 Anti-GPRC5D-TCB mediated T cell activation

In order to mechanically influence the modes of action of anti-GPRC5D-TCB, T cell activation was measured after co-culture with multiple myeloma target cell lines in the presence of anti-GPRC5D-TCB. A similar experiment is described in Example 4 and in FIG. 5A-E, cell lines co-cultured at a target:effector ratio of 1:10 with 3.105 allogeneic T cells isolated from peripheral blood mononuclear cells (PBMCs) (buffy coat from Blutspende Schlieren) using a human Pan T cell isolation kit. (Miltenyi Biotec) in IMDM Medium (Gibco) enriched with 10% FST (Gibco) + 1% PS (Gibco). Human anti-GPRC5D-TCB antibodies (5E11-TCB, 5F11-TCB, ET150-5-TCB or DP47-TCB) are added to the co-culture at various concentrations ranging from 1 µg/mL to 0.000001 µg/mL with serial dilution of 0.1 or 0 µg/ml. After 20 h incubation at 37° C. with 5% CO ₂ , cells are stained to assess T cell activation. Cells are first stained with Live blue (Life Technologies) diluted 1:800 in PBS (Gibco) for 20 minutes at 4°C. The cells were then stained with AF700 human anti-CD4 (clone OKT4), BV711 human anti-CD8 (clone SKI), BV605 anti-human CD25 (clone BC96), APC-Cy7 human anti-CD69 (clone FN50), all from BioLegend, and PE-Cy5.5 human anti-CD3 (clone SK7; eBioscience) in liquid cytometry staining buffer (eBioscience) for 30 min at 4°C. Flow cytometry was performed on a custom designed BD Biosciences Fortessa cytometer and analyzed using FlowJo software (Tree Star, Ashland, OR) and GraphPad Prism.

In FIG. 6 shows that 5F11-TCB induces T cell activation in co-cultures with NCI-H929 cells by upregulating the activation marker CD25 and CD69, while controls, such as untargeted DP47-TCB without any TCB, do not induce T activation. -cells. As another negative control, 5F11-TCB treated T cells were co-cultured with WSU-DLCL2 cells, and the T cells were also not activated. These activation profiles are consistent across many cell lines studied, eg AMO-1, NCI-H929, RPMI-8226, L363 (FIGS. 7A-K). Consistent with low killing efficiency, ET150-5-TCB does not induce T cell activation except at the highest concentration tested of 1 mg/kg.

Example 6 Localization and Internalization of Anti-GRKC5D-TSV

NCI-H929 cells were stained with CMFDA (Invitrogen) and plated on poly-E-lysine-coated round coverslips (Sigma) in 24-well plates. Antibodies (5E11-IgG, 5E11-TCB, 5F11-IgG, 5F11-TCB) are labeled with Alexa Fluor 647 succinimidyl ester (InVitrogen, catalog number A201106) at a molar ratio of 2.5. Cells are allowed to attach overnight at 37° C. before adding fluorescently labeled antibodies (Alexa Fluor 647, labeled with 5E11-IgG, -5E11-TCB, -5F11-IgG, -5F11-TCB) directly to the growth medium at various times. and temperature (30 min on ice, 1 hour at 37°C and 3 hours at 37°C). Cold PBS (Lonza) is used to quench the reaction and wash off unbound antibodies after each checkpoint. The cells are then fixed with Cytofix (BD) for 20 min at 4° C. and washed twice with PBS. The coverslips are then transferred to Fluoromount G slides (eBioscience) and stored in the dark at 4° C. overnight prior to imaging. Fluorescent confocal microscopy is performed on an inverted microscope LSM 700 from Zeiss with a 60x immersion objective. Images were collected using Zen software (Zeiss) built into the microscope and visualized using IMARIS software (Bitplane). In FIG. 8A shows that all antibodies stain the surface (plasma membrane) of a multiple myeloma cell line at 4°C or 37°C. If the antibodies are internalized by the cells, fluorescent staining appears in the cytoplasm when cultured at 37°C. No internalization of GPRC5D-binding IgG or GRKC5D-binding TCB by GPRC5D cell lines was observed. This was further confirmed by assessing the total intensity for certain areas of cells defined by the membrane and cytoplasm (after three hours). The IMARIS software is used to analyze and quantify the ratio of signals from the membrane to the cytoplasm. Fig. 8B shows that 3 h after incubation with various antibodies, the ratio of membrane to cytoplasm intensity did not change and was ~4, which means that the fluorescent signals are concentrated on the surface and not in the cytoplasm.

Example 7 Description of GPRC5D binders: Recombinant cell binding as determined by ELISA

Stably transfected CHO clones expressing human GPRC5D or cynomolgus macaque GPRC5D or rodent GPRC5D or human GPRC5A are used to assay the binding of potential leader candidate IgG antibodies. More specifically, 10 ⁴ cells (survival ≥98%) were seeded in 384-well microtiter plates (poly D-lysine, BD, catalog number 356662; volume: 25 μl/well) using fresh culture medium. After overnight incubation at 37°C, 25 µl/well dilutions of antibodies (15×1:3 dilutions in 1×pBS, concentrations start at 30 µg/ml) are added to the cells for 2 h at 4°C . After a washing step using 90 µl/well PBST (10x PBS, Roche, #11666789001 + 0.1% Tween 20), cells are sequentially fixed with 50 µl/well 0.05% glutaraldehyde (Sigma catalog number G5882 ; in 1×pSB) for 10 min at room temperature (RT). After three additional washes with 90 µl/well PBST, secondary antibodies are added for detection: for human antibodies, use (25 µl/well) a goat anti-human Ig antibody conjugated to HPR (Millipore, catalog number AP502P) is used diluted 1:2000 in blocking buffer (1 x PSB (Roche, cat. no. 11666789001) + 2% BSA (Bovine Serum Albumin Fraction V, free of fatty acids; Roche, cat. no. 10735086001) + 0.05% Tween 20). For rat antibodies, a mixture of a goat anti-rat IgG2a antibody conjugated to HPR (Bethyl, catalog number A110-106P), a goat anti-rat IgG2a antibody conjugated to HPR (Bethyl, catalog number A110-109P), and goat anti-rat IgG2b antibody conjugated to HPR (Bethyl, catalog number A110-111P) at a 1:10,000 dilution of each antibody in blocking buffer (25 μl/well). After incubation for 1 hour at RT and three additional washing steps using 90 µl/well PBST, add 25 µl/well TMB substrate (Roche, cat. no. ) is determined by measurement at 370 nm/492 nm.

All antibodies tested showed positive binding to human GPRC5D with EC ₅₀ values (reflecting avidity) in the pM range. Only 10B10 and 07A04 rat IgGs show cross-reactivity on CHO cells expressing cynomolgus macaque GPRC5D with EC ₅₀ values comparable to the human receptor version (FIG. 9). Cynomolgus cross-reactivity was also found for all other antibodies, but at lower levels compared to 10B10 and 07A04 (FIG. 9). No significant binding was found to CHO cells expressing mouse GPRC5D and no binding was found to CHO cells expressing the human version of GPRC5A (FIG. 9). EC ₅₀ binding values are summarized in Table 1. 2.

Example 8 GPRC5D Bindings: GPRC5D-TCB recombinant mediates T cell cytotoxicity in the MM cell line.

To compare the functionality of GPRC5D-TCB or other targeted TCBs, an in vitro T-cell cytotoxicity assay was performed on multiple myeloma (MM) cell lines: MOLP-2 (Fig. 10B), AMO-1 (Fig. 10C), EJM (Fig. 10D). ) and NCI-H929 (Fig. 10G). Briefly, cell lines were cultured in RPMI 1640 + GlutaMax (Gibco) supplemented with 20% heat-inactivated fetal calf serum (FBS; Gibco) and 1% penicillin-streptomycin 100X (PS; Gibco). MOLP-2 is cultured in this medium enriched with GlutaMax IX (Gibco). OPM-2 (FIG. 10A), RPMI-8226 (FIG. 10D) and L-363 (FIG. 10E), the cell line was cultured in the same medium supplemented with only 10% PBS. NCI-H929 is cultured in this medium supplemented with 50 μM mercaptoethanol (Gibco), 1 mM sodium pyruvate (Gibco) and GlutaMax 1X (Gibco). EJM is cultured in IMDM (Gibco) + 10% PBS (Gibco) and 1% PS (Gibco). All cell lines are cultured in 75 cm ² flasks (TPP) at two passages per week.

The cell lines are co-cultured at a 10:1 effector/target ratio using 0.3 million allogeneic T cells isolated from PBMC (buffy coat from Blutspende Schlieren) using a human Pan T cell isolation kit (Miltenyi Biotec) in RPMI (Gibco) supplemented with 10% PS (Gibco) + 1% PS (Gibco). The human anti-GRKC5D construct TCB (5E11-TCB, 5F11-TCB, 10 B10-TCB, B72-TCB, BCMA-TCB and DP47-TCB) is added to the co-culture at various concentrations, from 12.5 nM to 0.0000125 nM with a serial dilution of 1/10 and compared with untreated samples. After 20 hours of incubation at 37° C. with 5% CO ₂ 75 μl of the supernatant per well was transferred to a 96-well white plate (Greiner bio-one) with 25 μl per well of the CytoTox-Glo Cytotoxicity Assay (Promega). Luminescence acquisition was performed on a PerkinElmer En Vision after 15 min incubation at room temperature and analyzed using GraphPad Prism and XL fit software. The data is plotted as a luminescence signal for LDH release (FIG. 10). In FIG. 10A-G summarize data showing that both 5E11-TCB and 5F11-TCB mediate stronger T cell cytotoxicity against MM cell lines than BCMA-TCB, 10B10-TCB, and B72-TCB. EU ₅₀ mediated TSV destruction is shown in table. 3 and calculated as the mean value from different experiments with different donor T cells (n=2 or n=3).

Example 9 In Vitro Activation of T Cells in Healthy Human Bone Marrow Cells.

Fresh unprocessed bone marrow from four different healthy donors (Lonza, catalog number 1M-105, lots 0000739254; 0000739255; 0000739256 and 0000734008) are processed after 1 or 2 days after sampling. After a quick red blood cell lysis using BD Pharm Lysis buffer (BD catalog number 555899; 1X in sterile water) for 5 minutes at CT; cells are washed twice, centrifuged and buffer exchanged at 126g and 443g, respectively. Cells are counted and resuspended to 300,000 cells/ml in RPMI 1640 Glutamax + 20% HI fetal calf serum + 2% human serum + 1% penicillin/streptomycin (all reagents from Gibco) and 100 µl of the cell suspension are seeded into wells in 96-well round bottom plate (TPP). 50 µl of medium or medium enriched with B72-TSB, 5F11-TCB, 5E11-TSB, BCMA-TSB, 10B10-TSB or DP47-TCB, from 200 nM (4X) to 20 pM in a sterile 1/10 dilution is added to the wells . Finally, 50 µl of allogeneic T cells isolated using Pan T cells (Miltenyi Biotec, cat. no. 130-096-535) from healthy donor PBMCs were added at 6 million/ml (ratio of effector T cells and healthy bone marrow target cells 10:1). After an overnight incubation at 37°C in a humidified incubator, the cells are washed once with PBS and stained for 20 min at 4°C with 50 µl of Live blue dye (Invitrogen, catalog number L23105) diluted 1/800 in PBS. After washing, the cells are incubated for 30 min at 4° with the following mixture of antibodies diluted in PAC buffer (PBS 1X, 2% fetal calf serum; 1% 0.5 M EDTA pH 8; 0.25% NaNs sodium azide (20% )): CD25 BV605, CD69 APC-Cy7, BCMA BV421, CD38 BV510, CD138 FITC, FcRH5 PE diluted 1/100, and CD8 BV711, CD3 PE-Cy5 and CD4 AlexaFluor 700 diluted 1/300 (all reagents from BioLegend), and GPRC5D AlexaFluor 647 (in-house, clone 5E11 IgG). After washing, the cells are resuspended in 100 μl of PAC buffer and harvested with a Fortessa fluorimeter (BD Biosciences).

The data shown in FIG. 11A-E show that B72-TSV induces non-specific T cell activation (as measured by CD69 activation) in healthy bone marrow, but not any other TSV tested. The non-specific activation induced by B72-TSV was shown to be concentration dependent and more pronounced at 50 nm than at 5 nm (FIGS. 12A and 12B).

Example 10 In Vivo Efficacy of TCB

In an efficacy study, different TCB constructs (GPRC5D 5F110-TCB, 5E11-TCB, BCMA-TCB, and B72-TCB) were compared for tumor regression in fully humanized NSG mice with multiple myeloma. NCI-H929 cells were initially obtained from the ATCC collection and OPM-2 cells from the DSMZ collection. Cells of both lines proliferated. Cells are cultured in RPMI medium containing 10% FST and 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate. Cells are cultured at 37°C in a humid atmosphere at 5% CO ₂ . 2.5×10 ⁶ NCI-H929 and 5×10 ⁶ OPM-2 cells per animal were injected subcutaneously in the right flank in cell culture medium RPMI (Gibco) and GFR matrigel (1:1, total volume 100 μl) at viability >95.0%.

Female NSG mice (NOD.Cg-Prkdcscid Il2rgtmlWjl/SzJ) 4-5 weeks old from the start of the experiment (breeding at Charles River, Lyon, France) are maintained under pathogen-free conditions with a daily cycle of 12 h light/12 h darkness in accordance with established recommendations (GV-Solas; Felasa; TierschG). The pilot study protocol has been reviewed and approved by the local authorities (ROB-55.2-2532. Vet_03-16-10). After arrival, the animals are kept for one week to get used to the new environment and for observation. Continuous health monitoring is carried out on a regular basis.

According to the protocol, female NSG mice are intraperitoneally injected at a rate of 15 mg/kg of busulfan, and then 1×10 ⁵ human hematopoietic stem cells isolated from umbilical cord blood are intravenously injected a day later. At 16-20 weeks after stem cell injection, mice are bled and analyzed by liquid cytometry for successful humanization. Effectively vaccinated mice are randomized according to the frequency of human T-lymphocytes in different treatment groups (n=10/group). At this time, mice are subcutaneously injected with tumor cells as described above and treated once a week with the compounds or PBS (solvent) when the tumor size reached approximately 200 mm ³ . All mice were intravenously injected with various doses of TCB molecules (see FIGS. 13A-D and 14A-D).

Stock solutions are diluted with histidine buffer (20 mM histidine, 140 mM NaCl, pH 6.0) to obtain the required amount of compounds. Tumor growth is measured twice a week with a caliper and the tumor volume is calculated as follows:

Tumor volume = (W ² /2)×D

(W: Width, L: Length)

The study is terminated and all mice are sacrificed after four compound injections, tumors are explanted and weighed.

Fig. 13A-D show the kinetics of tumor growth in all animals that received NC1-H929 injections after treatment. 5F11-TSV-induced complete tumor remission in all animals at 1 mg/kg or 0.1 mg/kg (Fig. 13A), although B72-TSV induces only partial tumor remission at 1 mg/kg, in the absence of effect at a dose of 0.1 mg/kg (Fig. 13B). BCMA-TSV also induces partial tumor remission at 1 mg/kg (FIG. 13B).

Fig. 14A-D show the kinetics of tumor growth in all animals that received ORM-2 injections after treatment. 5F11-TCB (Fig. 14A, upper panel) and 5E11-TCB (Fig. 14B, upper panel) induce complete tumor remission in most animals at 0.1 mg/kg, while B72-TCB (Fig. 14C, upper panel) panel) at a dose of 0.1 mg/kg shows less activity in stopping tumor growth. At a dose of 0.01 mg/kg, 5F11-TCB (Fig. 14A, lower panel) and 5E11-TCB (Fig. 14B, upper panel) suppressed tumor growth more than B72-TCB (Fig. 14C, lower panel) .

Example 11 Humanization of Anti-GRKC5D Antibodies

Suitable human acceptor frameworks are identified by querying the BLASTp database for sequences of human V and J regions to enter rodent sequences (truncated to variable portion). Selective criteria for selection of the human acceptor framework were sequence homology, CDR length proximity or identity, and putative human germline frequency, as well as retention of certain amino acids at the VH-VL domain interface. After the germline identification step, the CDRs of the introduced rodent sequences are transferred into human acceptor frameworks. Each amino acid difference between these original CDR grafts and the original antibodies is evaluated for possible impact on the structural integrity of the respective variable region, and "backmutations" to the original sequence are introduced as appropriate. Structural evaluation is based on Fv region homology models of both the parental antibody and humanized variants developed by the inventors' antibody structure homology modeling protocol implemented using the BIOVIA Discovery Studio Environment, version 17R2. Some humanized variants include "forward mutations", i.e., amino acid substitutions that change the original amino acid present at a given CDR position of the original binder to an amino acid found at an equal position in the human germline acceptor. The aim is to enhance the overall human properties of the humanized variants (outside of the framework regions) to further reduce the risk of immunogenicity.

The in silico tool developed by the present inventors is used to predict the orientation of the VH-VL domain of paired VH and VL humanized variants (similar to that set forth in WO 2016/062734 A1, the spirit of which is incorporated herein by reference). The results are compared with the prediction of the orientation of the VH-VL domain of the original binders to select combinations of framework regions that are close in geometry to the original antibodies. It is rational to discover possible amino acid exchanges in the region of the VH-VL interface, which can lead to disruptive changes in the pairing of the two domains, which in turn can have detrimental effects on binding properties.

Choice of Acceptor Framework and Its Adaptation for GPRC5D Binder 5F11

Acceptor framework regions are selected from those presented in table. 4.

Post-CDR3 framework regions are adapted from the human IGHJ germline IGHJ3*02 (DAFDIWGQGTMVTVSS) and the human IGKJ germline IGKJ5*01 (ITFGQGTRLEIK). The portion relevant to the acceptor framework is shown in bold.

Based on structure analysis, backmutations from the human acceptor framework for the amino acid in the parent binder are introduced at specific positions in the humanized 5E11 variants (Tables 5 and 6). In addition, certain positions are identified as promising direct mutation candidates where an amino acid in the CDR of the original binder is replaced with an amino acid found in the human acceptor germline. The changes are detailed in the table below.

Note. Backmutations are prefixed with b, forward mutations with f, for example, bS49A denotes a backmutation (human germline amino acid relative to the amino acid of the parent antibody) from serine to alanine at position 49. All residues are numbered according to the Kabat system.

Choice of Acceptor Framework and Its Adaptation for GPRC5D Binder 5F11

Acceptor framework regions are selected from those presented in table. 7.

Human V-region germline identity

Post-CDR3 frameworks are adapted from the human IGHJ germline IGHJ3*02 (DAFDIWGQGTMVTVSS) and the human IGKJ germline IGKJ5*01 (YTFGQGTKLEIK). The portion relevant to the acceptor framework is shown in bold.

Based on structure analysis, backmutations from the human acceptor framework for the amino acid in the parent binder are introduced at specific positions in the humanized 5E11 variants (Tables 8 and 9). In addition, certain positions are identified as promising direct mutation candidates where an amino acid in the CDR of the original binder is replaced with an amino acid found in the human acceptor germline. The changes are detailed in the table below.

Note. Backmutations are prefixed with b, forward mutations with f, for example, bA93T denotes a backmutation (human germline amino acid relative to the amino acid of the parent antibody) from alanine to threonine at position 93. All residues are numbered according to the Kabat system.

Description of Humanized Variants by ELISA To characterize humanized variants of the VH and VL domains of GPRC5D binders, the ELISA protocol described above is used (see example 7). The data are summarized in Table. 10 for humanized variants 5E11 and in table. 11 for humanized variants of 5F11. In table. 12 shows the CDR sequences of parental 5E11 and parental 5E11 and selected humanization variants.

Example 12 In Vitro Activation of CAR-J Cells in the Presence of Various Humanized Variants of Selected Anti-GRKC5D IgGs

The ability of various humanized anti-GPRC5D IgGs to activate PGLALA-CAR-J effector cells was evaluated as described below. GRKC5D-expressing L363 multiple myeloma target cells (Diehl et al., Blut 36, 1978, 331-338) are co-cultured with anti-PGLALA-CAR-J effector cells (human acute lymphatic leukemia reporter cell line Jurkat-NFAT expressing TCR -directed against PGLALA (P329G L234A L235A) mutation in the Fc part of IgG molecules and containing the NFAT promoter, as described in PCT/EP2018/086038 and PCT/EP2018/086067.With simultaneous binding of the IgG molecule to GPRC5D on L363 cells and PGLALA-CAR cells -J, the NFAT promoter is activated and results in the expression of active firefly luciferase.

For analysis, humanized IgG variants are diluted in RPMI 1640 medium (containing Glutamax, 15% fetal calf serum HI, 1% penicillin-streptomycin; all reagents are from GIBCO) and transferred to round-bottom 96-well plates (final concentration ranging from 0, 2 pg/ml to 10 µg/ml). 20,000 L363 cells per well and anti-PGLALA-CAR-J effector cells were added for a final effector (anti-PGLALA-CAR-J) to target (L363) ratio of 5:1 and a final volume of 200 μg/well. Cells are incubated for about 16 hours at 37° C. in a humidified incubator. At the end of the incubation, transfer 100 µl of the supernatant to the wells of a white flat-bottomed 96-well plate (Costar) and incubate with another 100 µl/well ONE-Glo luciferase substrate (Promega) for 5 min before reading the luminescence readings using a PerkinElmer Envision reader. . Row data is plotted as relative luminescence signals (RLU) versus IgG concentration using GraphPad Prism software and EC ₅₀ calculated using XL-fit software.

As shown in FIG. 15A-B and in the table. 13, all of the anti-GPRC5D IgGs assessed induce CAR-J activation while binding to target cells expressing GPRC5D and anti-PGLALA-CAR-J cells. For both anti-GPRC5D binders, 5F11 and 5E11, humanized variants can be identified with similar or even improved EC ₅₀ values compared to the original non-humanized antibodies. For the 5F11 binder, the strongest activation could be caused by the P1AE5741 molecule (FIG. 15A). For the 5E11 binder, the strongest activation could be caused by P1AE5730 and P1AE5723 molecules (Fig. 15B).

Although the foregoing invention has been described in detail by means of illustrations and examples for clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the present invention. The gist of all patent and scientific literature cited is incorporated herein by reference in its entirety.

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<220>

<223> Synthetic Construct

<400> 18

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 19

<211> 447

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 19

Glu Leu Gln Leu Glu Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

Ala Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Gln Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Thr His Thr Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 20

<211> 672

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 20

Glu Leu Gln Leu Glu Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

Ala Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Gln Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Thr His Thr Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly

210 215 220

Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Pro Ser

225 230 235 240

Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser

245 250 255

Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys

260 265 270

Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala

275 280 285

Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala

290 295 300

Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr

305 310 315 320

Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys

325 330 335

Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

340 345 350

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

355 360 365

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

370 375 380

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

385 390 395 400

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

405 410 415

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

420 425 430

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

435 440 445

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

450 455 460

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

465 470 475 480

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

485 490 495

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

500 505 510

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

515 520 525

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

530 535 540

Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr

545 550 555 560

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

565 570 575

Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

580 585 590

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

595 600 605

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

610 615 620

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

625 630 635 640

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

645 650 655

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665 670

<210> 21

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 21

Asp Ile Val Met Thr Gln Ala Pro Leu Ser Val Ser Val Thr Pro Gly

1 5 10 15

Glu Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Val Tyr Trp Tyr Phe Gln Lys Pro Gly Lys Ser

35 40 45

Pro Gln Val Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Glu Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr His Cys Gly Gln Leu

85 90 95

Leu Glu Asn Pro Tyr Thr Phe Gly Ala Gly Thr Glu Leu Glu Leu Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg

115 120 125

Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 22

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 22

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 23

<211> 446

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 23

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr

20 25 30

Gly Met Ala Trp Val Arg Gln Ala Ala Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Ile Val Ser Arg Asp Asn Ala Lys Asn Thr Gln Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Arg His Asp Arg Gly Gly Leu Tyr Trp Gly Gln Gly Val Met Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 24

<211> 671

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr

20 25 30

Gly Met Ala Trp Val Arg Gln Ala Ala Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Ile Val Ser Arg Asp Asn Ala Lys Asn Thr Gln Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Arg His Asp Arg Gly Gly Leu Tyr Trp Gly Gln Gly Val Met Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Pro Ser Leu

225 230 235 240

Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr

245 250 255

Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro

260 265 270

Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro

275 280 285

Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala

290 295 300

Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys

305 310 315 320

Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu

325 330 335

Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

340 345 350

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

355 360 365

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

370 375 380

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

385 390 395 400

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

405 410 415

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

420 425 430

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

435 440 445

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

450 455 460

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

465 470 475 480

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

485 490 495

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

500 505 510

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

515 520 525

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

530 535 540

Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile

545 550 555 560

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

565 570 575

Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

580 585 590

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

595 600 605

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

610 615 620

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

625 630 635 640

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

645 650 655

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665 670

<210> 25

<211> 216

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 25

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Val Gly Gly Asn

20 25 30

Tyr Val Phe Trp Tyr Gln Gln Val Pro Gly Ala Thr Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Ser Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ala

50 55 60

Gly Ser Lys Ser Gly Ser Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Ser Leu

85 90 95

Ser Gly Phe Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Ser Ser Ser Lys Lys

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 26

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 26

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 27

<211> 447

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 27

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Lys Ala Tyr Asp Gln Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 28

<211> 672

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 28

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Lys Ala Tyr Asp Gln Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly

210 215 220

Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Pro Ser

225 230 235 240

Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser

245 250 255

Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys

260 265 270

Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala

275 280 285

Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala

290 295 300

Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr

305 310 315 320

Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys

325 330 335

Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

340 345 350

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

355 360 365

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

370 375 380

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

385 390 395 400

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

405 410 415

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

420 425 430

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

435 440 445

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

450 455 460

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

465 470 475 480

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

485 490 495

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

500 505 510

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

515 520 525

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

530 535 540

Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr

545 550 555 560

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

565 570 575

Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

580 585 590

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

595 600 605

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

610 615 620

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

625 630 635 640

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

645 650 655

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665 670

<210> 29

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 29

Thr Tyr Ala Met Asn

15

<210> 30

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 30

Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser

1 5 10 15

Val Lys Gly

<210> 31

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 31

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr

1 5 10

<210> 32

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 32

Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

1 5 10

<210> 33

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 33

Gly Thr Asn Lys Arg Ala Pro

15

<210> 34

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 34

Ala Leu Trp Tyr Ser Asn Leu Trp Val

15

<210> 35

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 35

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 36

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 36

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 37

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 37

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 38

<211> 105

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 38

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 39

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 39

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 40

<211> 207

<212> PRT

<213> Homo sapiens

<400> 40

Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr

20 25 30

Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr

35 40 45

Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys

50 55 60

Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp

65 70 75 80

His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr

85 90 95

Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu

100 105 110

Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met

115 120 125

Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu

130 135 140

Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys

145 150 155 160

Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn

165 170 175

Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg

180 185 190

Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile

195 200 205

<210> 41

<211> 198

<212> PRT

<213> Macaca fascicularis

<400> 41

Met Gln Ser Gly Thr Arg Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Ile Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr

20 25 30

Gln Thr Pro Tyr Gln Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr

35 40 45

Cys Ser Gln His Leu Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys

50 55 60

Asn Lys Glu Asp Ser Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu

65 70 75 80

Met Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro

85 90 95

Glu Asp Ala Ser His His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn

100 105 110

Cys Met Glu Met Asp Val Met Ala Val Ala Thr Ile Val Ile Val Asp

115 120 125

Ile Cys Ile Thr Leu Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys

130 135 140

Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly

145 150 155 160

Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn

165 170 175

Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Gln Asp Leu Tyr Ser Gly

180 185 190

Leu Asn Gln Arg Arg Ile

195

<210> 42

<211> 225

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 42

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro

225

<210> 43

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 43

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 44

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 44

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 45

<211> 345

<212> PRT

<213> Homo sapiens

<400> 45

Met Tyr Lys Asp Cys Ile Glu Ser Thr Gly Asp Tyr Phe Leu Leu Cys

1 5 10 15

Asp Ala Glu Gly Pro Trp Gly Ile Ile Leu Glu Ser Leu Ala Ile Leu

20 25 30

Gly Ile Val Val Thr Ile Leu Leu Leu Leu Ala Phe Leu Phe Leu Met

35 40 45

Arg Lys Ile Gln Asp Cys Ser Gln Trp Asn Val Leu Pro Thr Gln Leu

50 55 60

Leu Phe Leu Leu Ser Val Leu Gly Leu Phe Gly Leu Ala Phe Ala Phe

65 70 75 80

Ile Ile Glu Leu Asn Gln Gln Thr Ala Pro Val Arg Tyr Phe Leu Phe

85 90 95

Gly Val Leu Phe Ala Leu Cys Phe Ser Cys Leu Leu Ala His Ala Ser

100 105 110

Asn Leu Val Lys Leu Val Arg Gly Cys Val Ser Phe Ser Trp Thr Thr

115 120 125

Ile Leu Cys Ile Ala Ile Gly Cys Ser Leu Leu Gln Ile Ile Ile Ala

130 135 140

Thr Glu Tyr Val Thr Leu Ile Met Thr Arg Gly Met Met Phe Val Asn

145 150 155 160

Met Thr Pro Cys Gln Leu Asn Val Asp Phe Val Val Leu Leu Val Tyr

165 170 175

Val Leu Phe Leu Met Ala Leu Thr Phe Phe Val Ser Lys Ala Thr Phe

180 185 190

Cys Gly Pro Cys Glu Asn Trp Lys Gln His Gly Arg Leu Ile Phe Ile

195 200 205

Thr Val Leu Phe Ser Ile Ile Ile Trp Val Val Trp Ile Ser Met Leu

210 215 220

Leu Arg Gly Asn Pro Gln Phe Gln Arg Gln Pro Gln Trp Asp Asp Pro

225 230 235 240

Val Val Cys Ile Ala Leu Val Thr Asn Ala Trp Val Phe Leu Leu Leu

245 250 255

Tyr Ile Val Pro Glu Leu Cys Ile Leu Tyr Arg Ser Cys Arg Gln Glu

260 265 270

Cys Pro Leu Gln Gly Asn Ala Cys Pro Val Thr Ala Tyr Gln His Ser

275 280 285

Phe Gln Val Glu Asn Gln Glu Leu Ser Arg Ala Arg Asp Ser Asp Gly

290 295 300

Ala Glu Glu Asp Val Ala Leu Thr Ser Tyr Gly Thr Pro Ile Gln Pro

305 310 315 320

Gln Thr Val Asp Pro Thr Gln Glu Cys Phe Ile Pro Gln Ala Lys Leu

325 330 335

Ser Pro Gln Gln Asp Ala Gly Gly Val

340 345

<210> 46

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 46

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

Ala Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr His Thr Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser

115

<210> 47

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 47

Glu Leu Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

Ala Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr His Thr Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser

115

<210> 48

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 48

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

Ala Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr His Thr Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser

115

<210> 49

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 49

Glu Leu Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

Ala Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr His Thr Gly Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser

115

<210> 50

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 50

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Ser Ile Ser

20 25 30

Gly Ile Asn Leu Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Ile Leu Ala Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Glu Ser Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 51

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 51

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Ser Ile Ser

20 25 30

Gly Ile Asn Leu Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Ile Leu Ala Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Glu Ser Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 52

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 52

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Ser

20 25 30

Gly Ile Asn Leu Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro

35 40 45

Arg Leu Leu Ile Tyr His Ala Ser Ile Leu Ala Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Glu Ser Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 53

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 53

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Ser

20 25 30

Gly Ile Asn Leu Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Ile Leu Ala Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Glu Ser Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 54

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 54

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ile Ser

20 25 30

Gly Ile Asn Leu Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Gln Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Ile Leu Ala Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Glu Ser Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 55

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 55

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ile Ser

20 25 30

Gly Ile Asn Leu Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Ile Leu Ala Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Glu Ser Pro Leu Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 56

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 56

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr

20 25 30

Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg His Asp Arg Gly Gly Leu Tyr Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser

115

<210> 57

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 57

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr

20 25 30

Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg His Asp Arg Gly Gly Leu Tyr Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser

115

<210> 58

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 58

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr

20 25 30

Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg His Asp Arg Gly Gly Leu Tyr Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser

115

<210> 59

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 59

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr

20 25 30

Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg His Asp Arg Gly Gly Leu Tyr Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser

115

<210> 60

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 60

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr

20 25 30

Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg His Asp Arg Gly Gly Leu Tyr Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser

115

<210> 61

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 61

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr

20 25 30

Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg His Asp Arg Gly Gly Leu Tyr Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser

115

<210> 62

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 62

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Val Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Val Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr His Cys Gly Gln Leu

85 90 95

Leu Glu Asn Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 63

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 63

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Val Tyr Trp Tyr Leu Gln Lys Pro Gly Lys Ser

35 40 45

Pro Gln Val Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr His Cys Gly Gln Leu

85 90 95

Leu Glu Asn Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 64

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 64

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Val Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr His Cys Gly Gln Leu

85 90 95

Leu Glu Asn Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 65

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 65

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Val Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr His Cys Gly Gln Leu

85 90 95

Leu Glu Asn Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 66

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 66

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Ile Thr Tyr Val Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Ile Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr His Cys Gly Gln Leu

85 90 95

Leu Glu Asn Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 67

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 67

Asp Ile Gln Leu Thr Gln Ser Pro His Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Gly Ile Ser Asn Tyr

20 25 30

Leu Ala Trp Phe His Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Ser Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Asn Met Gln Pro

65 70 75 80

Glu Asp Glu Gly Val Tyr Tyr Cys Gln Gln Gly Tyr Lys Tyr Pro Leu

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 68

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 68

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 69

<211> 452

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 69

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Phe

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Ala Leu Glu Trp Val

35 40 45

Ala Ser Ile Asn Thr Gly Gly Gly Tyr Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Thr Arg Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Glu Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Leu Thr Tyr Tyr Gly Arg Tyr Tyr Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

355 360 365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 70

<211> 677

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 70

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Phe

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Ala Leu Glu Trp Val

35 40 45

Ala Ser Ile Asn Thr Gly Gly Gly Tyr Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Thr Arg Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Glu Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Leu Thr Tyr Tyr Gly Arg Tyr Tyr Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val

225 230 235 240

Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu

245 250 255

Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

260 265 270

Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly

275 280 285

Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu

290 295 300

Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp

305 310 315 320

Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe

325 330 335

Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ser Ala Ser Thr Lys Gly

340 345 350

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

355 360 365

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

370 375 380

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

385 390 395 400

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

405 410 415

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

420 425 430

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

435 440 445

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala

450 455 460

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

465 470 475 480

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

485 490 495

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

500 505 510

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

515 520 525

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

530 535 540

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala

545 550 555 560

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

565 570 575

Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln

580 585 590

Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

595 600 605

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

610 615 620

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

625 630 635 640

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

645 650 655

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

660 665 670

Leu Ser Pro Gly Lys

675

<210> 71

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 71

Asp Val Gln Met Thr Gln Ser Pro Tyr Asn Leu Ala Ala Ser Pro Gly

1 5 10 15

Glu Ser Val Ser Ile Asn Cys Lys Ala Ser Lys Ser Ile Ser Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Asn Lys Leu Leu Ile

35 40 45

Tyr Asp Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Gly Leu Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Leu

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 72

<211> 454

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 72

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser His

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Met Gly Val Asn Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala Ser Ile Trp Trp Asn Gly Asn Thr Tyr Asn Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Val Ser Lys Asp Thr Ser Asn Asn Gln Ala

65 70 75 80

Phe Leu Lys Val Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Val His Thr Arg Gly Ile Ile Arg Gly Arg Gly Leu Phe Phe Asp

100 105 110

Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 73

<211> 441

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 73

Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ser Ala

100 105 110

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

115 120 125

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

130 135 140

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

145 150 155 160

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

165 170 175

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

180 185 190

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

195 200 205

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

210 215 220

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

225 230 235 240

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

245 250 255

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

260 265 270

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

275 280 285

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

290 295 300

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

305 310 315 320

Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

325 330 335

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Cys Arg Asp Glu Leu

340 345 350

Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro

355 360 365

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

370 375 380

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

385 390 395 400

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

405 410 415

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

420 425 430

Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 74

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 74

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 75

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 75

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Ser Asp Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Ala Leu Arg Val Ala Leu Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

355 360 365

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 76

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 76

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Ala Thr His

20 25 30

Val Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asn Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 77

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 77

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Thr Ala Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Pro Met Ser Leu Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 78

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 78

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ala Tyr

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Met Tyr Asp Ala Ser Ile Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Glu Arg Trp Pro

85 90 95

Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Arg Lys Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 79

<211> 670

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 79

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Thr Ala Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Pro Met Ser Leu Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser

210 215 220

Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr

225 230 235 240

Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly

245 250 255

Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly

260 265 270

Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly

275 280 285

Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu

290 295 300

Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala

305 310 315 320

Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr

325 330 335

Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

340 345 350

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

355 360 365

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

370 375 380

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

385 390 395 400

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

405 410 415

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

420 425 430

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

435 440 445

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

450 455 460

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

465 470 475 480

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

485 490 495

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

500 505 510

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

515 520 525

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

530 535 540

Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser

545 550 555 560

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

565 570 575

Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

580 585 590

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

595 600 605

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

610 615 620

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

625 630 635 640

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

645 650 655

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

660 665 670

<210> 80

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 80

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 81

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 81

Asp Ile Gln Leu Thr Gln Ser Pro His Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Thr Val Ser Ile Glu Cys Leu Ala Ser Glu Gly Ile Ser Asn Tyr

20 25 30

Leu Ala Trp Phe His Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Ser Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Ser Asn Met Gln Pro

65 70 75 80

Glu Asp Glu Gly Val Tyr Tyr Cys Gln Gln Gly Tyr Lys Tyr Pro Leu

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 82

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 82

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Phe

20 25 30

Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Ala Leu Glu Trp Val

35 40 45

Ala Ser Ile Asn Thr Gly Gly Gly Tyr Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Thr Arg Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Glu Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Leu Thr Tyr Tyr Gly Arg Tyr Tyr Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Val Met Val Thr Val Ser Ser

115 120

<210> 83

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 83

Gly Phe Thr Phe Ser Lys Tyr Ala Met Ala

1 5 10

<210> 84

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 84

Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Arg Asp Ser Val Lys

1 5 10 15

Ala

<210> 85

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 85

Ser Ile Ser Thr Gly Gly Val Asn Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 86

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 86

His Thr Gly Asp Tyr Phe Asp Tyr

15

<210> 87

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 87

Arg Ala Ser Gln Ser Val Ser Ile Ser Gly Ile Asn Leu Met Asn

1 5 10 15

<210> 88

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 88

His Ala Ser Ile Leu Ala Ser

15

<210> 89

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 89

Gln Gln Thr Arg Glu Ser Pro Leu Thr

15

<210> 90

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 90

Gly Phe Ser Phe Ser Asn Tyr Gly Met Ala

1 5 10

<210> 91

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 91

Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Arg Asp Ser Val Lys

1 5 10 15

gly

<210> 92

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 92

Ser Ile Ser Thr Gly Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 93

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 93

His Asp Arg Gly Gly Leu Tyr

15

<210> 94

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 94

Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Val Tyr

1 5 10 15

<210> 95

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 95

Arg Met Ser Asn Leu Ala Ser

15

<210> 96

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 96

Arg Met Ser Asn Arg Ala Ser

15

<210> 97

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic Construct

<400> 97

Gly Gln Leu Leu Glu Asn Pro Tyr Thr

15

<---

Claims (61)

1. An antibody or antigen-binding fragment thereof that binds to a class C G-protein receptor of a representative of group 5 D (GPRC5D) and includes: (i) 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: 84, 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, (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 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 a 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, (iv) a heavy chain variable region (VH) comprising a 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 a 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, or (v) a heavy chain variable region (VH) comprising a 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 a 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. 2. The antibody or its antigen-binding fragment according to claim 1, (i) wherein VH includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13, and VL includes an amino acid sequence that is at least least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14; or (ii) wherein VH includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15, and VL includes an amino acid sequence that is at least least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16; or (iii) wherein VH includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48, and VL includes an amino acid sequence that is at least least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53; or (iv) wherein VH includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 49, and VL includes an amino acid sequence that is at least least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 52; or (v) wherein VH includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57, and VL includes an amino acid sequence that is at least least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64; or (vi) wherein VH 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 VL includes an amino acid sequence that is at least at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 63. 3. An antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is an IgG antibody. 4. An antibody or antigen-binding fragment thereof according to claim 3, wherein the antibody is an IgG ₁ antibody. 5. An antibody or antigen-binding fragment according to any one of paragraphs. 1-4, where the antibody is a full length antibody. 6. An antibody or antigen-binding fragment according to any one of paragraphs. 1-4, wherein the antigen binding fragment is selected from the group consisting of an Fv molecule, an scFv molecule, a Fab molecule, and an F(ab') ₂ molecule. 7. A bispecific antigen-binding molecule that specifically binds to the G-protein receptor class C, representative of group 5 D (GPRC5D) and CD3, including: (a) a first antigen-binding fragment that binds to a first antigen, wherein the first antigen is GPRC5D and the first antigen-binding fragment comprises: (i) 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: 84, 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, (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, 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 complementarity determining region (HCDR) 1 of SEQ ID NO: 90, HCDR 2 of SEQ ID NO: 91, HCDR 3 of SEQ ID NO: 93, and a light chain variable region (VL) comprising a 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, (iv) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 90, HCDR 2 of SEQ ID NO: 91, HCDR 3 of SEQ ID NO: 93, and a light chain variable region (VL) comprising a 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, or (v) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 90, HCDR 2 of SEQ ID NO: 92, HCDR 3 of SEQ ID NO: 93, and a light chain variable region (VL) comprising a 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 (b) a second antigen-binding fragment that specifically binds to the second antigen. 8. The bispecific antigen-binding molecule according to claim 7, (i) wherein the VH of the first antigen-binding fragment 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 the fragment includes 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 fragment 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 the fragment includes 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 fragment 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 the fragment includes 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 fragment 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 the fragment includes 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 fragment 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 the fragment includes 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 fragment 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 the fragment includes 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. 9. The bispecific antigen-binding molecule according to claim 7 or 8, wherein the second antigen is CD3. 10. The bispecific antigen-binding molecule of claim 9, wherein the second antigen is CD3ε. 11. The bispecific antigen-binding molecule according to claim 9 or 10, wherein the second antigen-binding fragment comprises a VH region containing HCDR 1 with the sequence of SEQ ID NO: 29, HCDR 2 with the sequence of SEQ ID NO: 30, HCDR 3 with the sequence of SEQ ID NO: 31, as well as a VL region containing LCDR 1 with SEQ ID NO: 32, LCDR 2 with SEQ ID NO: 33, and LCDR 3 with SEQ ID NO: 34. 12. The bispecific antigen-binding molecule of claim 11, wherein the VH of the second antigen-binding fragment 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 wherein the VL of the second antigen-binding fragment 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: 36. 13. Bispecific antigennegative molecule according to any one of paragraphs. 7-12, in which the first and/or second antigen-binding fragment is a Fab molecule. 14. Bispecific antigennegative molecule according to any one of paragraphs. 7-13, in which the second antigen-binding fragment is a Fab molecule, wherein the VL and VH variable domains or the CL and CH1 constant domains, in particular the VL and VH variable domains of the Fab light chain and the Fab heavy chain, are replaced with each other. 15. Bispecific antigennegative molecule according to any one of paragraphs. 7-14, in which the first antigen-binding fragment is a Fab molecule, and in the constant domain the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) - Kabat numbering and the amino acid at position 123 is independently substituted with lysine ( K), arginine (R) or histidine (H) - Kabat numbering and in the CH1 constant domain the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) - Kabat numbering EU index and the amino acid at position 213 independently substituted with glutamic acid (E) or aspartic acid (D) - numbering according to the Kabat EU index. 16. Bispecific antigennegative molecule according to any one of paragraphs. 7-15, in which the first and second antigen binding fragments are fused to each other, optionally via a peptide linker. 17. Bispecific antigen-binding molecule according to any one of paragraphs. 7-16, wherein each first and second antigen-binding fragment is a Fab molecule, and wherein either (i) the second antigen-binding fragment 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 fragment, or (ii) the first antigen-binding fragment fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding fragment. 18. Bispecific antigen-binding molecule according to any one of paragraphs. 7-17 containing an Fc domain consisting of a first and a second subunit. 19. The bispecific antigen-binding molecule of claim 18, wherein each of the first and second antigen-binding fragments is a Fab molecule, and wherein either (i) the second antigen-binding fragment 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 fragment, and the first antigen-binding fragment 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 fragment 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 fragment and the second antigen-binding fragment is fused at The C-terminus of the Fab heavy chain with the N-terminus of the first subunit of the Fc domain. 20. The bispecific antigen-binding molecule of claim 18 or 19, wherein the Fc domain is an IgG Fc domain. 21. The bispecific antigen-binding molecule of claim 20, wherein the Fc domain is an IgG ₁ Fc domain. 22. Bispecific antigennegative molecule according to any one of paragraphs. 18-21, in which the Fc domain is a human Fc domain. 23. Bispecific antigen-binding molecule according to any one of paragraphs. 18-22, in which an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced by an amino acid residue having a larger side chain, thereby forming a ridge in the CH3 domain of the first subunit that can be located in a recess in the CH3 domain of the second subunit, and an amino acid residue in CH3 domain of the second subunit of the Fc domain is substituted with an amino acid residue having a smaller side chain, thereby forming a trough in the CH3 domain of the second subunit, within which the overhang of the first subunit CH3 domain can be located. 24. Bispecific antigennegative molecule according to any one of paragraphs. 18-23, in which the Fc domain contains one or more amino acid substitutions that reduce Fc receptor binding and/or effector function. 25. Selected polynucleotide encoding an antibody according to any one of paragraphs. 1-6. 26. An isolated polynucleotide encoding a bispecific antigen-binding molecule according to any one of paragraphs. 7-24. 27. An expression vector containing a polynucleotide according to claim 25. 28. An expression vector containing a polynucleotide according to claim 26. 29. A host cell designed to produce an antibody according to any one of paragraphs. 1-6, containing the vector according to item 27. 30. A host cell designed to obtain a bispecific antigen-binding molecule according to any one of paragraphs. 7-24 containing the vector of claim 28. 31. A method of obtaining an antibody that binds to GPRC5D according to any one of paragraphs. 1-6, which includes the steps of a) culturing the host cells according to claim 29 under conditions that ensure the expression of the antibody, and b) isolating the antibody. 32. Pharmaceutical composition for the treatment of an autoimmune disease, comprising an antibody according to any one of paragraphs. 1-6 or bispecific antigennegative molecule according to any one of paragraphs. 7-24 and a pharmaceutically acceptable carrier. 33. The use of antibodies according to any one of paragraphs. 1-6 for the treatment of multiple myeloma. 34. The use of a bispecific antigen-binding molecule according to any one of paragraphs. 7-24 for the treatment of multiple myeloma. 35. The use of an antibody according to any one of paragraphs. 1-6 for the preparation of a medicament for the treatment of multiple myeloma. 36. The use of a bispecific antigen-binding molecule according to any one of paragraphs. 7-24 to obtain a medicament for the treatment of multiple myeloma. 37. A method of treating multiple myeloma in an individual, comprising administering to said individual a therapeutically effective amount of a pharmaceutical composition according to claim 32.

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